Contribution of proton linkage to the thermodynamic stability of the majorcold-shock protein of Escherichia coli CspA

The stability of protein is defined not only by the hydrogen bonding, hydro phobic effect, van der Waals interactions, and salt bridges. Additional, mu ch more subtle contributions to protein stability can arise from surface re sidues that change their properties upon unfolding. The recombinant major c old shock protein of Escherichia coli CspA an all-beta protein unfolds reve rsible in a two-state manner, and behaves in all other respects as typical globular protein. However, the enthalpy of CspA unfolding strongly depends on the pH and buffer composition. Detailed analysis of the unfolding enthal pies as a function of pH and buffers with different heats of ionization sho ws that CspA unfolding in the pH ranges 5.5-9.0 is linked to protonation of an amino group. This amino group appears to be the N-terminal alpha-amino group of the CspA molecule. It undergoes a 1.6 U shift in pK(a) values betw een native and unfolded slates. Although this shift in pK(a) is expected to contribute similar to 5 kJ/mol to CspA stabilization energy the experiment ally observed stabilization is only similar to 1 kJ/mol. This discrepancy i s related to a strong enthalpy-entropy compensation due, most likely, to th e differences in hydration of the protonated and deprotonated forms of the alpha-amino group.