Individual amide proton exchange rates in thermally unfolded basic pancreatic trypsin inhibitor
- PMID: 2417626
- DOI: 10.1021/bi00346a056
Individual amide proton exchange rates in thermally unfolded basic pancreatic trypsin inhibitor
Abstract
A novel experiment is described for measurements of amide proton exchange rates in proteins with a time resolution of about 1 s. A flow apparatus was used to expose protein solutions in 2H2O first to high temperature for a predetermined time period, during which 1H-2H exchange proceeded, and then to ice-water. The technique was applied for exchange studies in thermally unfolded, selectively reduced basic pancreatic trypsin inhibitor. Measurements were made by 1H nuclear magnetic resonance after the exchange was quenched by rapid cooling. Thereby, the sequence-specific resonance assignments for the folded protein could be used, which had been previously obtained. The results of this study indicate that the exchange rates in the thermally unfolded protein are close to those expected for a random chain and that the NH exchange is catalyzed by 2H+ and O2H- up to high temperature, with no significant contributions from p2H-independent catalysis. We conclude that the parameters derived by Molday et al. [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158] from measurements with small model peptides can be used to calculate intrinsic exchange rates in unfolded proteins and thus provide a reliable reference for the interpretation of exchange rates measured under native conditions.
Similar articles
-
Amide proton exchange in proteins by EX1 kinetics: studies of the basic pancreatic trypsin inhibitor at variable p2H and temperature.Biochemistry. 1985 Dec 3;24(25):7396-407. doi: 10.1021/bi00346a055. Biochemistry. 1985. PMID: 2417625
-
Hydrogen exchange in thermally denatured ribonuclease A.Biochemistry. 1991 Oct 15;30(41):9907-14. doi: 10.1021/bi00105a014. Biochemistry. 1991. PMID: 1911782
-
Kinetics of amide proton exchange in helical peptides of varying chain lengths. Interpretation by the Lifson-Roig equation.Biochemistry. 1992 Feb 11;31(5):1263-9. doi: 10.1021/bi00120a001. Biochemistry. 1992. PMID: 1310608
-
NMR characterization of partially folded and unfolded conformational ensembles of proteins.Biopolymers. 1999;51(3):191-207. doi: 10.1002/(SICI)1097-0282(1999)51:3<191::AID-BIP3>3.0.CO;2-B. Biopolymers. 1999. PMID: 10516571 Review.
-
Measuring the hydrogen/deuterium exchange of proteins at high spatial resolution by mass spectrometry: overcoming gas-phase hydrogen/deuterium scrambling.Acc Chem Res. 2014 Oct 21;47(10):3018-27. doi: 10.1021/ar500194w. Epub 2014 Aug 29. Acc Chem Res. 2014. PMID: 25171396 Review.
Cited by
-
The contribution of electrostatics to hydrogen exchange in the unfolded protein state.Biophys J. 2021 Sep 21;120(18):4107-4114. doi: 10.1016/j.bpj.2021.08.003. Epub 2021 Aug 8. Biophys J. 2021. PMID: 34370996 Free PMC article.
-
Carbon-13 NMR method for the detection of correlated hydrogen exchange at adjacent backbone peptide amides and its application to hydrogen exchange in five antiparallel beta strands within the hydrophobic core of Streptomyces subtilisin inhibitor (SSI).Biochemistry. 2005 Sep 6;44(35):11811-20. doi: 10.1021/bi050467s. Biochemistry. 2005. PMID: 16128582 Free PMC article.
-
Protein dynamics viewed by hydrogen exchange.Protein Sci. 2012 Jul;21(7):996-1005. doi: 10.1002/pro.2081. Epub 2012 Jun 11. Protein Sci. 2012. PMID: 22544544 Free PMC article.
-
Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation.Protein Sci. 1993 Apr;2(4):522-31. doi: 10.1002/pro.5560020404. Protein Sci. 1993. PMID: 8390883 Free PMC article.
-
The pH dependence of hydrogen-deuterium exchange in trp repressor: the exchange rate of amide protons in proteins reflects tertiary interactions, not only secondary structure.Protein Sci. 1996 Apr;5(4):653-62. doi: 10.1002/pro.5560050409. Protein Sci. 1996. PMID: 8845754 Free PMC article.