Salt bridge as a gatekeeper against partial unfolding.

Area of Science:

• Protein structure and dynamics
• Biophysical chemistry
• Enzymology

Background:

• Salt bridges are common in protein structures and are thought to influence structural specificity more than global stability.
• The energetic contribution of salt bridges is highly context-dependent.
• Investigating salt bridges in Escherichia coli ribonuclease H (RNase H*) can elucidate their role in protein structural specificity.

Purpose of the Study:

• To investigate the role of a specific salt bridge in enhancing structural specificity by examining its contribution to the energetics of native-state partial unfolding in RNase H*.
• To determine if this salt bridge affects global stability or partial unfolding energetics.

Main Methods:

• Utilized a cysteine-free mutant of RNase H* (RNase H*).
• Assessed global stability of wild-type and mutant (K86Q/D108N) RNase H*.
• Employed native-state proteolysis using thermolysin to probe partial unfolding energetics.
• Performed double mutant cycles to analyze interaction energy changes upon partial unfolding.

Main Results:

• The salt bridge between Lys86 and Asp108 in RNase H* does not significantly contribute to global protein stability.
• Mutants lacking this salt bridge (K86Q/D108N RNase H*) are significantly more susceptible to thermolysin-mediated proteolysis, indicating increased susceptibility to partial unfolding.
• The salt bridge significantly increases the energy barrier for partial unfolding by destabilizing the partially unfolded state.

Conclusions:

• Salt bridges can function as critical "gatekeepers" that prevent partial unfolding, thereby contributing to structural specificity even without significantly impacting global stability.
• The energetic contribution of this salt bridge to structural specificity arises from its role in destabilizing partially unfolded states.
• This finding highlights a nuanced role for salt bridges in protein structure and function, emphasizing their importance in maintaining specific conformations.