Functional Tuning of Photoactive Yellow Protein by Active Site Residue 46

Abstract

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.

Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

“…The protein environment, however, can suppress the unwanted major channels observed in solution (64) and enhance minor reactions, such as all-trans retinal isomerizing into 13-cis (and nothing else) with 64% yield (vs. 1%) in bacteriorhodopsin, by adjusting the potential energy landscape of the embedded chromophores. Protein matrices have major effects on redistributing the quantum yields, which indicates that it is possible to attain desired properties by adjusting the protein environment without changing the chromophore.…”

“…Furthermore, the overall structure, folding efficiency, and chromophore maturation efficiency have to be preserved on mutations, complicating the protein design problem. Second, structure-energetics knowledge is necessary and can be obtained either from computational predictions using X-ray structures (44,49) or by characterizing exhaustive mutations on important residues of model systems for specific photochemistry (64,72).…”

Robustness and evolvability in the functional anatomy of a PER-ARNT-SIM (PAS) domain

“…We have developed 96-well-based methods for the heterologous expression, purification, and spectroscopic characterization of PYP mutants (32). Here, we use this microscale high-throughput approach to study a complete Ala scan library of PYP mutants.…”

“…The growth of the 125 E. coli strains overproducing the PYP mutants, their purification, and most of their spectroscopic analysis were performed in 96-well format. The proteins were purified using Ni-NTA agarose as described in (32), with the modifications described in the SI Text. Selected purified samples were examined by MALDI-TOF mass spectrometry.…”

Hydrogen bond dynamics in the active site of photoactive yellow protein

“…1) (15). Disruption of this network via site-directed mutagenesis alters the photoabsorption properties of the chromophore and perturbs the kinetics of several steps within the photocycle (16)(17)(18), implying a role for these residues in tuning the ground state photophysics of PYP and the energetics of its conformational transitions during the photocycle. While these mutagenesis studies provide strong evidence for the functional importance of hydrogen bonds, they do not reveal the physical properties of the parent, intact hydrogen bonds that underlie their functional roles during the PYP photocycle.…”