39.
Salima Bahri; Ravi Shankar Palani; Robert Silvers; Brian Michael; Veronica Lattanzi; Ingemar André; Sara Linse; Robert G Griffin
High-resolution structure of monomorphic Aβ fibrils
In: Proc Natl Acad Sci U S A, 123 (28), pp. e2603575123, 2026, ISSN: 1091-6490.
Abstract | Links:
@article{pmid42418485b,
title = {High-resolution structure of monomorphic Aβ fibrils},
author = {Salima Bahri and Ravi Shankar Palani and Robert Silvers and Brian Michael and Veronica Lattanzi and Ingemar André and Sara Linse and Robert G Griffin},
doi = {10.1073/pnas.2603575123},
issn = {1091-6490},
year = {2026},
date = {2026-07-01},
journal = {Proc Natl Acad Sci U S A},
volume = {123},
number = {28},
pages = {e2603575123},
abstract = {Amyloid-β (Aβ) fibrils primarily composed of Aβ and Aβ form the core of senile plaques in Alzheimer's disease. Aβ fibrils may exhibit significant polymorphism influenced by sample preparation conditions, complicating atomic resolution structural characterization. To establish a reliable structural baseline, we developed a protocol for expressing and purifying recombinant Aβ that forms monomorphic fibrils under physiological conditions (pH 7.4). We present a high-resolution structure of these unseeded, monomorphic Aβ fibrils obtained using magic-angle spinning NMR spectroscopy (PDB ID 12GB). We obtained unambiguous chemical shift assignments for approximately 90% of the residues and measured over 500 distance and torsion angle restraints. The resolved structure, with a backbone RMSD of 0.63 ± 0.06 Å, shows two monomers per filament plane, with two distinct β-sheets (residues E11-E22 and K28-V39, respectively) running along the fibril axis with H-bonding between each plane, and the two strands linked by a flexible loop region. This structure reveals three continuous hydrophobic cores inside each filament which bury 24 hydrophobic side chains per filament plane: those of L17, F19, A21, V24, A30, I32, M35, V40 between the two β-strands within each monomer and I31, L34, V36, V39 between the two monomers. Small angle X-ray scattering reveals the size and geometry of the fibril cross-section, which is compatible with a two-filament arrangement with a total of 4 monomers per fibril plane.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Amyloid-β (Aβ) fibrils primarily composed of Aβ and Aβ form the core of senile plaques in Alzheimer's disease. Aβ fibrils may exhibit significant polymorphism influenced by sample preparation conditions, complicating atomic resolution structural characterization. To establish a reliable structural baseline, we developed a protocol for expressing and purifying recombinant Aβ that forms monomorphic fibrils under physiological conditions (pH 7.4). We present a high-resolution structure of these unseeded, monomorphic Aβ fibrils obtained using magic-angle spinning NMR spectroscopy (PDB ID 12GB). We obtained unambiguous chemical shift assignments for approximately 90% of the residues and measured over 500 distance and torsion angle restraints. The resolved structure, with a backbone RMSD of 0.63 ± 0.06 Å, shows two monomers per filament plane, with two distinct β-sheets (residues E11-E22 and K28-V39, respectively) running along the fibril axis with H-bonding between each plane, and the two strands linked by a flexible loop region. This structure reveals three continuous hydrophobic cores inside each filament which bury 24 hydrophobic side chains per filament plane: those of L17, F19, A21, V24, A30, I32, M35, V40 between the two β-strands within each monomer and I31, L34, V36, V39 between the two monomers. Small angle X-ray scattering reveals the size and geometry of the fibril cross-section, which is compatible with a two-filament arrangement with a total of 4 monomers per fibril plane.
38.
S Shirin Kamalaldinezabadi; Joshua I Santiago; Johanna E Papa; Yu Wang; Patrick A Frantom; Hong Li; Robert Silvers; A Carl Whittington; Brian G Miller
Evolution of Protein Regulation in the Vertebrate Glucose Sensor
In: bioRxiv, 2026, ISSN: 2692-8205.
Abstract | Links:
@article{pmid42146401,
title = {Evolution of Protein Regulation in the Vertebrate Glucose Sensor},
author = {S Shirin Kamalaldinezabadi and Joshua I Santiago and Johanna E Papa and Yu Wang and Patrick A Frantom and Hong Li and Robert Silvers and A Carl Whittington and Brian G Miller},
doi = {10.64898/2026.05.05.723016},
issn = {2692-8205},
year = {2026},
date = {2026-05-01},
journal = {bioRxiv},
abstract = {Protein regulation is essential for cellular function and mis-regulation commonly causes disease. Despite this fact, we know little about how new regulatory strategies first emerge and how they evolve to act in concert to control complex physiological processes. Glucokinase (GCK), the body's glucose sensor, lies at the heart of vertebrate glucose homeostasis and its activity is tightly controlled by multiple regulatory mechanisms. In the pancreas and liver, GCK is regulated by a unique form of monomeric allostery originating from the unliganded enzyme's conformational dynamics. In the liver, GCK and GKRP form an inhibitory protein-protein interaction that sequesters GCK within the hepatocyte nucleus. Using a vertical, evolutionary approach, we resurrected extinct GCKs and GKRPs along correlated evolutionary trajectories. Using enzyme kinetics, limited proteolysis, hydrogen-deuterium exchange, high resolution NMR, and X-ray crystallography we determined the historical and molecular origins of protein regulation. Prior to the emergence of jawed vertebrates, a non-regulated GCK ancestor underwent a conformational expansion leading to monomeric allostery. This novel conformation includes an intrinsically disordered substrate binding loop. Paradoxically, the emergence of disorder did not require sequence change in the loop. The new GCK conformation also exposed a hydrophobic cleft. In the jawed vertebrate GKRP ancestor, a loop insertion enabled exaptation of the pre-existing hydrophobic patch in GCK. Our results demonstrate how multiple, distinct regulatory strategies can arise at a central homeostatic control point through evolutionary addition of novel conformations. Additionally, our results provide a general mechanism for the emergence of heteromeric protein-protein interactions.
SIGNIFICANCE STATEMENT: Glucose homeostasis was a key innovation in vertebrate evolution. Here, we uncover the evolutionary basis of regulation in two key homeostatic proteins, glucokinase (GCK) and glucokinase regulatory protein (GKRP). We find that the unique cooperativity of vertebrate GCK resulted from an expansion of this enzyme's conformational landscape. This expansion included sampling a new state and the emergence of intrinsic disorder, which did not require substitutions in the disordered region itself. We also discover that the GCK-GKRP interaction emerged when a pre-existing hydrophobic surface - a structural spandrel resulting from prior conformational expansion - was co-opted by loop insertion in GKRP, facilitating a new, inhibitory heteromeric interaction. Our results demonstrate how multiple, mechanistically distinct regulatory strategies arise from an ability to sample new protein conformations.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Protein regulation is essential for cellular function and mis-regulation commonly causes disease. Despite this fact, we know little about how new regulatory strategies first emerge and how they evolve to act in concert to control complex physiological processes. Glucokinase (GCK), the body's glucose sensor, lies at the heart of vertebrate glucose homeostasis and its activity is tightly controlled by multiple regulatory mechanisms. In the pancreas and liver, GCK is regulated by a unique form of monomeric allostery originating from the unliganded enzyme's conformational dynamics. In the liver, GCK and GKRP form an inhibitory protein-protein interaction that sequesters GCK within the hepatocyte nucleus. Using a vertical, evolutionary approach, we resurrected extinct GCKs and GKRPs along correlated evolutionary trajectories. Using enzyme kinetics, limited proteolysis, hydrogen-deuterium exchange, high resolution NMR, and X-ray crystallography we determined the historical and molecular origins of protein regulation. Prior to the emergence of jawed vertebrates, a non-regulated GCK ancestor underwent a conformational expansion leading to monomeric allostery. This novel conformation includes an intrinsically disordered substrate binding loop. Paradoxically, the emergence of disorder did not require sequence change in the loop. The new GCK conformation also exposed a hydrophobic cleft. In the jawed vertebrate GKRP ancestor, a loop insertion enabled exaptation of the pre-existing hydrophobic patch in GCK. Our results demonstrate how multiple, distinct regulatory strategies can arise at a central homeostatic control point through evolutionary addition of novel conformations. Additionally, our results provide a general mechanism for the emergence of heteromeric protein-protein interactions.
SIGNIFICANCE STATEMENT: Glucose homeostasis was a key innovation in vertebrate evolution. Here, we uncover the evolutionary basis of regulation in two key homeostatic proteins, glucokinase (GCK) and glucokinase regulatory protein (GKRP). We find that the unique cooperativity of vertebrate GCK resulted from an expansion of this enzyme's conformational landscape. This expansion included sampling a new state and the emergence of intrinsic disorder, which did not require substitutions in the disordered region itself. We also discover that the GCK-GKRP interaction emerged when a pre-existing hydrophobic surface - a structural spandrel resulting from prior conformational expansion - was co-opted by loop insertion in GKRP, facilitating a new, inhibitory heteromeric interaction. Our results demonstrate how multiple, mechanistically distinct regulatory strategies arise from an ability to sample new protein conformations.
SIGNIFICANCE STATEMENT: Glucose homeostasis was a key innovation in vertebrate evolution. Here, we uncover the evolutionary basis of regulation in two key homeostatic proteins, glucokinase (GCK) and glucokinase regulatory protein (GKRP). We find that the unique cooperativity of vertebrate GCK resulted from an expansion of this enzyme's conformational landscape. This expansion included sampling a new state and the emergence of intrinsic disorder, which did not require substitutions in the disordered region itself. We also discover that the GCK-GKRP interaction emerged when a pre-existing hydrophobic surface - a structural spandrel resulting from prior conformational expansion - was co-opted by loop insertion in GKRP, facilitating a new, inhibitory heteromeric interaction. Our results demonstrate how multiple, mechanistically distinct regulatory strategies arise from an ability to sample new protein conformations.
37.
Nolan Blackford; Saileena Nepal; Lianqing Zheng; Wei Yang; Robert Silvers
Molecular Structure and DNA Binding Mode of Unsymmetric Cyanine Dyes RiboGreen and OliGreen
In: bioRxiv, 2026, ISSN: 2692-8205.
Abstract | Links:
@article{pmid42146639,
title = {Molecular Structure and DNA Binding Mode of Unsymmetric Cyanine Dyes RiboGreen and OliGreen},
author = {Nolan Blackford and Saileena Nepal and Lianqing Zheng and Wei Yang and Robert Silvers},
doi = {10.64898/2026.05.04.722657},
issn = {2692-8205},
year = {2026},
date = {2026-05-01},
journal = {bioRxiv},
abstract = {The binding of fluorescent dyes to nucleic acids and their fluorogenic properties are indispensable tools for nucleic acid detection, quantification, and imaging, yet the molecular structures of several widely used commercial dyes have remained unknown. Here, we determined the molecular structures of RiboGreen and OliGreen and confirmed the previously proposed structure of PicoGreen using high-field NMR spectroscopy. All three dyes were identified as unsymmetric cyanine dyes, where a benzoxazole/benzothiazole moiety is linked to a 4-quinoline by a monomethine bridge. Complete H and C resonance assignments enabled us to expand the existing chemical shift reference set for this important class of dyes. Photophysical characterization with standardized single- and double-stranded DNA and RNA targets indicated that all dyes performed similarly upon binding despite being marketed towards different nucleic acid types. NMR spectroscopy and long-timescale molecular dynamics simulations showed that RiboGreen interacts with double-stranded DNA predominantly by two binding modes, electrostatic interactions with the phosphodiester backbone and π-π stacking with the ultimate and penultimate base pairs of the DNA molecule. These results establish the molecular structures of three widely used commercial dyes and provide a structural and mechanistic framework for understanding the fluorogenic properties of this class of dyes.
HIGHLIGHTS: Determination of the molecular structures of nucleic acid dyes RiboGreen, OliGreen, and PicoGreenNMR spectroscopic characterization of all three dyes.NMR and MD data indicate binding to be dominated by electrostatic and π-π stacking interactions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The binding of fluorescent dyes to nucleic acids and their fluorogenic properties are indispensable tools for nucleic acid detection, quantification, and imaging, yet the molecular structures of several widely used commercial dyes have remained unknown. Here, we determined the molecular structures of RiboGreen and OliGreen and confirmed the previously proposed structure of PicoGreen using high-field NMR spectroscopy. All three dyes were identified as unsymmetric cyanine dyes, where a benzoxazole/benzothiazole moiety is linked to a 4-quinoline by a monomethine bridge. Complete H and C resonance assignments enabled us to expand the existing chemical shift reference set for this important class of dyes. Photophysical characterization with standardized single- and double-stranded DNA and RNA targets indicated that all dyes performed similarly upon binding despite being marketed towards different nucleic acid types. NMR spectroscopy and long-timescale molecular dynamics simulations showed that RiboGreen interacts with double-stranded DNA predominantly by two binding modes, electrostatic interactions with the phosphodiester backbone and π-π stacking with the ultimate and penultimate base pairs of the DNA molecule. These results establish the molecular structures of three widely used commercial dyes and provide a structural and mechanistic framework for understanding the fluorogenic properties of this class of dyes.
HIGHLIGHTS: Determination of the molecular structures of nucleic acid dyes RiboGreen, OliGreen, and PicoGreenNMR spectroscopic characterization of all three dyes.NMR and MD data indicate binding to be dominated by electrostatic and π-π stacking interactions.
HIGHLIGHTS: Determination of the molecular structures of nucleic acid dyes RiboGreen, OliGreen, and PicoGreenNMR spectroscopic characterization of all three dyes.NMR and MD data indicate binding to be dominated by electrostatic and π-π stacking interactions.
36.
Nazifa Tasnim Ahmad; Jhinuk Saha; Yimin Mao; Robert Silvers; Zaid Abulaban; Joshua Mysona; Ayyalusamy Ramamoorthy
Gangliosides GM3 And GD3 Modulate Insulin Aggregation Pathways and Reduce Cytotoxicity Through Structural Remodeling
In: bioRxiv, 2026, ISSN: 2692-8205.
Abstract | Links:
@article{pmid41676560,
title = {Gangliosides GM3 And GD3 Modulate Insulin Aggregation Pathways and Reduce Cytotoxicity Through Structural Remodeling},
author = {Nazifa Tasnim Ahmad and Jhinuk Saha and Yimin Mao and Robert Silvers and Zaid Abulaban and Joshua Mysona and Ayyalusamy Ramamoorthy},
doi = {10.64898/2026.02.03.703542},
issn = {2692-8205},
year = {2026},
date = {2026-02-01},
journal = {bioRxiv},
abstract = {Insulin amyloid aggregation is a key pathological and pharmaceutical concern, particularly in the context of Type-2 Diabetes (T2D), where amyloid deposition of protein can impair therapeutic efficacy and contribute to cell death leading to local tissue damage. Although gangliosides-glycosphingolipids containing sialic acid residues-are known to modulate amyloid formation in neurodegenerative disorders, their influence on insulin aggregation remains largely unexplored. In this study, we investigate the effects of gangliosides GM3 and GD3 on insulin aggregation. Using Thioflavin-T (ThT) based fluorescence kinetics, Fourier Transform Infrared (FTIR) spectroscopy, Circular Dichroism (CD) spectroscopy, Small Angle X-ray Scattering (SAXS), Nuclear Magnetic Resonance (NMR) spectroscopy, and Transmission Electron Microscopy (TEM), the aggregation pathway, changes in the secondary structure and morphology of insulin aggregates have been characterized. Our results show that both GM3 and GD3 lipids accelerated insulin aggregation in a concentration-dependent manner while steering the pathway away from classical fibril formation, producing short, beaded structures distinct from the extended fibrils observed under lipid-free conditions. CD and FTIR data analyses revealed that insulin in the presence of gangliosides formed non-fibrillar intermediates with distinct secondary structures: β-sheet-rich globular clusters in presence of GD3 and α-helical intermediates in GM3-treated samples. Cytotoxicity assays further demonstrated that ganglioside-induced aggregates are significantly less toxic to cells when compared to insulin-only aggregates. Furthermore, ganglioside-bound insulin oligomers retain seeding capacity, suggesting that they can nucleate further aggregation despite their non-fibrillar morphology. These findings underscore the role of gangliosides in modulating insulin amyloid polymorphism and toxicity, offering new insights into their potential impact on the pathology of T2D and treatment strategies.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Insulin amyloid aggregation is a key pathological and pharmaceutical concern, particularly in the context of Type-2 Diabetes (T2D), where amyloid deposition of protein can impair therapeutic efficacy and contribute to cell death leading to local tissue damage. Although gangliosides-glycosphingolipids containing sialic acid residues-are known to modulate amyloid formation in neurodegenerative disorders, their influence on insulin aggregation remains largely unexplored. In this study, we investigate the effects of gangliosides GM3 and GD3 on insulin aggregation. Using Thioflavin-T (ThT) based fluorescence kinetics, Fourier Transform Infrared (FTIR) spectroscopy, Circular Dichroism (CD) spectroscopy, Small Angle X-ray Scattering (SAXS), Nuclear Magnetic Resonance (NMR) spectroscopy, and Transmission Electron Microscopy (TEM), the aggregation pathway, changes in the secondary structure and morphology of insulin aggregates have been characterized. Our results show that both GM3 and GD3 lipids accelerated insulin aggregation in a concentration-dependent manner while steering the pathway away from classical fibril formation, producing short, beaded structures distinct from the extended fibrils observed under lipid-free conditions. CD and FTIR data analyses revealed that insulin in the presence of gangliosides formed non-fibrillar intermediates with distinct secondary structures: β-sheet-rich globular clusters in presence of GD3 and α-helical intermediates in GM3-treated samples. Cytotoxicity assays further demonstrated that ganglioside-induced aggregates are significantly less toxic to cells when compared to insulin-only aggregates. Furthermore, ganglioside-bound insulin oligomers retain seeding capacity, suggesting that they can nucleate further aggregation despite their non-fibrillar morphology. These findings underscore the role of gangliosides in modulating insulin amyloid polymorphism and toxicity, offering new insights into their potential impact on the pathology of T2D and treatment strategies.
35.
Ravi Shankar Palani; Christopher G Williams; Dev Thacker; Robert Silvers; Fang Qian; Paul H Weinreb; Leonard J Mueller; Sara Linse; Robert G Griffin
Aducanumab binding to Aβ fibrils alters dynamics of the N-terminal tail while preserving the fibril core
In: Proc Natl Acad Sci U S A, 122 (52), pp. e2515673122, 2025, ISSN: 1091-6490.
Abstract | Links:
@article{pmid41428870,
title = {Aducanumab binding to Aβ fibrils alters dynamics of the N-terminal tail while preserving the fibril core},
author = {Ravi Shankar Palani and Christopher G Williams and Dev Thacker and Robert Silvers and Fang Qian and Paul H Weinreb and Leonard J Mueller and Sara Linse and Robert G Griffin},
doi = {10.1073/pnas.2515673122},
issn = {1091-6490},
year = {2025},
date = {2025-12-01},
journal = {Proc Natl Acad Sci U S A},
volume = {122},
number = {52},
pages = {e2515673122},
abstract = {Aducanumab, a human IgG1 antibody with plaque-clearing effects and modest clinical benefit, binds selectively to aggregated Aβ via the N-terminal region. Yet, the molecular details of how the antibody engages Aβ fibrils remain unresolved. Using magic-angle spinning NMR, we show that binding of aducanumab preserves the overall architecture of the Aβ fibril core while inducing significant structural and dynamic perturbations in the N-terminal region. Antibody binding markedly reduces flexibility in this domain, with the appearance of side-chain resonances from residues D1, E3, and histidine (likely H6) in dipolar-based experiments. These side chains-previously observed in scalar-coupling spectra of the unbound state-indicate rigidification of residues that were dynamic. The interaction extends to S8 and Y10, indicating broader fibril engagement than the minimal epitope (residues 3 to 7) defined in fragment-based studies. Perturbations in the C-terminal segment (G37-A42) are consistent with its spatial proximity to the antibody-bound N termini of neighboring monomers. Cryo-TEM images reveal fibrils bundling in the presence of aducanumab, consistent with lateral association via antibody cross-linking, supporting a model where surface coating and steric hindrance suppress secondary nucleation. This mode of action restricts monomer access to catalytic sites on the fibril surface, resulting in partial inhibition (~threefold reduction) of secondary nucleation. The effect depends on high avidity and relatively high stoichiometry but is ultimately limited by antibody size relative to N-terminal spacing along the fibril. These findings provide atomic-level insights into aducanumab's binding mode and supply a structural framework for understanding antibody-mediated fibril recognition and for guiding next-generation therapies targeting Aβ aggregates in Alzheimer's disease.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Aducanumab, a human IgG1 antibody with plaque-clearing effects and modest clinical benefit, binds selectively to aggregated Aβ via the N-terminal region. Yet, the molecular details of how the antibody engages Aβ fibrils remain unresolved. Using magic-angle spinning NMR, we show that binding of aducanumab preserves the overall architecture of the Aβ fibril core while inducing significant structural and dynamic perturbations in the N-terminal region. Antibody binding markedly reduces flexibility in this domain, with the appearance of side-chain resonances from residues D1, E3, and histidine (likely H6) in dipolar-based experiments. These side chains-previously observed in scalar-coupling spectra of the unbound state-indicate rigidification of residues that were dynamic. The interaction extends to S8 and Y10, indicating broader fibril engagement than the minimal epitope (residues 3 to 7) defined in fragment-based studies. Perturbations in the C-terminal segment (G37-A42) are consistent with its spatial proximity to the antibody-bound N termini of neighboring monomers. Cryo-TEM images reveal fibrils bundling in the presence of aducanumab, consistent with lateral association via antibody cross-linking, supporting a model where surface coating and steric hindrance suppress secondary nucleation. This mode of action restricts monomer access to catalytic sites on the fibril surface, resulting in partial inhibition (~threefold reduction) of secondary nucleation. The effect depends on high avidity and relatively high stoichiometry but is ultimately limited by antibody size relative to N-terminal spacing along the fibril. These findings provide atomic-level insights into aducanumab's binding mode and supply a structural framework for understanding antibody-mediated fibril recognition and for guiding next-generation therapies targeting Aβ aggregates in Alzheimer's disease.
34.
Ravi Shankar Palani; Christopher G Williams; Dev Thacker; Robert Silvers; Fang Qian; Paul H Weinreb; Leonard J Mueller; Sara Linse; Robert G Griffin
Aducanumab Binding to Aβ Fibrils Alters Dynamics of the N-Terminal Tail While Preserving the Fibril Core
In: bioRxiv, 2025, ISSN: 2692-8205.
Abstract | Links:
@article{pmid41279514,
title = {Aducanumab Binding to Aβ Fibrils Alters Dynamics of the N-Terminal Tail While Preserving the Fibril Core},
author = {Ravi Shankar Palani and Christopher G Williams and Dev Thacker and Robert Silvers and Fang Qian and Paul H Weinreb and Leonard J Mueller and Sara Linse and Robert G Griffin},
doi = {10.1101/2025.11.01.686027},
issn = {2692-8205},
year = {2025},
date = {2025-11-01},
journal = {bioRxiv},
abstract = {Aducanumab, a human IgG1 antibody with plaque-clearing effects and modest clinical benefit, binds selectively to aggregated Aβ via the N-terminal region. Yet, the molecular details of how the antibody engages Aβ fibrils remain unresolved. Using magic-angle spinning nuclear magnetic resonance, we show that binding of aducanumab preserves the overall architecture of the Aβ fibril core while inducing significant structural and dynamic perturbations in the N-terminal region. Antibody binding markedly reduces flexibility in this domain, with the appearance of sidechain resonances from residues D1, E3, and histidine (likely H6) in dipolar-based experiments. These sidechains-previously observed only in scalar-coupling spectra of the unbound state-indicate rigidification of residues that were dynamic. The interaction extends to S8 and Y10, indicating broader fibril engagement than the minimal epitope (residues 3-7) defined in fragment-based studies. Perturbations in the C-terminal segment (G37-A42) are consistent with its spatial proximity to the antibody-bound N-termini of neighboring monomers. Cryo-TEM images reveal fibrils bundling in the presence of aducanumab, consistent with lateral association via antibody cross-linking, supporting a model where surface coating and steric hindrance suppress secondary nucleation. This mode of action restricts monomer access to catalytic sites on fibril surface, resulting in partial inhibition (~three-fold reduction) of secondary nucleation. The effect depends on high avidity and relatively high stoichiometry, but is ultimately limited by antibody size relative to N-terminal spacing along the fibril. These findings provide atomic-level insights into aducanumab's binding mode and supply a structural framework for understanding antibody-mediated fibril recognition and for guiding next-generation therapies targeting Aβ aggregates in Alzheimer's disease.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Aducanumab, a human IgG1 antibody with plaque-clearing effects and modest clinical benefit, binds selectively to aggregated Aβ via the N-terminal region. Yet, the molecular details of how the antibody engages Aβ fibrils remain unresolved. Using magic-angle spinning nuclear magnetic resonance, we show that binding of aducanumab preserves the overall architecture of the Aβ fibril core while inducing significant structural and dynamic perturbations in the N-terminal region. Antibody binding markedly reduces flexibility in this domain, with the appearance of sidechain resonances from residues D1, E3, and histidine (likely H6) in dipolar-based experiments. These sidechains-previously observed only in scalar-coupling spectra of the unbound state-indicate rigidification of residues that were dynamic. The interaction extends to S8 and Y10, indicating broader fibril engagement than the minimal epitope (residues 3-7) defined in fragment-based studies. Perturbations in the C-terminal segment (G37-A42) are consistent with its spatial proximity to the antibody-bound N-termini of neighboring monomers. Cryo-TEM images reveal fibrils bundling in the presence of aducanumab, consistent with lateral association via antibody cross-linking, supporting a model where surface coating and steric hindrance suppress secondary nucleation. This mode of action restricts monomer access to catalytic sites on fibril surface, resulting in partial inhibition (~three-fold reduction) of secondary nucleation. The effect depends on high avidity and relatively high stoichiometry, but is ultimately limited by antibody size relative to N-terminal spacing along the fibril. These findings provide atomic-level insights into aducanumab's binding mode and supply a structural framework for understanding antibody-mediated fibril recognition and for guiding next-generation therapies targeting Aβ aggregates in Alzheimer's disease.
33.
Nolan Blackford; Victoria S Ogunkunle; Robert Silvers
Fluorescent Dyes for Differential Scanning Fluorimetry of RNA
In: Chembiochem, 26 (20), pp. e202500560, 2025, ISSN: 1439-7633.
Abstract | Links:
@article{pmid41147167,
title = {Fluorescent Dyes for Differential Scanning Fluorimetry of RNA},
author = {Nolan Blackford and Victoria S Ogunkunle and Robert Silvers},
doi = {10.1002/cbic.202500560},
issn = {1439-7633},
year = {2025},
date = {2025-10-01},
journal = {Chembiochem},
volume = {26},
number = {20},
pages = {e202500560},
abstract = {Differential scanning fluorimetry (DSF) to determine the thermal stability of RNA has only recently been adapted from proteins using the nucleic acid dye RiboGreen. Here, we investigate the suitability of seven commercially available nucleic acid dyes for use in DSF on RNA-acridine orange, ethidium bromide, RiboGreen, OliGreen, SYBR Green II, SYBR Gold, and TOTO-1 iodide-using a double-stranded RNA model and the 27-nt neomycin-sensing riboswitch aptamer. Concentration-dependent DSF curves revealed pronounced dye-specific effects on both initial fluorescence and apparent melting temperatures. RiboGreen and OliGreen yielded concentration-independent melting temperatures within experimental error, whereas SYBR derivatives, acridine orange, and ethidium bromide stabilized RNA progressively with increasing dye concentration, shifting the melting temperature by up to +9 °C. Taken together, the data identify RiboGreen and OliGreen as probes for use in DSF on RNA that minimize dye-induced artifacts while preserving robust signal-to-noise, and they provide a framework for tailoring DSF conditions to specific RNA targets and ligand-binding screens. Other tested dyes displaying concentration-dependent melting temperatures are suitable as well but require appropriate care when utilizing them in DSF on RNA.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Differential scanning fluorimetry (DSF) to determine the thermal stability of RNA has only recently been adapted from proteins using the nucleic acid dye RiboGreen. Here, we investigate the suitability of seven commercially available nucleic acid dyes for use in DSF on RNA-acridine orange, ethidium bromide, RiboGreen, OliGreen, SYBR Green II, SYBR Gold, and TOTO-1 iodide-using a double-stranded RNA model and the 27-nt neomycin-sensing riboswitch aptamer. Concentration-dependent DSF curves revealed pronounced dye-specific effects on both initial fluorescence and apparent melting temperatures. RiboGreen and OliGreen yielded concentration-independent melting temperatures within experimental error, whereas SYBR derivatives, acridine orange, and ethidium bromide stabilized RNA progressively with increasing dye concentration, shifting the melting temperature by up to +9 °C. Taken together, the data identify RiboGreen and OliGreen as probes for use in DSF on RNA that minimize dye-induced artifacts while preserving robust signal-to-noise, and they provide a framework for tailoring DSF conditions to specific RNA targets and ligand-binding screens. Other tested dyes displaying concentration-dependent melting temperatures are suitable as well but require appropriate care when utilizing them in DSF on RNA.
32.
Blaine H Gordon; Victoria S Ogunkunle; Robert Silvers
Noncanonical RNA binding of human La-related protein 6
In: Nucleic Acids Res, 53 (14), 2025, ISSN: 1362-4962.
Abstract | Links:
@article{pmid40705921,
title = {Noncanonical RNA binding of human La-related protein 6},
author = {Blaine H Gordon and Victoria S Ogunkunle and Robert Silvers},
doi = {10.1093/nar/gkaf682},
issn = {1362-4962},
year = {2025},
date = {2025-07-01},
journal = {Nucleic Acids Res},
volume = {53},
number = {14},
abstract = {La-related proteins (LARPs) are RNA-binding proteins that are involved in a variety of disease-related processes. Most LARPs recognize short single-stranded poly(U/A) motifs via a conserved hydrophobic pocket. Human LARP6 (HsLARP6) is an exception, binding a structured 5' stem-loop (5'SL) that controls type I collagen translation and fibroproliferative disease progression. Here, we present the de novo solution nuclear magnetic resonance structure of the La domain of HsLARP6 in the bound state. Chemical shift perturbation, solvent paramagnetic relaxation enhancement, intermolecular nuclear Overhauser effects, and targeted mutagenesis converge on a previously unknown binding interface that integrates electrostatic and hydrophobic contacts with shape complementarity in 5'SL binding. This noncanonical interface enables the La domain to discriminate 5'SL RNA from homopolymeric or purely helical hairpin RNAs with low-nanomolar affinity, overturning earlier views that the adjacent RNA recognition motif is required for recognition. The structure provides the first molecular model for 5'SL recognition and expands the paradigm of La-mediated RNA binding beyond 3'-terminal oligo-U/A motifs. These insights provide the biophysical framework for molecular recognition of 5'SL by LARP6 that is related to collagen biosynthesis in fibrosis and associated pathologies.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
La-related proteins (LARPs) are RNA-binding proteins that are involved in a variety of disease-related processes. Most LARPs recognize short single-stranded poly(U/A) motifs via a conserved hydrophobic pocket. Human LARP6 (HsLARP6) is an exception, binding a structured 5' stem-loop (5'SL) that controls type I collagen translation and fibroproliferative disease progression. Here, we present the de novo solution nuclear magnetic resonance structure of the La domain of HsLARP6 in the bound state. Chemical shift perturbation, solvent paramagnetic relaxation enhancement, intermolecular nuclear Overhauser effects, and targeted mutagenesis converge on a previously unknown binding interface that integrates electrostatic and hydrophobic contacts with shape complementarity in 5'SL binding. This noncanonical interface enables the La domain to discriminate 5'SL RNA from homopolymeric or purely helical hairpin RNAs with low-nanomolar affinity, overturning earlier views that the adjacent RNA recognition motif is required for recognition. The structure provides the first molecular model for 5'SL recognition and expands the paradigm of La-mediated RNA binding beyond 3'-terminal oligo-U/A motifs. These insights provide the biophysical framework for molecular recognition of 5'SL by LARP6 that is related to collagen biosynthesis in fibrosis and associated pathologies.
31.
Blaine H Gordon; Robert Silvers
H, C, and N resonance assignment of the 5'SL-bound La domain of the human La-related protein 6
In: Biomol NMR Assign, 19 (1), pp. 165–173, 2025, ISSN: 1874-270X.
Abstract | Links:
@article{pmid40304844,
title = {H, C, and N resonance assignment of the 5'SL-bound La domain of the human La-related protein 6},
author = {Blaine H Gordon and Robert Silvers},
doi = {10.1007/s12104-025-10232-7},
issn = {1874-270X},
year = {2025},
date = {2025-06-01},
journal = {Biomol NMR Assign},
volume = {19},
number = {1},
pages = {165--173},
abstract = {Human La-related protein 6 (HsLARP6) participates in the post-transcriptional regulation of type I collagen biosynthesis and is involved in the onset and progression of fibroproliferative disease. The RNA-binding protein HsLARP6 recognizes a hairpin structure known as the 5' stem-loop (5'SL) located at the junction of 5' untranslated and coding regions of type I collagen mRNA. Despite extensive biochemical and functional studies of the interaction between HsLARP6 and the 5'SL motif, the lack of high-resolution molecular data significantly hampers our understanding of the binding mechanism. Here, we introduced a shorter 5'SL model, named A2M5, reducing the molecular size of the protein-RNA complex as well as spectral overlap in RNA-based spectra. Furthermore, we reported the near-complete backbone and side chain resonance assignment of the La domain of HsLARP6 in a 1:1 complex with the A2M5 model RNA. These results will provide a significant platform for future NMR spectroscopic studies of 5'SL binding to the La domain of HsLARP6.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Human La-related protein 6 (HsLARP6) participates in the post-transcriptional regulation of type I collagen biosynthesis and is involved in the onset and progression of fibroproliferative disease. The RNA-binding protein HsLARP6 recognizes a hairpin structure known as the 5' stem-loop (5'SL) located at the junction of 5' untranslated and coding regions of type I collagen mRNA. Despite extensive biochemical and functional studies of the interaction between HsLARP6 and the 5'SL motif, the lack of high-resolution molecular data significantly hampers our understanding of the binding mechanism. Here, we introduced a shorter 5'SL model, named A2M5, reducing the molecular size of the protein-RNA complex as well as spectral overlap in RNA-based spectra. Furthermore, we reported the near-complete backbone and side chain resonance assignment of the La domain of HsLARP6 in a 1:1 complex with the A2M5 model RNA. These results will provide a significant platform for future NMR spectroscopic studies of 5'SL binding to the La domain of HsLARP6.
30.
Blaine H Gordon; Nolan Blackford; Robert Silvers
A Robust Expression and Purification Protocol for the Production of the La Domain of Human LARP6
In: ACS Omega, 10 (12), pp. 12699–12709, 2025, ISSN: 2470-1343.
Abstract | Links:
@article{pmid40191362,
title = {A Robust Expression and Purification Protocol for the Production of the La Domain of Human LARP6},
author = {Blaine H Gordon and Nolan Blackford and Robert Silvers},
doi = {10.1021/acsomega.5c01045},
issn = {2470-1343},
year = {2025},
date = {2025-04-01},
journal = {ACS Omega},
volume = {10},
number = {12},
pages = {12699--12709},
abstract = {Human La-related protein 6 (HsLARP6) regulates the highly organized biosynthesis of type I procollagen polypeptides and affects the proper assembly of procollagen peptides into heterotrimers of type I procollagen. HsLARP6-mediated regulation of collagen biosynthesis is mediated through interaction with the 5' stem loop (5'SL) motif found in type I and III collagen mRNA. Recent studies highlight the involvement of HsLARP6 in fibroproliferative diseases and its potential as a target for therapeutic intervention. The intrinsic propensity of the La domain of HsLARP6 to aggregate hampers studies probing the molecular basis of biologically and disease-relevant structure-function relationships, particularly when high concentrations are required. This work provides detailed procedures to produce milligram amounts of RNase-free and functional La domain of HsLARP6. Furthermore, we investigated the effects of the protein construct length and RNA binding on protein stability. C-terminal truncations greatly impact protein stability, while N-terminal truncations have little to no effect on protein aggregation and RNA binding. When in complex with its cognate 5'SL RNA, the La domain shows unprecedented stability compared to the aggregation-prone unbound state. The protein-RNA complex remains stable for at least 50 times longer than the unbound state under identical conditions. These results provide a significant platform for further studies of the molecular recognition of 5'SL by HsLARP6.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Human La-related protein 6 (HsLARP6) regulates the highly organized biosynthesis of type I procollagen polypeptides and affects the proper assembly of procollagen peptides into heterotrimers of type I procollagen. HsLARP6-mediated regulation of collagen biosynthesis is mediated through interaction with the 5' stem loop (5'SL) motif found in type I and III collagen mRNA. Recent studies highlight the involvement of HsLARP6 in fibroproliferative diseases and its potential as a target for therapeutic intervention. The intrinsic propensity of the La domain of HsLARP6 to aggregate hampers studies probing the molecular basis of biologically and disease-relevant structure-function relationships, particularly when high concentrations are required. This work provides detailed procedures to produce milligram amounts of RNase-free and functional La domain of HsLARP6. Furthermore, we investigated the effects of the protein construct length and RNA binding on protein stability. C-terminal truncations greatly impact protein stability, while N-terminal truncations have little to no effect on protein aggregation and RNA binding. When in complex with its cognate 5'SL RNA, the La domain shows unprecedented stability compared to the aggregation-prone unbound state. The protein-RNA complex remains stable for at least 50 times longer than the unbound state under identical conditions. These results provide a significant platform for further studies of the molecular recognition of 5'SL by HsLARP6.
29.
Benjamin C Smith; Robert Silvers
H, C, and N resonance assignments of the La Motif of the human La-related protein 1
In: Biomol NMR Assign, 18 (1), pp. 111–118, 2024, ISSN: 1874-270X.
Abstract | Links:
@article{pmid38691336,
title = {H, C, and N resonance assignments of the La Motif of the human La-related protein 1},
author = {Benjamin C Smith and Robert Silvers},
doi = {10.1007/s12104-024-10176-4},
issn = {1874-270X},
year = {2024},
date = {2024-06-01},
journal = {Biomol NMR Assign},
volume = {18},
number = {1},
pages = {111--118},
abstract = {Human La-related protein 1 (HsLARP1) is involved in post-transcriptional regulation of certain 5' terminal oligopyrimidine (5'TOP) mRNAs as well as other mRNAs and binds to both the 5'TOP motif and the 3'-poly(A) tail of certain mRNAs. HsLARP1 is heavily involved in cell proliferation, cell cycle defects, and cancer, where HsLARP1 is significantly upregulated in malignant cells and tissues. Like all LARPs, HsLARP1 contains a folded RNA binding domain, the La motif (LaM). Our current understanding of post-transcriptional regulation that emanates from the intricate molecular framework of HsLARP1 is currently limited to small snapshots, obfuscating our understanding of the full picture on HsLARP1 functionality in post-transcriptional events. Here, we present the nearly complete resonance assignment of the LaM of HsLARP1, providing a significant platform for future NMR spectroscopic studies.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Human La-related protein 1 (HsLARP1) is involved in post-transcriptional regulation of certain 5' terminal oligopyrimidine (5'TOP) mRNAs as well as other mRNAs and binds to both the 5'TOP motif and the 3'-poly(A) tail of certain mRNAs. HsLARP1 is heavily involved in cell proliferation, cell cycle defects, and cancer, where HsLARP1 is significantly upregulated in malignant cells and tissues. Like all LARPs, HsLARP1 contains a folded RNA binding domain, the La motif (LaM). Our current understanding of post-transcriptional regulation that emanates from the intricate molecular framework of HsLARP1 is currently limited to small snapshots, obfuscating our understanding of the full picture on HsLARP1 functionality in post-transcriptional events. Here, we present the nearly complete resonance assignment of the LaM of HsLARP1, providing a significant platform for future NMR spectroscopic studies.
28.
Blaine H Gordon; Peilu Liu; A Carl Whittington; Robert Silvers; Brian G Miller
Biochemical methods to map and quantify allosteric motions in human glucokinase
In: Methods Enzymol, 685 , pp. 433–459, 2023, ISSN: 1557-7988.
Abstract | Links:
@article{pmid37245911,
title = {Biochemical methods to map and quantify allosteric motions in human glucokinase},
author = {Blaine H Gordon and Peilu Liu and A Carl Whittington and Robert Silvers and Brian G Miller},
doi = {10.1016/bs.mie.2023.03.009},
issn = {1557-7988},
year = {2023},
date = {2023-01-01},
journal = {Methods Enzymol},
volume = {685},
pages = {433--459},
abstract = {Allosteric regulation of protein function is ubiquitous in biology. Allostery originates from ligand-mediated alterations in polypeptide structure and/or dynamics, which produce a cooperative kinetic or thermodynamic response to changing ligand concentrations. Establishing a mechanistic description of individual allosteric events requires both mapping the relevant changes in protein structure and quantifying the rates of differential conformational dynamics in the absence and presence of effectors. In this chapter, we describe three biochemical approaches to understand the dynamic and structural signatures of protein allostery using the well-established cooperative enzyme glucokinase as a case study. The combined application of pulsed proteolysis, biomolecular nuclear magnetic resonance spectroscopy and hydrogen-deuterium exchange mass spectrometry offers complementary information that can used to establish molecular models for allosteric proteins, especially when differential protein dynamics are involved.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Allosteric regulation of protein function is ubiquitous in biology. Allostery originates from ligand-mediated alterations in polypeptide structure and/or dynamics, which produce a cooperative kinetic or thermodynamic response to changing ligand concentrations. Establishing a mechanistic description of individual allosteric events requires both mapping the relevant changes in protein structure and quantifying the rates of differential conformational dynamics in the absence and presence of effectors. In this chapter, we describe three biochemical approaches to understand the dynamic and structural signatures of protein allostery using the well-established cooperative enzyme glucokinase as a case study. The combined application of pulsed proteolysis, biomolecular nuclear magnetic resonance spectroscopy and hydrogen-deuterium exchange mass spectrometry offers complementary information that can used to establish molecular models for allosteric proteins, especially when differential protein dynamics are involved.
27.
Lela Stefanovic; Blaine H. Gordon; Robert Silvers; Branko Stefanovic
Characterization of sequence specific binding of LARP6 to the 5' stem-loop of type I collagen mRNAs and implications for rational design of antifibrotic drugs
In: Journal of Molecular Biology, 434 (2), pp. 167394, 2022.
Abstract | Links:
@article{Stefanovic_2022,
title = {Characterization of sequence specific binding of LARP6 to the 5' stem-loop of type I collagen mRNAs and implications for rational design of antifibrotic drugs},
author = {Lela Stefanovic and Blaine H. Gordon and Robert Silvers and Branko Stefanovic},
doi = {10.1016/j.jmb.2021.167394},
year = {2022},
date = {2022-01-30},
urldate = {2022-01-30},
journal = {Journal of Molecular Biology},
volume = {434},
number = {2},
pages = {167394},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Excessive synthesis of type I collagen is a hallmark of fibrotic diseases. Binding of La-related protein 6 (LARP6) to the 5′ stem-loop (5′SL) of collagen mRNAs regulates their translation leading to an unnaturally elevated rate of collagen biosynthesis in fibrosis. Previous work suggested that LARP6 needs two domains to form stable complex with 5′SL RNA, the La domain and the juxtaposed RNA recognition motif (RRM), jointly called the La-module. Here we describe that La domain of LARP6 is necessary and sufficient for recognition of 5′SL in RNA sequence specific manner. A three-amino-acid motif located in the flexible loop connecting the second α-helix to the β-sheet of the La domain, called the RNK-motif, is critical for binding. Mutation of any of these three amino acids abolishes the binding of the La domain to 5′SL. The major site of crosslinking of LARP6 to 5′SL RNA was mapped to this motif, as well. The RNK-motif is not found in other LARPs, which cannot bind 5′SL. Presence of RRM increases the stability of complex between La domain and 5′SL RNA and RRM domain does not make extensive contacts with 5′SL RNA. We propose a model in which the initial recognition of 5′SL by LARP6 is mediated by the RNK epitope and further stabilized by the RRM domain. This discovery suggests that the interaction between LARP6 and collagen mRNAs can be blocked by small molecules that target the RNK epitope and will help rational design of the LARP6 binding inhibitors as specific antifibrotic drugs.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Excessive synthesis of type I collagen is a hallmark of fibrotic diseases. Binding of La-related protein 6 (LARP6) to the 5′ stem-loop (5′SL) of collagen mRNAs regulates their translation leading to an unnaturally elevated rate of collagen biosynthesis in fibrosis. Previous work suggested that LARP6 needs two domains to form stable complex with 5′SL RNA, the La domain and the juxtaposed RNA recognition motif (RRM), jointly called the La-module. Here we describe that La domain of LARP6 is necessary and sufficient for recognition of 5′SL in RNA sequence specific manner. A three-amino-acid motif located in the flexible loop connecting the second α-helix to the β-sheet of the La domain, called the RNK-motif, is critical for binding. Mutation of any of these three amino acids abolishes the binding of the La domain to 5′SL. The major site of crosslinking of LARP6 to 5′SL RNA was mapped to this motif, as well. The RNK-motif is not found in other LARPs, which cannot bind 5′SL. Presence of RRM increases the stability of complex between La domain and 5′SL RNA and RRM domain does not make extensive contacts with 5′SL RNA. We propose a model in which the initial recognition of 5′SL by LARP6 is mediated by the RNK epitope and further stabilized by the RRM domain. This discovery suggests that the interaction between LARP6 and collagen mRNAs can be blocked by small molecules that target the RNK epitope and will help rational design of the LARP6 binding inhibitors as specific antifibrotic drugs.
26.
Salima Bahri; Robert Silvers; Brian Michael; Kristaps Jaudzems; Daniela Lalli; Gilles Casano; Olivier Ouari; Anne Lesage; Guido Pintacuda; Sara Linse; Robert G. Griffin
1H detection and dynamic nuclear polarization-enhanced NMR of Aβ1-42 fibrils
In: Proc Natl Acad Sci U S A, 119 (1), pp. e2114413119, 2022, ISSN: 1091-6490.
Abstract | Links:
@article{bahri_2022,
title = {1H detection and dynamic nuclear polarization-enhanced NMR of Aβ1-42 fibrils},
author = {Salima Bahri and Robert Silvers and Brian Michael and Kristaps Jaudzems and Daniela Lalli and Gilles Casano and Olivier Ouari and Anne Lesage and Guido Pintacuda and Sara Linse and Robert G. Griffin},
doi = {10.1073/pnas.2114413119},
issn = {1091-6490},
year = {2022},
date = {2022-01-04},
urldate = {2022-01-04},
journal = {Proc Natl Acad Sci U S A},
volume = {119},
number = {1},
pages = {e2114413119},
abstract = {Several publications describing high-resolution structures of amyloid-β (Aβ) and other fibrils have demonstrated that magic-angle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments-namely, H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive two-dimensional (2D) and three-dimensional (3D) correlations obtained on C,N-enriched, and fully protonated samples of MAβ fibrils by high-field H-detected NMR at 23.4 T and 18.8 T, and C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of MAβ (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently ∼6.5 times more sensitive than H detection. These results suggest that H detection and DNP may be the spectroscopic approaches of choice for future studies of Aβ and other amyloid systems.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Several publications describing high-resolution structures of amyloid-β (Aβ) and other fibrils have demonstrated that magic-angle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments-namely, H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive two-dimensional (2D) and three-dimensional (3D) correlations obtained on C,N-enriched, and fully protonated samples of MAβ fibrils by high-field H-detected NMR at 23.4 T and 18.8 T, and C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of MAβ (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently ∼6.5 times more sensitive than H detection. These results suggest that H detection and DNP may be the spectroscopic approaches of choice for future studies of Aβ and other amyloid systems.
25.
Robert Silvers; Jules R. Stephan; Robert G. Griffin; Elizabeth M. Nolan
Molecular Basis of Ca(II)-Induced Tetramerization and Transition-Metal Sequestration in Human Calprotectin
In: Journal of the American Chemical Society, 143 (43), pp. 18073-18090, 2021, ISSN: 1520-5126.
Abstract | Links:
@article{silvers_2021,
title = {Molecular Basis of Ca(II)-Induced Tetramerization and Transition-Metal Sequestration in Human Calprotectin},
author = {Robert Silvers and Jules R. Stephan and Robert G. Griffin and Elizabeth M. Nolan},
doi = {10.1021/jacs.1c06402},
issn = {1520-5126},
year = {2021},
date = {2021-10-26},
urldate = {2021-10-26},
journal = {Journal of the American Chemical Society},
volume = {143},
number = {43},
pages = {18073-18090},
abstract = {Human calprotectin (CP, S100A8/S100A9 oligomer, MRP8/MRP14 oligomer) is an abundant innate immune protein that contributes to the host metal-withholding response. Its ability to sequester transition metal nutrients from microbial pathogens depends on a complex interplay of Ca(II) binding and self-association, which converts the αβ heterodimeric apo protein into a Ca(II)-bound (αβ) heterotetramer that displays enhanced transition metal affinities, antimicrobial activity, and protease stability. A paucity of structural data on the αβ heterodimer has hampered molecular understanding of how Ca(II) binding enables CP to exert its metal-sequestering innate immune function. We report solution NMR data that reveal how Ca(II) binding affects the structure and dynamics of the CP αβ heterodimer. These studies provide a structural model in which the apo αβ heterodimer undergoes conformational exchange and switches between two states, a tetramerization-incompetent or "inactive" state and a tetramerization-competent or "active" state. Ca(II) binding to the EF-hands of the αβ heterodimer causes the active state to predominate, resulting in self-association and formation of the (αβ) heterotetramer. Moreover, Ca(II) binding causes local and allosteric ordering of the HisAsp and His metal-binding sites. Ca(II) binding to the noncanonical EF-hand of S100A9 positions (A9)D30 and organizes the HisAsp site. Remarkably, Ca(II) binding causes allosteric effects in the C-terminal region of helix α of S100A9, which stabilize the α-helicity at positions H91 and H95 and thereby organize the functionally versatile His site. Collectively, this study illuminates the molecular basis for how CP responds to high extracellular Ca(II) concentrations, which enables its metal-sequestering host-defense function.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Human calprotectin (CP, S100A8/S100A9 oligomer, MRP8/MRP14 oligomer) is an abundant innate immune protein that contributes to the host metal-withholding response. Its ability to sequester transition metal nutrients from microbial pathogens depends on a complex interplay of Ca(II) binding and self-association, which converts the αβ heterodimeric apo protein into a Ca(II)-bound (αβ) heterotetramer that displays enhanced transition metal affinities, antimicrobial activity, and protease stability. A paucity of structural data on the αβ heterodimer has hampered molecular understanding of how Ca(II) binding enables CP to exert its metal-sequestering innate immune function. We report solution NMR data that reveal how Ca(II) binding affects the structure and dynamics of the CP αβ heterodimer. These studies provide a structural model in which the apo αβ heterodimer undergoes conformational exchange and switches between two states, a tetramerization-incompetent or "inactive" state and a tetramerization-competent or "active" state. Ca(II) binding to the EF-hands of the αβ heterodimer causes the active state to predominate, resulting in self-association and formation of the (αβ) heterotetramer. Moreover, Ca(II) binding causes local and allosteric ordering of the HisAsp and His metal-binding sites. Ca(II) binding to the noncanonical EF-hand of S100A9 positions (A9)D30 and organizes the HisAsp site. Remarkably, Ca(II) binding causes allosteric effects in the C-terminal region of helix α of S100A9, which stabilize the α-helicity at positions H91 and H95 and thereby organize the functionally versatile His site. Collectively, this study illuminates the molecular basis for how CP responds to high extracellular Ca(II) concentrations, which enables its metal-sequestering host-defense function.
24.
Robert Silvers; Matthew T. Eddy
NMR Spectroscopic Studies of Ion Channels in Lipid Bilayers: Sample Preparation Strategies Exemplified by the Voltage Dependent Anion Channel
In: Schmidt-Krey, Ingeborg; Gumbart, James C. (Ed.): Structure and Function of Membrane Proteins, 2302 , pp. 201-217, Humana, New York, NY, 2021, ISSN: 1940-6029.
Abstract | Links:
@inbook{silvers_2021_2,
title = {NMR Spectroscopic Studies of Ion Channels in Lipid Bilayers: Sample Preparation Strategies Exemplified by the Voltage Dependent Anion Channel},
author = {Robert Silvers and Matthew T. Eddy},
editor = {Ingeborg Schmidt-Krey and James C. Gumbart},
doi = {10.1007/978-1-0716-1394-8_11},
issn = {1940-6029},
year = {2021},
date = {2021-04-21},
urldate = {2021-04-21},
booktitle = {Structure and Function of Membrane Proteins},
journal = {Methods in Molecular Biology},
volume = {2302},
pages = {201-217},
publisher = {Humana},
address = {New York, NY},
series = {Methods In Molecular Biology},
abstract = {We describe approaches for the preparation of membrane proteins in detergent micelles and lipid bilayers for solution and magic angle spinning NMR studies, respectively, as exemplified by the human voltage dependent anion channel 1 (hVDAC1). Here, we report protocols for the preparation of homogenous samples of recombinant hVDAC1 in detergent micelles and lipid two-dimensional crystals yielding high resolution NMR spectra. Procedures are described for the recombinant production of stable-isotope labeled hVDAC1 in E. coli, the isolation of hVDAC1 from inclusion bodies and the refolding into detergent micelles, as well as the reconstitution of hVDAC1 into lipids to form 2D crystals.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}
We describe approaches for the preparation of membrane proteins in detergent micelles and lipid bilayers for solution and magic angle spinning NMR studies, respectively, as exemplified by the human voltage dependent anion channel 1 (hVDAC1). Here, we report protocols for the preparation of homogenous samples of recombinant hVDAC1 in detergent micelles and lipid two-dimensional crystals yielding high resolution NMR spectra. Procedures are described for the recombinant production of stable-isotope labeled hVDAC1 in E. coli, the isolation of hVDAC1 from inclusion bodies and the refolding into detergent micelles, as well as the reconstitution of hVDAC1 into lipids to form 2D crystals.
23.
Matthew G. Iadanza; Robert Silvers; Joshua Boardman; Hugh I. Smith; Theodoros K. Karamanos; Galia T. Debelouchina; Yongchao Su; Robert G. Griffin; Neil A. Ranson; Sheena E. Radford
The structure of a β2-microglobulin fibril suggests a molecular basis for its amyloid polymorphism
In: Nature Communications, 9 (1), 2018.
Abstract | Links:
@article{Iadanza_2018,
title = {The structure of a β2-microglobulin fibril suggests a molecular basis for its amyloid polymorphism},
author = {Matthew G. Iadanza and Robert Silvers and Joshua Boardman and Hugh I. Smith and Theodoros K. Karamanos and Galia T. Debelouchina and Yongchao Su and Robert G. Griffin and Neil A. Ranson and Sheena E. Radford},
doi = {10.1038/s41467-018-06761-6},
year = {2018},
date = {2018-10-01},
urldate = {2018-10-01},
journal = {Nature Communications},
volume = {9},
number = {1},
publisher = {Springer Science and Business Media LLC},
abstract = {All amyloid fibrils contain a cross-β fold. How this structure differs in fibrils formed from proteins associated with different diseases remains unclear. Here, we combine cryo-EM and MAS-NMR to determine the structure of an amyloid fibril formed in vitro from β2-microglobulin (β2m), the culprit protein of dialysis-related amyloidosis. The fibril is composed of two identical protofilaments assembled from subunits that do not share β2m’s native tertiary fold, but are formed from similar β-strands. The fibrils share motifs with other amyloid fibrils, but also contain unique features including π-stacking interactions perpendicular to the fibril axis and an intramolecular disulfide that stabilises the subunit fold. We also describe a structural model for a second fibril morphology and show that it is built from the same subunit fold. The results provide insights into the mechanisms of fibril formation and the commonalities and differences within the amyloid fold in different protein sequences.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
All amyloid fibrils contain a cross-β fold. How this structure differs in fibrils formed from proteins associated with different diseases remains unclear. Here, we combine cryo-EM and MAS-NMR to determine the structure of an amyloid fibril formed in vitro from β2-microglobulin (β2m), the culprit protein of dialysis-related amyloidosis. The fibril is composed of two identical protofilaments assembled from subunits that do not share β2m’s native tertiary fold, but are formed from similar β-strands. The fibrils share motifs with other amyloid fibrils, but also contain unique features including π-stacking interactions perpendicular to the fibril axis and an intramolecular disulfide that stabilises the subunit fold. We also describe a structural model for a second fibril morphology and show that it is built from the same subunit fold. The results provide insights into the mechanisms of fibril formation and the commonalities and differences within the amyloid fold in different protein sequences.
22.
Florian Lehner; Denis Kudlinzki; Christian Richter; Henrike M Müller-Werkmeister; Katharina B Eberl; Jens Bredenbeck; Harald Schwalbe; Robert Silvers
Impact of Azidohomoalanine Incorporation on Protein Structure and Ligand Binding
In: ChemBioChem, 18 (23), pp. 2340–2350, 2017.
@article{Lehner_2017b,
title = {Impact of Azidohomoalanine Incorporation on Protein Structure and Ligand Binding},
author = {Florian Lehner and Denis Kudlinzki and Christian Richter and Henrike M Müller-Werkmeister and Katharina B Eberl and Jens Bredenbeck and Harald Schwalbe and Robert Silvers},
url = {https://doi.org/10.1002%2Fcbic.201700437},
doi = {10.1002/cbic.201700437},
year = {2017},
date = {2017-11-01},
urldate = {2017-11-01},
journal = {ChemBioChem},
volume = {18},
number = {23},
pages = {2340--2350},
publisher = {Wiley-Blackwell},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
21.
Kevin J Donovan; Robert Silvers; Sara Linse; Robert G Griffin
3D MAS NMR Experiment Utilizing Through-Space 15N–15N Correlations
In: Journal of the American Chemical Society, 139 (19), pp. 6518–6521, 2017.
@article{Donovan_2017,
title = {3D MAS NMR Experiment Utilizing Through-Space 15N–15N Correlations},
author = {Kevin J Donovan and Robert Silvers and Sara Linse and Robert G Griffin},
url = {https://doi.org/10.1021%2Fjacs.7b01159},
doi = {10.1021/jacs.7b01159},
year = {2017},
date = {2017-05-01},
urldate = {2017-05-01},
journal = {Journal of the American Chemical Society},
volume = {139},
number = {19},
pages = {6518--6521},
publisher = {American Chemical Society (ACS)},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
20.
Robert Silvers; Michael T. Colvin; Kendra K. Frederick; Angela C. Jacavone; Susan Lindquist; Sara Linse; Robert G. Griffin
Aggregation and Fibril Structure of AβM01-42 and Aβ1-42
In: Biochemistry, 56 (36), pp. 4850-4859, 2017, ISSN: 1520-4995.
Abstract | Links:
@article{silvers_2017,
title = {Aggregation and Fibril Structure of AβM01-42 and Aβ1-42},
author = {Robert Silvers and Michael T. Colvin and Kendra K. Frederick and Angela C. Jacavone and Susan Lindquist and Sara Linse and Robert G. Griffin},
doi = {10.1021/acs.biochem.7b00729},
issn = {1520-4995},
year = {2017},
date = {2017-01-01},
urldate = {2017-01-01},
journal = {Biochemistry},
volume = {56},
number = {36},
pages = {4850-4859},
abstract = {A mechanistic understanding of Aβ aggregation and high-resolution structures of Aβ fibrils and oligomers are vital to elucidating relevant details of neurodegeneration in Alzheimer's disease, which will facilitate the rational design of diagnostic and therapeutic protocols. The most detailed and reproducible insights into structure and kinetics have been achieved using Aβ peptides produced by recombinant expression, which results in an additional methionine at the N-terminus. While the length of the C-terminus is well established to have a profound impact on the peptide's aggregation propensity, structure, and neurotoxicity, the impact of the N-terminal methionine on the aggregation pathways and structure is unclear. For this reason, we have developed a protocol to produce recombinant Aβ, sans the N-terminal methionine, using an N-terminal small ubiquitin-like modifier-Aβ fusion protein in reasonable yield, with which we compared aggregation kinetics with Aβ containing the additional methionine residue. The data revealed that Aβ and Aβ aggregate with similar rates and by the same mechanism, in which the generation of new aggregates is dominated by secondary nucleation of monomers on the surface of fibrils. We also recorded magic angle spinning nuclear magnetic resonance spectra that demonstrated that excellent spectral resolution is maintained with both Aβ and Aβ and that the chemical shifts are virtually identical in dipolar recoupling experiments that provide information about rigid residues. Collectively, these results indicate that the structure of the fibril core is unaffected by N-terminal methionine. This is consistent with the recent structures of Aβ in which M0 is located at the terminus of a disordered 14-amino acid N-terminal tail.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
A mechanistic understanding of Aβ aggregation and high-resolution structures of Aβ fibrils and oligomers are vital to elucidating relevant details of neurodegeneration in Alzheimer's disease, which will facilitate the rational design of diagnostic and therapeutic protocols. The most detailed and reproducible insights into structure and kinetics have been achieved using Aβ peptides produced by recombinant expression, which results in an additional methionine at the N-terminus. While the length of the C-terminus is well established to have a profound impact on the peptide's aggregation propensity, structure, and neurotoxicity, the impact of the N-terminal methionine on the aggregation pathways and structure is unclear. For this reason, we have developed a protocol to produce recombinant Aβ, sans the N-terminal methionine, using an N-terminal small ubiquitin-like modifier-Aβ fusion protein in reasonable yield, with which we compared aggregation kinetics with Aβ containing the additional methionine residue. The data revealed that Aβ and Aβ aggregate with similar rates and by the same mechanism, in which the generation of new aggregates is dominated by secondary nucleation of monomers on the surface of fibrils. We also recorded magic angle spinning nuclear magnetic resonance spectra that demonstrated that excellent spectral resolution is maintained with both Aβ and Aβ and that the chemical shifts are virtually identical in dipolar recoupling experiments that provide information about rigid residues. Collectively, these results indicate that the structure of the fibril core is unaffected by N-terminal methionine. This is consistent with the recent structures of Aβ in which M0 is located at the terminus of a disordered 14-amino acid N-terminal tail.
19.
W Y Zhu; R Silvers; H Schwalbe; T A Keiderling
Reduced and mutant lysozyme refolding with lipid vesicles. Model study of disulfide impact on equilibria and dynamics
In: Biochimica Et Biophysica Acta-Proteins and Proteomics, 1864 (9), pp. 1083-1092, 2016.
@article{RID:0918161225351-30,
title = {Reduced and mutant lysozyme refolding with lipid vesicles. Model study of disulfide impact on equilibria and dynamics},
author = {W Y Zhu and R Silvers and H Schwalbe and T A Keiderling},
year = {2016},
date = {2016-01-01},
urldate = {2016-01-01},
journal = {Biochimica Et Biophysica Acta-Proteins and Proteomics},
volume = {1864},
number = {9},
pages = {1083-1092},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
18.
Monu Kaushik; Thorsten Bahrenberg; Thach V Can; Marc A Caporini; Robert Silvers; Jorg Heiliger; Albert A Smith; Harald Schwalbe; Robert G Griffin; Bjorn Corzilius
Gd(iii) and Mn(ii) complexes for dynamic nuclear polarization: small molecular chelate polarizing agents and applications with site-directed spin labeling of proteins
In: Physical Chemistry Chemical Physics, 2016.
@article{RID:0918161225351-29,
title = {Gd(iii) and Mn(ii) complexes for dynamic nuclear polarization: small molecular chelate polarizing agents and applications with site-directed spin labeling of proteins},
author = {Monu Kaushik and Thorsten Bahrenberg and Thach V Can and Marc A Caporini and Robert Silvers and Jorg Heiliger and Albert A Smith and Harald Schwalbe and Robert G Griffin and Bjorn Corzilius},
year = {2016},
date = {2016-01-01},
urldate = {2016-01-01},
journal = {Physical Chemistry Chemical Physics},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
17.
F Sochor; R Silvers; D Muller; C Richter; B Furtig; H Schwalbe
F-19-labeling of the adenine H-2-site to study large RNAs by NMR spectroscopy
In: Journal of Biomolecular Nmr, 64 (1), pp. 63-74, 2016.
@article{RID:0918161225350-31,
title = {F-19-labeling of the adenine H-2-site to study large RNAs by NMR spectroscopy},
author = {F Sochor and R Silvers and D Muller and C Richter and B Furtig and H Schwalbe},
year = {2016},
date = {2016-01-01},
urldate = {2016-01-01},
journal = {Journal of Biomolecular Nmr},
volume = {64},
number = {1},
pages = {63-74},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
16.
Michael T. Colvin; Robert Silvers; Birgitta Frohm; Yongchao Su; Sara Linse; Robert G. Griffin
High Resolution Structural Characterization of Aβ42 Amyloid Fibrils by Magic Angle Spinning NMR
In: Journal of the American Chemical Society, 137 (23), pp. 7509-7518, 2015.
Abstract | Links:
@article{RID:0918161225350-28,
title = {High Resolution Structural Characterization of Aβ42 Amyloid Fibrils by Magic Angle Spinning NMR},
author = {Michael T. Colvin and Robert Silvers and Birgitta Frohm and Yongchao Su and Sara Linse and Robert G. Griffin},
doi = {10.1021/jacs.5b03997},
year = {2015},
date = {2015-05-22},
urldate = {2015-05-22},
journal = {Journal of the American Chemical Society},
volume = {137},
number = {23},
pages = {7509-7518},
abstract = {The presence of amyloid plaques composed of amyloid beta (Aβ) fibrils is a hallmark of Alzheimer’s disease (AD). The Aβ peptide is present as several length variants with two common alloforms consisting of 40 and 42 amino acids, denoted Aβ1–40 and Aβ1–42, respectively. While there have been numerous reports that structurally characterize fibrils of Aβ1–40, very little is known about the structure of amyloid fibrils of Aβ1–42, which are considered the more toxic alloform involved in AD. We have prepared isotopically 13C/15N labeled AβM01–42 fibrils in vitro from recombinant protein and examined their 13C–13C and 13C–15N magic angle spinning (MAS) NMR spectra. In contrast to several other studies of Aβ fibrils, we observe spectra with excellent resolution and a single set of chemical shifts, suggesting the presence of a single fibril morphology. We report the initial structural characterization of AβM01–42 fibrils utilizing 13C and 15N shift assignments of 38 of the 43 residues, including the backbone and side chains, obtained through a series of cross-polarization based 2D and 3D 13C–13C, 13C–15N MAS NMR experiments for rigid residues along with J-based 2D TOBSY experiments for dynamic residues. We find that the first ∼5 residues are dynamic and most efficiently detected in a J-based TOBSY spectrum. In contrast, residues 16–42 are easily observed in cross-polarization experiments and most likely form the amyloid core. Calculation of ψ and φ dihedral angles from the chemical shift assignments indicate that 4 β-strands are present in the fibril’s secondary structure.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The presence of amyloid plaques composed of amyloid beta (Aβ) fibrils is a hallmark of Alzheimer’s disease (AD). The Aβ peptide is present as several length variants with two common alloforms consisting of 40 and 42 amino acids, denoted Aβ1–40 and Aβ1–42, respectively. While there have been numerous reports that structurally characterize fibrils of Aβ1–40, very little is known about the structure of amyloid fibrils of Aβ1–42, which are considered the more toxic alloform involved in AD. We have prepared isotopically 13C/15N labeled AβM01–42 fibrils in vitro from recombinant protein and examined their 13C–13C and 13C–15N magic angle spinning (MAS) NMR spectra. In contrast to several other studies of Aβ fibrils, we observe spectra with excellent resolution and a single set of chemical shifts, suggesting the presence of a single fibril morphology. We report the initial structural characterization of AβM01–42 fibrils utilizing 13C and 15N shift assignments of 38 of the 43 residues, including the backbone and side chains, obtained through a series of cross-polarization based 2D and 3D 13C–13C, 13C–15N MAS NMR experiments for rigid residues along with J-based 2D TOBSY experiments for dynamic residues. We find that the first ∼5 residues are dynamic and most efficiently detected in a J-based TOBSY spectrum. In contrast, residues 16–42 are easily observed in cross-polarization experiments and most likely form the amyloid core. Calculation of ψ and φ dihedral angles from the chemical shift assignments indicate that 4 β-strands are present in the fibril’s secondary structure.
15.
M T Eddy; Y C Su; R Silvers; L Andreas; L Clark; G Wagner; G Pintacuda; L Emsley; R G Griffin
Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR
In: Journal of Biomolecular Nmr, 61 (3-4), pp. 299-310, 2015.
@article{RID:0918161225352-26,
title = {Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR},
author = {M T Eddy and Y C Su and R Silvers and L Andreas and L Clark and G Wagner and G Pintacuda and L Emsley and R G Griffin},
year = {2015},
date = {2015-01-01},
journal = {Journal of Biomolecular Nmr},
volume = {61},
number = {3-4},
pages = {299-310},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
14.
Michael T Colvin; Robert Silvers; Birgitta Frohm; Yongchao Su; Sara Linse; Robert G Griffin
High Resolution Structural Characterization of Abeta42 Amyloid Fibrils by Magic Angle Spinning NMR
In: Journal of the American Chemical Society, 137 (23), pp. 7509-18, 2015.
@article{RID:0918161225351-27,
title = {High Resolution Structural Characterization of Abeta42 Amyloid Fibrils by Magic Angle Spinning NMR},
author = {Michael T Colvin and Robert Silvers and Birgitta Frohm and Yongchao Su and Sara Linse and Robert G Griffin},
year = {2015},
date = {2015-01-01},
journal = {Journal of the American Chemical Society},
volume = {137},
number = {23},
pages = {7509-18},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
13.
Robert Silvers; Heiko Keller; Harald Schwalbe; Martin Hengesbach
Differential Scanning Fluorimetry for Monitoring RNA Stability
In: ChemBioChem, 16 (7), pp. 1109-1114, 2015.
Abstract | Links:
@article{doi:10.1002/cbic.201500046,
title = {Differential Scanning Fluorimetry for Monitoring RNA Stability},
author = {Robert Silvers and Heiko Keller and Harald Schwalbe and Martin Hengesbach},
doi = {10.1002/cbic.201500046},
year = {2015},
date = {2015-01-01},
journal = {ChemBioChem},
volume = {16},
number = {7},
pages = {1109-1114},
abstract = {Abstract Cellular RNA function is closely linked to RNA structure. It is therefore imperative to develop methods that report on structural stability of RNA and how it is modulated by binding of ions, other osmolytes, and RNA-binding ligands. Here, we present a novel method to analyze the stability of virtually any structured RNA in a highly parallel fashion. This method can easily determine the influence of various additives on RNA stability, and even characterize ligand-induced stabilization of riboswitch RNA. Current approaches to assess RNA stability include thermal melting profiles (absorption or circular dichroism) and differential scanning calorimetry. These techniques, however, require a substantial amount of material and cannot be significantly parallelized. Current fluorescence spectroscopic methods rely on intercalating dyes, which alter the stability of RNA. We employ the commercial fluorescent dye RiboGreen, which discriminates between single-stranded (or unstructured regions) and double-stranded RNA. Binding leads to an increase in fluorescence quantum yield, and thus reports structural changes by a change in fluorescence intensity.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Abstract Cellular RNA function is closely linked to RNA structure. It is therefore imperative to develop methods that report on structural stability of RNA and how it is modulated by binding of ions, other osmolytes, and RNA-binding ligands. Here, we present a novel method to analyze the stability of virtually any structured RNA in a highly parallel fashion. This method can easily determine the influence of various additives on RNA stability, and even characterize ligand-induced stabilization of riboswitch RNA. Current approaches to assess RNA stability include thermal melting profiles (absorption or circular dichroism) and differential scanning calorimetry. These techniques, however, require a substantial amount of material and cannot be significantly parallelized. Current fluorescence spectroscopic methods rely on intercalating dyes, which alter the stability of RNA. We employ the commercial fluorescent dye RiboGreen, which discriminates between single-stranded (or unstructured regions) and double-stranded RNA. Binding leads to an increase in fluorescence quantum yield, and thus reports structural changes by a change in fluorescence intensity.
12.
J Stehle; R Silvers; K Werner; D Chatterjee; S Gande; F Scholz; A Dutta; J Wachtveitl; J Klein-Seetharaman; H Schwalbe
Characterization of the Simultaneous Decay Kinetics of Metarhodopsin States II and III in Rhodopsin by Solution-State NMR Spectroscopy
In: Angewandte Chemie-International Edition, 53 (8), pp. 2078-2084, 2014.
@article{RID:0918161225352-24,
title = {Characterization of the Simultaneous Decay Kinetics of Metarhodopsin States II and III in Rhodopsin by Solution-State NMR Spectroscopy},
author = {J Stehle and R Silvers and K Werner and D Chatterjee and S Gande and F Scholz and A Dutta and J Wachtveitl and J Klein-Seetharaman and H Schwalbe},
year = {2014},
date = {2014-01-01},
journal = {Angewandte Chemie-International Edition},
volume = {53},
number = {8},
pages = {2078-2084},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
11.
R Silvers; H Schwalbe
Detecting Intracellular Cysteine Redox States by in-Cell NMR Spectroscopy
In: Chembiochem, 14 (14), pp. 1705-1707, 2013.
@article{RID:0918161225352-22,
title = {Detecting Intracellular Cysteine Redox States by in-Cell NMR Spectroscopy},
author = {R Silvers and H Schwalbe},
year = {2013},
date = {2013-01-01},
journal = {Chembiochem},
volume = {14},
number = {14},
pages = {1705-1707},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
10.
Robert Silvers
Conformational dynamics of non-native states of proteins and intrinsically unstructured proteins by NMR spectroscopy
2012.
@phdthesis{nokey,
title = {Conformational dynamics of non-native states of proteins and intrinsically unstructured proteins by NMR spectroscopy},
author = {Robert Silvers},
year = {2012},
date = {2012-12-01},
keywords = {},
pubstate = {published},
tppubtype = {phdthesis}
}
9.
R Silvers; K Saxena; D Kudlinzki; H Schwalbe
Recombinant expression and purification of human TATA binding protein using a chimeric fusion
In: Protein Expression and Purification, 85 (1), pp. 142-147, 2012.
@article{RID:0918161225353-19,
title = {Recombinant expression and purification of human TATA binding protein using a chimeric fusion},
author = {R Silvers and K Saxena and D Kudlinzki and H Schwalbe},
year = {2012},
date = {2012-01-01},
journal = {Protein Expression and Purification},
volume = {85},
number = {1},
pages = {142-147},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
8.
R Silvers; F Sziegat; H Tachibana; S Segawa; S Whittaker; U L Gunther; F Gabel; J R Huang; M Blackledge; J Wirmer-Bartoschek; H Schwalbe
Modulation of Structure and Dynamics by Disulfide Bond Formation in Unfolded States
In: Journal of the American Chemical Society, 134 (15), pp. 6846-6854, 2012.
@article{RID:0918161225353-15,
title = {Modulation of Structure and Dynamics by Disulfide Bond Formation in Unfolded States},
author = {R Silvers and F Sziegat and H Tachibana and S Segawa and S Whittaker and U L Gunther and F Gabel and J R Huang and M Blackledge and J Wirmer-Bartoschek and H Schwalbe},
year = {2012},
date = {2012-01-01},
journal = {Journal of the American Chemical Society},
volume = {134},
number = {15},
pages = {6846-6854},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
7.
F Sziegat; R Silvers; M Hahnke; M R Jensen; M Blackledge; J Wirmer-Bartoschek; H Schwalbe
Disentangling the Coil: Modulation of Conformational and Dynamic Properties by Site-Directed Mutation in the Non-Native State of Hen Egg White Lysozyme
In: Biochemistry, 51 (16), pp. 3361-3372, 2012.
@article{RID:0918161225353-14,
title = {Disentangling the Coil: Modulation of Conformational and Dynamic Properties by Site-Directed Mutation in the Non-Native State of Hen Egg White Lysozyme},
author = {F Sziegat and R Silvers and M Hahnke and M R Jensen and M Blackledge and J Wirmer-Bartoschek and H Schwalbe},
year = {2012},
date = {2012-01-01},
journal = {Biochemistry},
volume = {51},
number = {16},
pages = {3361-3372},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
6.
Sina Reckel; Daniel Gottstein; Jochen Stehle; Frank Löhr; Mirka-Kristin Verhoefen; Mitsuhiro Takeda; Robert Silvers; Masatsune Kainosho; Clemens Glaubitz; Josef Wachtveitl; Frank Bernhard; Harald Schwalbe; Peter Güntert; Volker Dötsch
Solution NMR Structure of Proteorhodopsin
In: Angewandte Chemie International Edition, 50 (50), pp. 11942-11946, 2011.
@article{RID:0918161225354-5,
title = {Solution NMR Structure of Proteorhodopsin},
author = {Sina Reckel and Daniel Gottstein and Jochen Stehle and Frank Löhr and Mirka-Kristin Verhoefen and Mitsuhiro Takeda and Robert Silvers and Masatsune Kainosho and Clemens Glaubitz and Josef Wachtveitl and Frank Bernhard and Harald Schwalbe and Peter Güntert and Volker Dötsch},
year = {2011},
date = {2011-01-01},
journal = {Angewandte Chemie International Edition},
volume = {50},
number = {50},
pages = {11942-11946},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
5.
Robert Silvers; Kai Schlepckow; Julia Wirmer-Bartoschek; Harald Schwalbe
NMR-Spectroscopic Investigation of Disulfide Dynamics in Unfolded States of Proteins Folding of Disulfide Proteins
In: Chang, Rowen J Y; Ventura, Salvador (Ed.): 2011.
@inbook{RID:0918161225354-1,
title = {NMR-Spectroscopic Investigation of Disulfide Dynamics in Unfolded States of Proteins
Folding of Disulfide Proteins},
author = {Robert Silvers and Kai Schlepckow and Julia Wirmer-Bartoschek and Harald Schwalbe},
editor = {Rowen J Y Chang and Salvador Ventura},
year = {2011},
date = {2011-01-01},
series = {Protein Reviews},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}
4.
Delphine B Salvatore; Nicolas Duraffourg; Adrien Favier; Björn A Persson; Mikael Lund; Marie-Madeleine Delage; Robert Silvers; Harald Schwalbe; Thomas Croguennec; Saïd Bouhallab; Vincent Forge
Investigation at Residue Level of the Early Steps during the Assembly of Two Proteins into Supramolecular Objects
In: Biomacromolecules, 12 (6), pp. 2200-2210, 2011.
@article{RID:0918161225354-6,
title = {Investigation at Residue Level of the Early Steps during the Assembly of Two Proteins into Supramolecular Objects},
author = {Delphine B Salvatore and Nicolas Duraffourg and Adrien Favier and Björn A Persson and Mikael Lund and Marie-Madeleine Delage and Robert Silvers and Harald Schwalbe and Thomas Croguennec and Saïd Bouhallab and Vincent Forge},
year = {2011},
date = {2011-01-01},
journal = {Biomacromolecules},
volume = {12},
number = {6},
pages = {2200-2210},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
3.
R Silvers; F Buhr; H Schwalbe
The Molecular Mechanism of Spider-Silk Formation
In: Angewandte Chemie-International Edition, 49 (32), pp. 5410-5412, 2010.
@article{RID:0918161225355-12,
title = {The Molecular Mechanism of Spider-Silk Formation},
author = {R Silvers and F Buhr and H Schwalbe},
year = {2010},
date = {2010-01-01},
journal = {Angewandte Chemie-International Edition},
volume = {49},
number = {32},
pages = {5410-5412},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
2.
Christian Gerum; Robert Silvers; Julia Wirmer-Bartoschek; Harald Schwalbe
Unfolded-State Structure and Dynamics Influence the Fibril Formation of Human Prion Protein
In: Angewandte Chemie International Edition, 48 (50), pp. 9452-9456, 2009.
@article{RID:0918161225355-8,
title = {Unfolded-State Structure and Dynamics Influence the Fibril Formation of Human Prion Protein},
author = {Christian Gerum and Robert Silvers and Julia Wirmer-Bartoschek and Harald Schwalbe},
year = {2009},
date = {2009-01-01},
journal = {Angewandte Chemie International Edition},
volume = {48},
number = {50},
pages = {9452-9456},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
1.
Senada Nozinovic; Boris Nachtsheim; Sebastian Scheuermann; Jan Schnorr; Robert Silvers; Matthias Wagner; Harald Schwalbe
Learning problem-solving thinking in group work
In: Nachrichten aus der Chemie, 53 (9), pp. 978-980, 2005.
Abstract | Links:
@article{nozinovic_2005,
title = {Learning problem-solving thinking in group work},
author = {Senada Nozinovic and Boris Nachtsheim and Sebastian Scheuermann and Jan Schnorr and Robert Silvers and Matthias Wagner and Harald Schwalbe},
doi = {10.1002/nadc.20050530955},
year = {2005},
date = {2005-09-01},
urldate = {2005-09-01},
journal = {Nachrichten aus der Chemie},
volume = {53},
number = {9},
pages = {978-980},
abstract = {In the midst of the discussion about elite promotion and cutting-edge research, two liaison lecturers from the German National Academic Foundation developed and tested a new research college with talented chemistry and biochemistry students.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In the midst of the discussion about elite promotion and cutting-edge research, two liaison lecturers from the German National Academic Foundation developed and tested a new research college with talented chemistry and biochemistry students.