2022
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 Journal Article
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 Journal Article
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.
2021
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 Journal Article
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 Book Chapter
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.
2018
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 Journal Article
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.
2017
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 Journal Article
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 Journal Article
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 Journal Article
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.
2016
19.
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 Journal Article
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}
}
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 Journal Article
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.
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 Journal Article
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}
}
2015
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 Journal Article
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.
Robert Silvers; Heiko Keller; Harald Schwalbe; Martin Hengesbach
Differential Scanning Fluorimetry for Monitoring RNA Stability Journal Article
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.
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 Journal Article
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.
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 Journal Article
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}
}
2014
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 Journal Article
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}
}
2013
11.
R Silvers; H Schwalbe
Detecting Intracellular Cysteine Redox States by in-Cell NMR Spectroscopy Journal Article
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}
}
2012
10.
Robert Silvers
Conformational dynamics of non-native states of proteins and intrinsically unstructured proteins by NMR spectroscopy PhD Thesis
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.
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 Journal Article
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}
}
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 Journal Article
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.
R Silvers; K Saxena; D Kudlinzki; H Schwalbe
Recombinant expression and purification of human TATA binding protein using a chimeric fusion Journal Article
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}
}
2011
6.
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 Journal Article
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}
}
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 Book Chapter
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.
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 Journal Article
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}
}
2010
3.
R Silvers; F Buhr; H Schwalbe
The Molecular Mechanism of Spider-Silk Formation Journal Article
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}
}
2009
2.
Christian Gerum; Robert Silvers; Julia Wirmer-Bartoschek; Harald Schwalbe
Unfolded-State Structure and Dynamics Influence the Fibril Formation of Human Prion Protein Journal Article
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}
}
2005
1.
Senada Nozinovic; Boris Nachtsheim; Sebastian Scheuermann; Jan Schnorr; Robert Silvers; Matthias Wagner; Harald Schwalbe
Learning problem-solving thinking in group work Journal Article
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.