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Salt bridge as a gatekeeper against partial unfolding
Author(s) -
Hinzman Mark W.,
Essex Morgan E.,
Park Chiwook
Publication year - 2016
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2908
Subject(s) - thermolysin , salt bridge , rnase p , chemistry , native state , proteolysis , context (archaeology) , protein folding , crystallography , salt (chemistry) , biophysics , mutant , biochemistry , enzyme , biology , trypsin , rna , paleontology , gene
Salt bridges are frequently observed in protein structures. Because the energetic contribution of salt bridges is strongly dependent on the environmental context, salt bridges are believed to contribute to the structural specificity rather than the stability. To test the role of salt bridges in enhancing structural specificity, we investigated the contribution of a salt bridge to the energetics of native‐state partial unfolding in a cysteine‐free version of Escherichia coli ribonuclease H (RNase H*). Thermolysin cleaves a protruding loop of RNase H * through transient partial unfolding under native conditions. Lys86 and Asp108 in RNase H * form a partially buried salt bridge that tethers the protruding loop. Investigation of the global stability of K86Q/D108N RNase H * showed that the salt bridge does not significantly contribute to the global stability. However, K86Q/D108N RNase H * is greatly more susceptible to proteolysis by thermolysin than wild‐type RNase H * is. The free energy for partial unfolding determined by native‐state proteolysis indicates that the salt bridge significantly increases the energy for partial unfolding by destabilizing the partially unfolded form. Double mutant cycles with single and double mutations of the salt bridge suggest that the partially unfolded form is destabilized due to a significant decrease in the interaction energy between Lys86 and Asp108 upon partial unfolding. This study demonstrates that, even in the case that a salt bridge does not contribute to the global stability, the salt bridge may function as a gatekeeper against partial unfolding that disturbs the optimal geometry of the salt bridge.