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

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• 作者：Ingemar André ; Sara Linse ; Frans A. A. Mulder
• 刊名：Journal of the American Chemical Society
• 出版年：2007
• 出版时间：December 26, 2007
• 年：2007
• 卷：129
• 期：51
• 页码：15805 - 15813
• 全文大小：214K
• 年卷期：v.129,no.51(December 26, 2007)
• ISSN：1520-5126

文摘

Electrostatic interactions in proteins can be probed experimentally through determination ofresidue-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 ondetection of nonexchangeable protons over the full range of pH and temperature and therefore are wellsuited for pK_a determination of individual amino acid side chains. The experiments follow the side-chain¹⁵N (lysine) and ¹⁵N or ¹³C (arginine) chemical shift, which changes due to sizable changes in theheteronuclear 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 NMRin terms of accuracy, sensitivity, and selectivity. Moreover, the ¹⁵N and ¹⁵N nuclei may be used to probechanges 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 weredetermined 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 chargeinteractions between the basic residues. These results are compared with earlier studies based on chemicalmodification.