Understanding Functional Tuning, Robustness, and Evolvability in Proteins By High-Throughput Biophysics

Abstract

Proteins consist of only 20 different amino acids with very modest chemical reactivity, but perform a breathtaking range of functions. A central question in biophysics is to understand how proteins achieve such functional versatility. A key recent advance in understanding protein structure-function relationships is the robustness of proteins against point mutations, which implies that only a small subset of residues determines functional properties. We tested this prediction using photoactive yellow protein (PYP), a 125-residue prototype of the PAS domain superfamily of signaling proteins. PAS domains are defined by a small number of conserved residues of unknown function. Our high-throughput biophysical measurements on a complete ala scan of purified PYP mutants characterizing active site properties, functional kinetics, stability, and production level reveal that 124 mutants retain the characteristic photocycle of PYP, but that the majority of substitutions significantly alter functional properties (Philip et al., PNAS 2010, early edition 10.1073/1006660107). Only 35% of substitutions that strongly affect function are located at the active site. Unexpectedly, most PAS-conserved residues are required for maintaining protein production. The photocycle kinetics are significantly altered by substitutions at 58 positions and span a 3,000-fold range, allowing us to identify conserved interactions governing allosteric switching in PYP. The data also shed light on two classic examples of functional tuning of active site groups, shifts in pKa value and shifts in absorbance maximum for spectral tuning, and result in a novel model for spectral tuning based on changes in the shape of the energy surfaces involved (Philip et al., 2010, PNAS 107: 5821-5826). The results show that PYP combines robustness with a high degree of evolvability, imply production level as an important factor in protein evolution, and provide new insights into the functional tuning of active site residues.