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Experimental p K a Value Determination of All Ionizable Groups of a Hyperstable Protein
Author(s) -
Raum Heiner N.,
Weininger Ulrich
Publication year - 2019
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201800628
Subject(s) - chemistry , dimer , crystallography , helix (gastropod) , organic chemistry , ecology , snail , biology
Electrostatic interactions significantly contribute to the stability and function of proteins. The stabilizing or destabilizing effect of local charge is reflected in the perturbation of the p K a value of an ionizable group from the intrinsic p K a value. Herein, the charge network of a hyperstable dimeric protein (ribbon–helix–helix (rhh) protein from plasmid pRN1 from Sulfolobus islandicus ) is studied through experimental determination of the p K a values of all ionizable groups. Transitions were monitored by multiple NMR signals per ionizable group between pH 0 and 12.5, prior to a global analysis, which accounted for the effects of neighboring residues. It is found that for several residues involved in salt bridges (four Asp and one Lys) the p K a values are shifted in favor of the charged state. Furthermore, the p K a values of residues C40 and Y47, both located in the hydrophobic dimer interface, are shifted beyond 13.7. The necessary energy for such a shift is about two‐thirds of the total stability of the protein, which confirms the importance of the hydrophobic core to the overall stability of the rhh protein.