Validated Determination of NRG1 Ig-like Domain Structure by Mass Spectrometry Coupled with Computational Modeling

High resolution hydroxyl radical protein footprinting (HR-HRPF) is a mass spectrometry-based method that measures the solvent exposure of multiple amino acids in a single experiment, offering constraints for experimentally-informed computational modeling. HR-HRPF-based modeling has previously been used to accurately model the structure of proteins of known structure, but the technique has never been used to determine the structure of a protein of unknown structure leaving questions of unintentional bias and applicability to unknown structures unresolved. Here, we present the use of HR-HRPF-based modeling to determine the structure of the Ig-like domain of NRG1, a protein with no close homolog of known structure. Independent determination of the protein structure by both HR-HRPF-based modeling and heteronuclear NMR was carried out, with results compared only after both processes were complete. The HR-HRPF-based model was highly similar to the lowest energy NMR model, with a backbone RMSD of 1.6 Å. To our knowledge, this is the first use of HR-HRPF-based modeling to determine a previously uncharacterized protein structure.

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Accurate protein structure prediction with hydroxyl radical protein footprinting data

Nature Communications ◽

10.1038/s41467-020-20549-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sarah E. Biehn ◽

Steffen Lindert

Keyword(s):

Protein Structure ◽

Hydroxyl Radical ◽

Protein Structure Prediction ◽

Structure Prediction ◽

Tertiary Structure ◽

Solvent Exposure ◽

Protein Footprinting ◽

Hydroxyl Radical Protein Footprinting ◽

Predict Protein Structure ◽

Ab Initio Models

AbstractHydroxyl radical protein footprinting (HRPF) in combination with mass spectrometry reveals the relative solvent exposure of labeled residues within a protein, thereby providing insight into protein tertiary structure. HRPF labels nineteen residues with varying degrees of reliability and reactivity. Here, we are presenting a dynamics-driven HRPF-guided algorithm for protein structure prediction. In a benchmark test of our algorithm, usage of the dynamics data in a score term resulted in notable improvement of the root-mean-square deviations of the lowest-scoring ab initio models and improved the funnel-like metric Pnear for all benchmark proteins. We identified models with accurate atomic detail for three of the four benchmark proteins. This work suggests that HRPF data along with side chain dynamics sampled by a Rosetta mover ensemble can be used to accurately predict protein structure.

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High-Resolution Hydroxyl Radical Protein Footprinting: Biophysics Tool for Drug Discovery

Annual Review of Biophysics ◽

10.1146/annurev-biophys-070317-033123 ◽

2018 ◽

Vol 47 (1) ◽

pp. 315-333 ◽

Cited By ~ 16

Author(s):

Janna Kiselar ◽

Mark R. Chance

Keyword(s):

Protein Structure ◽

High Resolution ◽

Hydroxyl Radical ◽

Solvent Accessibility ◽

Side Chains ◽

Protein Footprinting ◽

Hydroxyl Radical Protein Footprinting ◽

Wide Range ◽

Hydroxyl Radical Footprinting

Hydroxyl radical footprinting (HRF) of proteins with mass spectrometry (MS) is a widespread approach for assessing protein structure. Hydroxyl radicals react with a wide variety of protein side chains, and the ease with which radicals can be generated (by radiolysis or photolysis) has made the approach popular with many laboratories. As some side chains are less reactive and thus cannot be probed, additional specific and nonspecific labeling reagents have been introduced to extend the approach. At the same time, advances in liquid chromatography and MS approaches permit an examination of the labeling of individual residues, transforming the approach to high resolution. Lastly, advances in understanding of the chemistry of the approach have led to the determination of absolute protein topologies from HRF data. Overall, the technology can provide precise and accurate measures of side-chain solvent accessibility in a wide range of interesting and useful contexts for the study of protein structure and dynamics in both academia and industry.

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Rosetta Protein Structure Prediction from Hydroxyl Radical Protein Footprinting Mass Spectrometry Data

Analytical Chemistry ◽

10.1021/acs.analchem.8b01624 ◽

2018 ◽

Vol 90 (12) ◽

pp. 7721-7729 ◽

Cited By ~ 22

Author(s):

Melanie L. Aprahamian ◽

Emily E. Chea ◽

Lisa M. Jones ◽

Steffen Lindert

Keyword(s):

Mass Spectrometry ◽

Protein Structure ◽

Hydroxyl Radical ◽

Protein Structure Prediction ◽

Structure Prediction ◽

Mass Spectrometry Data ◽

Protein Footprinting ◽

Hydroxyl Radical Protein Footprinting

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Current Trends in Biotherapeutic Higher Order Structure Characterization by Irreversible Covalent Footprinting Mass Spectrometry

Protein and Peptide Letters ◽

10.2174/0929866526666181128141953 ◽

2019 ◽

Vol 26 (1) ◽

pp. 35-43 ◽

Cited By ~ 3

Author(s):

Natalie K. Garcia ◽

Galahad Deperalta ◽

Aaron T. Wecksler

Keyword(s):

Mass Spectrometry ◽

Protein Structure ◽

Protein Interactions ◽

Quaternary Structure ◽

Solvent Accessibility ◽

Higher Order ◽

Structure Characterization ◽

Order Structure ◽

Protein Footprinting ◽

Wide Range

Background: Biotherapeutics, particularly monoclonal antibodies (mAbs), are a maturing class of drugs capable of treating a wide range of diseases. Therapeutic function and solutionstability are linked to the proper three-dimensional organization of the primary sequence into Higher Order Structure (HOS) as well as the timescales of protein motions (dynamics). Methods that directly monitor protein HOS and dynamics are important for mapping therapeutically relevant protein-protein interactions and assessing properly folded structures. Irreversible covalent protein footprinting Mass Spectrometry (MS) tools, such as site-specific amino acid labeling and hydroxyl radical footprinting are analytical techniques capable of monitoring the side chain solvent accessibility influenced by tertiary and quaternary structure. Here we discuss the methodology, examples of biotherapeutic applications, and the future directions of irreversible covalent protein footprinting MS in biotherapeutic research and development. Conclusion: Bottom-up mass spectrometry using irreversible labeling techniques provide valuable information for characterizing solution-phase protein structure. Examples range from epitope mapping and protein-ligand interactions, to probing challenging structures of membrane proteins. By paring these techniques with hydrogen-deuterium exchange, spectroscopic analysis, or static-phase structural data such as crystallography or electron microscopy, a comprehensive understanding of protein structure can be obtained.

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Fast Photochemical Oxidation of Proteins Coupled with Mass Spectrometry

Protein and Peptide Letters ◽

10.2174/0929866526666181128124554 ◽

2019 ◽

Vol 26 (1) ◽

pp. 27-34 ◽

Cited By ~ 3

Author(s):

Liuqing Shi ◽

Michael L. Gross

Keyword(s):

Mass Spectrometry ◽

Hydroxyl Radical ◽

Protein Modification ◽

Solvent Accessibility ◽

Conformational Dynamics ◽

Photochemical Oxidation ◽

Structural Proteomics ◽

Sequence Coverage ◽

Protein Footprinting

Background: Determination of the composition and some structural features of macromolecules can be achieved by using structural proteomics approaches coupled with mass spectrometry (MS). One approach is hydroxyl radical protein footprinting whereby amino-acid side chains are modified with reactive reagents to modify irreversibly a protein side chain. The outcomes, when deciphered with mass-spectrometry-based proteomics, can increase our knowledge of structure, assembly, and conformational dynamics of macromolecules in solution. Generating the hydroxyl radicals by laser irradiation, Hambly and Gross developed the approach of Fast Photochemical Oxidation of Proteins (FPOP), which labels proteins on the sub millisecond time scale and provides, with MS analysis, deeper understanding of protein structure and protein-ligand and protein- protein interactions. This review highlights the fundamentals of FPOP and provides descriptions of hydroxyl-radical and other radical and carbene generation, of the hydroxyl labeling of proteins, and of determination of protein modification sites. We also summarize some recent applications of FPOP coupled with MS in protein footprinting. Conclusion: We survey results that show the capability of FPOP for qualitatively measuring protein solvent accessibility on the residue level. To make these approaches more valuable, we describe recent method developments that increase FPOP’s quantitative capacity and increase the spatial protein sequence coverage. To improve FPOP further, several new labeling reagents including carbenes and other radicals have been developed. These growing improvements will allow oxidative- footprinting methods coupled with MS to play an increasingly significant role in determining the structure and dynamics of macromolecules and their assemblies.

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