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A quantitative strategy to detect changes in accessibility of protein regions to chemical modification on heterodimerization

  1. Mathias Dreger1,†,
  2. Bo Wah Leung2,
  3. George G. Brownlee2,*,
  4. Tao Deng2,*

Article first published online: 13 MAY 2009

DOI: 10.1002/pro.159

Issue

Protein Science

Protein Science

Volume 18, Issue 7, pages 1448–1458, July 2009

Additional Information

How to Cite

Dreger, M., Leung, B. W., Brownlee, G. G. and Deng, T. (2009), A quantitative strategy to detect changes in accessibility of protein regions to chemical modification on heterodimerization. Protein Science, 18: 1448–1458. doi: 10.1002/pro.159

Author Information

  1. 1

    Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford OX1 3PT, United Kingdom

  2. 2

    Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom

  1. †

    Biochemistry Department, Caprotec Bioanalytics, Volmerstrasse 5, 12489 Berlin, Germany

*Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OXI 3RE, UK

Publication History

  1. Issue published online: 23 JUN 2009
  2. Article first published online: 13 MAY 2009
  3. Accepted manuscript online: 13 MAY 2009 12:00AM EST
  4. Manuscript Accepted: 30 APR 2009
  5. Manuscript Revised: 29 APR 2009
  6. Manuscript Received: 3 APR 2009

Funded by

  • MRC. Grant Numbers: G9523972, G9901312
  • Wellcome Trust (OXION Initiative on Ion Channels and Disease)

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Keywords:

  • Q-POP assay;
  • influenza virus polymerase;
  • PB1-PA heterodimer;
  • SILAC;
  • NHS-biotin;
  • LC-MS/MS

Abstract

We describe a method for studying quantitative changes in accessibility of surface lysine residues of the PB1 subunit of the influenza RNA polymerase as a result of association with the PA subunit to form a PB1-PA heterodimer. Our method combines two established methods: (i) the chemical modification of surface lysine residues of native proteins by N-hydroxysuccinimidobiotin (NHS-biotin) and (ii) the stable isotope labeling of amino acids in cell culture (SILAC) followed by tryptic digestion and mass spectrometry. By linking the chemical modification with the SILAC methodology for the first time, we obtain quantitative data on chemical modification allowing subtle changes in accessibility to be described. Five regions in the PB1 monomer showed altered reactivity to NHS-biotin when compared with the [PB1-PA] heterodimer. Mutational analysis of residues in two such regions—at K265 and K481 of PB1, which were about three- and twofold, respectively, less accessible to biotinylation in the PB1-PA heterodimer compared with the PB1 monomer, demonstrated that both K265 and K481 were crucial for polymerase function. This novel assay of quantitative profiling of biotinylation patterns (Q-POP assay) highlights likely conformational changes at important functional sites, as observed here for PB1, and may provide information on protein–protein interaction interfaces. The Q-POP assay should be a generally applicable approach and may detect novel functional sites suitable for targeting by drugs.

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