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Anal Chem. 2015 Apr 7;87(7):4015-4022. doi: 10.1021/acs.analchem.5b00372. Epub 2015 Mar 25.

Differential isotopic enrichment to facilitate characterization of asymmetric multimeric proteins using hydrogen/deuterium exchange mass spectrometry.

Author information

1
Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL 33458.USA.
2
Center for Advanced Biotechnology and Medicine, and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA.
3
Informatics Core, The Scripps Research Institute, Jupiter, FL 33458.USA.
#
Contributed equally

Abstract

Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry has emerged as a powerful tool for analyzing the conformational dynamics of protein-ligand and protein-protein interactions. Recent advances in instrumentation and methodology have expanded the utility of HDX for the analysis of large and complex proteins; however, asymmetric dimers with shared amino acid sequence present a unique challenge for HDX because assignment of peptides with identical sequence to their subunit of origin remains ambiguous. Here we report the use of differential isotopic labeling to facilitate HDX analysis of multimers using HIV-1 reverse transcriptase (RT) as a model. RT is an asymmetric heterodimer of 51 kDa (p51) and 66 kDa (p66) subunits. The first 440 residues of p51 and p66 are identical. In this study differentially labeled RT was reconstituted from isotopically enriched ((15)N-labeled) p51 and unlabeled p66. To enable detection of (15)N-deuterated RT peptides, the software HDX Workbench was modified to follow a 100% (15)N model. Our results demonstrated that (15)N enrichment of p51 did not affect its conformational dynamics compared to unlabeled p51, but (15)N-labeled p51 did show different conformational dynamics than p66 in the RT heterodimer. Differential HDX-MS of isotopically labeled RT in the presence of the non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz (EFV) showed subunit-specific perturbation in the rate of HDX consistent with previously published results and the RT-EFV cocrystal structure.

PMID:
25763479
PMCID:
PMC4445842
DOI:
10.1021/acs.analchem.5b00372
[Indexed for MEDLINE]
Free PMC Article

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