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Site-Specific Protonation Kinetics of Acidic Side Chains in Proteins Determined by pH-Dependent Carboxyl (13)C NMR Relaxation.

Wallerstein, Johan LU ; Weininger, Ulrich LU ; Khan, Ashhar LU ; Linse, Sara LU and Akke, Mikael LU (2015) In Journal of the American Chemical Society 137(8). p.3093-3101
Abstract
Proton-transfer dynamics plays a critical role in many biochemical processes, such as proton pumping across membranes and enzyme catalysis. The large majority of enzymes utilize acid-base catalysis and proton-transfer mechanisms, where the rates of proton transfer can be rate limiting for the overall reaction. However, measurement of proton-exchange kinetics for individual side-chain carboxyl groups in proteins has been achieved in only a handful of cases, which typically have involved comparative analysis of mutant proteins in the context of reaction network modeling. Here we describe an approach to determine site-specific protonation and deprotonation rate constants (kon and koff, respectively) of carboxyl side chains, based on (13)C NMR... (More)
Proton-transfer dynamics plays a critical role in many biochemical processes, such as proton pumping across membranes and enzyme catalysis. The large majority of enzymes utilize acid-base catalysis and proton-transfer mechanisms, where the rates of proton transfer can be rate limiting for the overall reaction. However, measurement of proton-exchange kinetics for individual side-chain carboxyl groups in proteins has been achieved in only a handful of cases, which typically have involved comparative analysis of mutant proteins in the context of reaction network modeling. Here we describe an approach to determine site-specific protonation and deprotonation rate constants (kon and koff, respectively) of carboxyl side chains, based on (13)C NMR relaxation measurements as a function of pH. We validated the method using an extensively studied model system, the B1 domain of protein G, for which we measured rate constants koff in the range (0.1-3) × 10(6) s(-1) and kon in the range (0.6-300) × 10(9) M(-1) s(-1), which correspond to acid-base equilibrium dissociation constants (Ka) in excellent agreement with previous results determined by chemical shift titrations. Our results further reveal a linear free-energy relationship between log kon and pKa, which provides information on the free-energy landscape of the protonation reaction, showing that the variability among residues in these parameters arises primarily from the extent of charge stabilization of the deprotonated state by the protein environment. We find that side-chain carboxyls with extreme values of koff or kon are involved in hydrogen bonding, thus providing a mechanistic explanation for the observed stabilization of the protonated or deprotonated state. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of the American Chemical Society
volume
137
issue
8
pages
3093 - 3101
publisher
The American Chemical Society
external identifiers
  • pmid:25665463
  • wos:000350614700044
  • scopus:84924301765
ISSN
1520-5126
DOI
10.1021/ja513205s
language
English
LU publication?
yes
id
afab6a3c-032d-46dd-8a8a-ce5a4f6f7582 (old id 5144986)
date added to LUP
2015-03-09 17:05:32
date last changed
2017-10-22 03:01:47
@article{afab6a3c-032d-46dd-8a8a-ce5a4f6f7582,
  abstract     = {Proton-transfer dynamics plays a critical role in many biochemical processes, such as proton pumping across membranes and enzyme catalysis. The large majority of enzymes utilize acid-base catalysis and proton-transfer mechanisms, where the rates of proton transfer can be rate limiting for the overall reaction. However, measurement of proton-exchange kinetics for individual side-chain carboxyl groups in proteins has been achieved in only a handful of cases, which typically have involved comparative analysis of mutant proteins in the context of reaction network modeling. Here we describe an approach to determine site-specific protonation and deprotonation rate constants (kon and koff, respectively) of carboxyl side chains, based on (13)C NMR relaxation measurements as a function of pH. We validated the method using an extensively studied model system, the B1 domain of protein G, for which we measured rate constants koff in the range (0.1-3) × 10(6) s(-1) and kon in the range (0.6-300) × 10(9) M(-1) s(-1), which correspond to acid-base equilibrium dissociation constants (Ka) in excellent agreement with previous results determined by chemical shift titrations. Our results further reveal a linear free-energy relationship between log kon and pKa, which provides information on the free-energy landscape of the protonation reaction, showing that the variability among residues in these parameters arises primarily from the extent of charge stabilization of the deprotonated state by the protein environment. We find that side-chain carboxyls with extreme values of koff or kon are involved in hydrogen bonding, thus providing a mechanistic explanation for the observed stabilization of the protonated or deprotonated state.},
  author       = {Wallerstein, Johan and Weininger, Ulrich and Khan, Ashhar and Linse, Sara and Akke, Mikael},
  issn         = {1520-5126},
  language     = {eng},
  number       = {8},
  pages        = {3093--3101},
  publisher    = {The American Chemical Society},
  series       = {Journal of the American Chemical Society},
  title        = {Site-Specific Protonation Kinetics of Acidic Side Chains in Proteins Determined by pH-Dependent Carboxyl (13)C NMR Relaxation.},
  url          = {http://dx.doi.org/10.1021/ja513205s},
  volume       = {137},
  year         = {2015},
}