Dataset Information

Accounting for Neighboring Residue Hydrophobicity in Diethylpyrocarbonate Labeling Mass Spectrometry Improves Rosetta Protein Structure Prediction.

ABSTRACT: Covalent labeling mass spectrometry allows for protein structure elucidation via covalent modification and identification of exposed residues. Diethylpyrocarbonate (DEPC) is a commonly used covalent labeling reagent that provides insight into structure through the labeling of lysine, histidine, serine, threonine, and tyrosine residues. We recently implemented a Rosetta algorithm that used binary DEPC labeling data to improve protein structure prediction efforts. In this work, we improved on our modeling efforts by accounting for the level of hydrophobicity of neighboring residues in the microenvironment of serine, threonine, and tyrosine residues to obtain a more accurate estimate of the hydrophobic neighbor count. This was incorporated into Rosetta functionality, along with considerations for solvent-exposed histidine and lysine residues. Overall, our new Rosetta score term successfully identified best scoring models with less than 2 Å root-mean-squared deviations (RMSDs) for five of the seven benchmark proteins tested. We additionally developed a confidence metric to measure prediction success for situations in which a native structure is unavailable.

SUBMITTER: Biehn SE

PROVIDER: S-EPMC8988852 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Accounting for Neighboring Residue Hydrophobicity in Diethylpyrocarbonate Labeling Mass Spectrometry Improves Rosetta Protein Structure Prediction.

Biehn Sarah E SE Picarello Danielle M DM Pan Xiao X Vachet Richard W RW Lindert Steffen S

Journal of the American Society for Mass Spectrometry 20220211 3

Covalent labeling mass spectrometry allows for protein structure elucidation via covalent modification and identification of exposed residues. Diethylpyrocarbonate (DEPC) is a commonly used covalent labeling reagent that provides insight into structure through the labeling of lysine, histidine, serine, threonine, and tyrosine residues. We recently implemented a Rosetta algorithm that used binary DEPC labeling data to improve protein structure prediction efforts. In this work, we improved on our ...[more]

PMID: 35147431

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Project description:Covalent labeling and mass spectrometry are seeing increased use together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g., diethylpyrocarbonate) and non-specific (e.g., hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. Here, we show that label loss is more significant and widespread than previously thought, especially for Ser, Thr, Tyr, and His residues, when relatively long protein digestion times are used. Such label loss ultimately decreases the amount of protein structural information that is obtainable with this reagent. We find, however, that the number of DEPC modified residues and, thus, protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g., 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g., microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Using short digestion times, we show that the percentage of solvent accessible residues that can be modified by DEPC increases from 44% to 67% for cytochrome c, 35% to 81% for myoglobin, and 76% to 95% for β-2-microglobulin. In effect, these increased numbers of modified residues improve the protein structural resolution available from this covalent labeling method. Compared with typical overnight digestion conditions, the short digestion times decrease the average distance between modified residues from 11 to 7 Å for myoglobin, 13 to 10 Å for cytochrome c, and 9 to 8 Å for β-2-microglobulin.

Dataset Information

Accounting for Neighboring Residue Hydrophobicity in Diethylpyrocarbonate Labeling Mass Spectrometry Improves Rosetta Protein Structure Prediction.

Publications

Accounting for Neighboring Residue Hydrophobicity in Diethylpyrocarbonate Labeling Mass Spectrometry Improves Rosetta Protein Structure Prediction.

Similar Datasets

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