Residue-Specific pKa Determination of Lysine and Arginine Side Chains by Indirect 15N and 13C NMR Spectroscopy: Application to apo Calmodulin

@article{citeulike:2313808, abstract = {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 pKa determination of individual amino acid side chains. The experiments follow the side-chain 15N (lysine) and 15N or 13C (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 1H-based NMR in terms of accuracy, sensitivity, and selectivity. Moreover, the 15N and 15N nuclei may be used to probe changes in the local electrostatic environment. Applications to three proteins are described: apo calmodulin, calbindin D9k, and FKBP12. For apo calmodulin, residue-specific pKa 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.}, address = {Contribution from the Department of Biophysical Chemistry, Lund University, P.O. Box 124, Sweden and Department of Biophysical Chemistry, University of Groningen, Nijenborgh 4 9747 AG Groningen, the Netherlands}, author = {Andre, I. and Linse, S. and Mulder, F. A. A. }, citeulike-article-id = {2313808}, doi = {10.1021/ja0721824}, journal = {J. Am. Chem. Soc.}, keywords = {nmr, pka}, month = {December}, number = {51}, pages = {15805--15813}, posted-at = {2008-01-31 12:52:27}, priority = {0}, title = {Residue-Specific pKa Determination of Lysine and Arginine Side Chains by Indirect 15N and 13C NMR Spectroscopy: Application to apo Calmodulin}, url = {http://dx.doi.org/10.1021/ja0721824}, volume = {129}, year = {2007} }

TY - JOUR ID - citeulike:2313808 L3 - citeulike-article-id:2313808 TI - Residue-Specific pKa Determination of Lysine and Arginine Side Chains by Indirect 15N and 13C NMR Spectroscopy: Application to apo Calmodulin JF - J. Am. Chem. Soc. VL - 129 IS - 51 SP - 15805 EP - 15813 AD - Contribution from the Department of Biophysical Chemistry, Lund University, P.O. Box 124, Sweden and Department of Biophysical Chemistry, University of Groningen, Nijenborgh 4 9747 AG Groningen, the Netherlands N2 - 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 pKa determination of individual amino acid side chains. The experiments follow the side-chain 15N (lysine) and 15N or 13C (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 1H-based NMR in terms of accuracy, sensitivity, and selectivity. Moreover, the 15N and 15N nuclei may be used to probe changes in the local electrostatic environment. Applications to three proteins are described: apo calmodulin, calbindin D9k, and FKBP12. For apo calmodulin, residue-specific pKa 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. KW - nmr KW - pka AU - Andre, I AU - Linse, S AU - Mulder, FAA PY - 2007/12/26/ UR - http://dx.doi.org/10.1021/ja0721824 ER -