Photochemical surface mapping of C14S-Sml1p for constrained computational modeling of protein structure

Joshua S. Sharp; Jun Tao Guo; Tomoaki Uchiki; Ying Xu; Chris Dealwis; Robert L. Hettich

doi:10.1016/j.ab.2005.02.005

Photochemical surface mapping of C14S-Sml1p for constrained computational modeling of protein structure

Joshua S. Sharp, Jun Tao Guo, Tomoaki Uchiki, Ying Xu, Chris Dealwis, Robert L. Hettich

Bioanalytical Mass Spectrometry

Research output: Contribution to journal › Article › peer-review

33 Scopus citations

Abstract

Photochemically generated hydroxyl radicals were used to map solvent-exposed regions in the C14S mutant of the protein Sml1p, a regulator of the ribonuclease reductase enzyme Rnr1p in Saccharomyces cerevisiae. By using high-performance mass spectrometry to characterize the oxidized peptides created by the hydroxyl radical reactions, amino acid solvent-accessibility data for native and denatured C14S Sml1p that revealed a solvent-excluding tertiary structure in the native state were obtained. The data on solvent accessibilities of various amino acids within the protein were then utilized to evaluate the de novo computational models generated by the HMMSTR/Rosetta server. The top five models initially generated by the server all disagreed with both published nuclear magnetic resonance (NMR) data and the solvent-accessibility data obtained in this study. A structural model adjusted to fit the previously reported NMR data satisfied most of the solvent-accessibility constraints. Through minor adjustment of the rotamers of two amino acid side chains for this latter structure, a model that not only provided a lower energy conformation but also completely satisfied previously reported data from NMR and tryptophan fluorescence measurements, in addition to the solvent-accessibility data presented here, was generated.

Original language	English
Pages (from-to)	201-212
Number of pages	12
Journal	Analytical Biochemistry
Volume	340
Issue number	2
DOIs	https://doi.org/10.1016/j.ab.2005.02.005
State	Published - May 15 2005

Funding

Research was conducted under an ORNL Exploratory Seed Project. J.S.S. acknowledges financial support from the Graduate School of Genome Science and Technology, University of Tennessee–Oak Ridge National Laboratory. J.G. and Y.X.’s work was supported in part by NSF fund DBI-0213840. ORNL is operated and managed by the University of Tennessee-Battelle, LLC, for the U.S. Department of Energy under Contract DE-AC05-00OR22725.

Funders	Funder number
Graduate School of Genome Science and Technology, University of Tennessee
University of Tennessee-Battelle
National Science Foundation	DBI-0213840
U.S. Department of Energy	DE-AC05-00OR22725
Oak Ridge National Laboratory

Keywords

Mass spectrometry
Photochemical surface mapping
Protein structure
Sml1p protein
Solvent accessibility
de novo computational modeling

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10.1016/j.ab.2005.02.005

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title = "Photochemical surface mapping of C14S-Sml1p for constrained computational modeling of protein structure",

abstract = "Photochemically generated hydroxyl radicals were used to map solvent-exposed regions in the C14S mutant of the protein Sml1p, a regulator of the ribonuclease reductase enzyme Rnr1p in Saccharomyces cerevisiae. By using high-performance mass spectrometry to characterize the oxidized peptides created by the hydroxyl radical reactions, amino acid solvent-accessibility data for native and denatured C14S Sml1p that revealed a solvent-excluding tertiary structure in the native state were obtained. The data on solvent accessibilities of various amino acids within the protein were then utilized to evaluate the de novo computational models generated by the HMMSTR/Rosetta server. The top five models initially generated by the server all disagreed with both published nuclear magnetic resonance (NMR) data and the solvent-accessibility data obtained in this study. A structural model adjusted to fit the previously reported NMR data satisfied most of the solvent-accessibility constraints. Through minor adjustment of the rotamers of two amino acid side chains for this latter structure, a model that not only provided a lower energy conformation but also completely satisfied previously reported data from NMR and tryptophan fluorescence measurements, in addition to the solvent-accessibility data presented here, was generated.",

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AU - Hettich, Robert L.

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AB - Photochemically generated hydroxyl radicals were used to map solvent-exposed regions in the C14S mutant of the protein Sml1p, a regulator of the ribonuclease reductase enzyme Rnr1p in Saccharomyces cerevisiae. By using high-performance mass spectrometry to characterize the oxidized peptides created by the hydroxyl radical reactions, amino acid solvent-accessibility data for native and denatured C14S Sml1p that revealed a solvent-excluding tertiary structure in the native state were obtained. The data on solvent accessibilities of various amino acids within the protein were then utilized to evaluate the de novo computational models generated by the HMMSTR/Rosetta server. The top five models initially generated by the server all disagreed with both published nuclear magnetic resonance (NMR) data and the solvent-accessibility data obtained in this study. A structural model adjusted to fit the previously reported NMR data satisfied most of the solvent-accessibility constraints. Through minor adjustment of the rotamers of two amino acid side chains for this latter structure, a model that not only provided a lower energy conformation but also completely satisfied previously reported data from NMR and tryptophan fluorescence measurements, in addition to the solvent-accessibility data presented here, was generated.

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Photochemical surface mapping of C14S-Sml1p for constrained computational modeling of protein structure

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