Functional tuning of photoactive yellow protein by active site residue 46.
Protein-ligand interactions alter the properties of active site groups to achieve specific biological functions. The active site of photoactive yellow protein (PYP) provides a model system for studying such functional tuning. PYP is a small bacterial photoreceptor with photochemistry based on its p-coumaric acid (pCA) chromophore. The absorbance maximum and pK(a) of the pCA in the active site of native PYP are shifted from 400 nm and 8.8 in water to 446 nm and 2.8 in the native protein milieu, respectively, by protein-ligand interactions. We report high-throughput microscale methods for the purification and spectroscopic investigation of PYP and use these to examine the role of active site residue Glu46 in PYP, which is hydrogen bonded to the pCA anion. The functional and structural attributes of the 19 substitution mutants of PYP at critical active site position 46 vary widely, with absorbance maxima from 441 to 478 nm, pCA fluorescence quantum yields from 0.19 to 1.4%, pCA pK(a) values from 3.0 to 9.0, and protein folding stabilities from 6.5 to 12.9 kcal/mol. The kinetics of the last photocycle transition vary by more than 4 orders of magnitude and are often strongly biphasic. Only E46Q PYP exhibits a greatly accelerated photocycling rate. All substitutions yield a folded, photoactive PYP, illustrating the robustness of protein structure and function. Correlations between side chain and mutant properties establish the importance of residue 46 in tuning the function of PYP and the significance of the strength of its hydrogen bond to the pCA. Native PYP exhibits the lowest values for pCA fluorescence quantum yield and pK(a), indicating their functional relevance. These results demonstrate the value of quantitative high-throughput biophysical studies of proteins.
Philip
Andrew F
AF
Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, USA.
Eisenman
Kaury T
KT
Papadantonakis
George A
GA
Hoff
Wouter D
WD
D001426:Bacterial Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D003373:Coumaric Acids / Q000737:chemistry*; D006860:Hydrogen Bonding; D008024:Ligands; D009154:Mutation; D010777:Photochemistry; D020283:Photoreceptors, Microbial / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D013057:Spectrum Analysis
D001426:Bacterial Proteins; D003373:Coumaric Acids; D008024:Ligands; D020283:Photoreceptors, Microbial; C050162:photoactive yellow protein, Bacteria