(607e) Understanding the Structural Basis of Protein Activation By Tuning the Pka of an Embedded Charge
Embedded charged residues play an important role in gating the functions of natural and engineered proteins. However, the pKaâs of buried charged residues are neither predictable nor deliberately tunable. The difficulties arise from a lack of general mechanistic understanding about how the surrounding environment in which protein resides affects the buried charges across relatively long distances. Here, proteorhodopsin (PR), a light-driven proton transport pump, was used as a model to study external factors that tune the protonation behavior of its embedded primary proton acceptor D97. This D97 residue of PR needs to be deprotonated to trigger the light activation of PR, and its pKa denotes the ratio of active PR under a specific pH. We found that the pKa of D97 shift significantly along with different electrostatic environments of PR-contained liposomes. Its value shifted from 7.6 to 5.6 for PR reconstituted in positively-charged liposomes (POPC/DOTAP, 80/20, mol/mol), compared to PR in negatively-charged liposomes (POPC/POPG, 80/20, mol/mol) in the absence of added salt. The addition of salt in buffer was found to diminish the pKa shift caused by the opposite net charge of liposomes. To further establish the mechanistic understanding of the pKa difference, DNP-enhanced magic angle spinning (MAS) NMR was applied to reveal the local structure around the embedded D97 when PR-contained liposomes were exposed to different electrostatic environments. The mechanistic understanding on the factors which control the pKa is critical for engineering transmembrane proteins whose functions are tuned by protonation/deprotonation at embedded sites.