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Residue-specific pK(a) determination of lysine and arginine side chains by indirect N-15 and C-13 NMR spectroscopy: Application to apo calmodulin

André, Ingemar LU ; Linse, Sara LU and Mulder, Frans LU (2007) In Journal of the American Chemical Society 129(51). p.15805-15813
Abstract
Electrostatic interactions in proteins can be probed experimentally through determination of residue-specific acidity constants, We describe here triple-resonance NMR techniques for direct determination of lysine and arginine side-chain protonation states in proteins. The experiments are based on detection of nonexchangeable protons over the full range of pH and temperature and therefore are well suited for pK(a) determination of individual amino acid side chains. The experiments follow the side-chain N-15(zeta) (lysine) and N-15(epsilon) or C-13(zeta) (arginine) chemical shift, which changes due to sizable changes in the heteronuclear electron distribution upon (de)protonation. Since heteronuclear chemical shifts are overwhelmed by the... (More)
Electrostatic interactions in proteins can be probed experimentally through determination of residue-specific acidity constants, We describe here triple-resonance NMR techniques for direct determination of lysine and arginine side-chain protonation states in proteins. The experiments are based on detection of nonexchangeable protons over the full range of pH and temperature and therefore are well suited for pK(a) determination of individual amino acid side chains. The experiments follow the side-chain N-15(zeta) (lysine) and N-15(epsilon) or C-13(zeta) (arginine) chemical shift, which changes due to sizable changes in the heteronuclear electron distribution upon (de)protonation. Since heteronuclear chemical shifts are overwhelmed by the charge state of the amino acid side chain itself, these methods supersede H-1-based NMR in terms of accuracy, sensitivity, and selectivity. Moreover, the N-15(zeta) and N-15(epsilon) nuclei may be used to probe changes in the local electrostatic environment. Applications to three proteins are described: apo calmodulin, calbindin D-9k, and FKBP12. For apo calmodulin, residue-specific pK(a) values of lysine side chains were determined to fall between 10.7 and 11.2 as a result of the high net negative charge on the protein surface. Ideal two-state titration behavior observed for all lysines indicates the absence of significant direct charge interactions between the basic residues. These results are compared with earlier studies based on chemical modification. (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
129
issue
51
pages
15805 - 15813
publisher
The American Chemical Society
external identifiers
  • wos:000251974000029
  • scopus:37549044716
ISSN
1520-5126
DOI
10.1021/ja0721824
language
English
LU publication?
yes
id
67735a31-0eb2-4dfb-9cbf-d6481875053e (old id 1409071)
date added to LUP
2009-06-01 16:37:02
date last changed
2017-08-27 05:22:36
@article{67735a31-0eb2-4dfb-9cbf-d6481875053e,
  abstract     = {Electrostatic interactions in proteins can be probed experimentally through determination of residue-specific acidity constants, We describe here triple-resonance NMR techniques for direct determination of lysine and arginine side-chain protonation states in proteins. The experiments are based on detection of nonexchangeable protons over the full range of pH and temperature and therefore are well suited for pK(a) determination of individual amino acid side chains. The experiments follow the side-chain N-15(zeta) (lysine) and N-15(epsilon) or C-13(zeta) (arginine) chemical shift, which changes due to sizable changes in the heteronuclear electron distribution upon (de)protonation. Since heteronuclear chemical shifts are overwhelmed by the charge state of the amino acid side chain itself, these methods supersede H-1-based NMR in terms of accuracy, sensitivity, and selectivity. Moreover, the N-15(zeta) and N-15(epsilon) nuclei may be used to probe changes in the local electrostatic environment. Applications to three proteins are described: apo calmodulin, calbindin D-9k, and FKBP12. For apo calmodulin, residue-specific pK(a) values of lysine side chains were determined to fall between 10.7 and 11.2 as a result of the high net negative charge on the protein surface. Ideal two-state titration behavior observed for all lysines indicates the absence of significant direct charge interactions between the basic residues. These results are compared with earlier studies based on chemical modification.},
  author       = {André, Ingemar and Linse, Sara and Mulder, Frans},
  issn         = {1520-5126},
  language     = {eng},
  number       = {51},
  pages        = {15805--15813},
  publisher    = {The American Chemical Society},
  series       = {Journal of the American Chemical Society},
  title        = {Residue-specific pK(a) determination of lysine and arginine side chains by indirect N-15 and C-13 NMR spectroscopy: Application to apo calmodulin},
  url          = {http://dx.doi.org/10.1021/ja0721824},
  volume       = {129},
  year         = {2007},
}