pH plays a crucial role in regulating biological activity, and recent studies have demonstrated the importance of transiently populated minor states in understanding protein dynamics and function. We hypothesize that pH-dependent transient states will be prevalent in proteins with buried ionizable groups, where the contrast between hydrophobic and hydrophilic environments provides the driving force for pH-mediated conformational rearrangements. To this end, we present the development and application of an explicit solvent constant pH molecular dynamics (CPHMD) framework, where the protonation states of ionizable residues are dynamically changing in response to the external pH and conformational dynamics. By applying CPHMD simulations to over a dozen staphylococcal nuclease (SNase) mutants with buried ionizable residues, we discovered that all �buried� residues with highly shifted pKa values had an open solvated state, which contributed a small but non-zero population of 1-2% at physiological pH. Analysis of the SNase mutants suggests that structural fluctuations are responsible for creating pathways for water entry, and further in silico mutations suggests that small proteins have high conformational plasticity for multiple water entry sites, and the role of internal salt-bridges was examined. Lastly, to demonstrate the functional relevance of pH-dependent transient states, we investigated a CFP with pH-dependent spectral properties that correlates with the protonation of its internal chromophore. Using CPHMD, we discovered the existence of a solvated open state, and demonstrate the interplay of conformation dynamics, solvation environment and internal salt-bridges in modulating the microscopic pKa of CFP. In conclusion, we provide the first validation that buried ionizable residues can readily trigger pH-mediated conformational fluctuations that may be observed as transient state structures at physiological pH. Moreover the role of both closed and open states in recapitulating macroscopic experimental pKa suggests that minor states need to be elucidated to understand the pH-dependent properties of internal ionizable groups.

Uncovering physiologically relevant hidden conformations of proteins using constant pH molecular dynamics

248th National Meeting (2014)

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