Structure-function relationships in the photoactive yellow protein family of photoreceptors
|Institution:||Oklahoma State University|
|Full text PDF:||http://pqdtopen.proquest.com/#viewpdf?dispub=10138974|
The amino acid sequence of a protein determines its three-dimensional structure, which in turn determines its functional properties. An intensively studied but still partially unresolved question is how the structure of a protein relates to its functional properties. Here we use photoactive yellow protein (PYP) as a model system to examine questions on protein structure-function relationships. PYP is a bacterial blue light photoreceptor, a prototype of the diverse PAS domain superfamily, and a model system for functional protein dynamics. The work in this thesis was directed at three aims: (1) developing tools to identify the structural change that triggers intramolecular proton transfer during the PYP photocycle; (2) the functional role of PAS-conserved residue Ile39 in PYP; and (3) determining to what extent the extensively studied structure-function relations in the PYP from Halorhodospira halophila apply to the PYP from Rhodospirillum centenum. (1) The molecular events that cause directional proton transfer in proteins are largely unknown. We develop tools to allow the testing of the specific hypothesis that the disruption of the Tyr42-pCA hydrogen bond during the PYP photocycle causes proton transfer. We developed an effective approach for obtaining Tyr-D4-labeled PYP that can be used in infrared studies to identify Tyr side chain signals. (2) The PAS domain superfamily is defined by weak but characteristic amino acid sequence conservation, but the functional role of PAS-conserved residues remains poorly understood. We examined PAS-conserved residue Ile39 through biophysical characterization of the I39A PYP mutant. This work revealed that Ile39 is at the core of a set of hydrophobic interactions conserved in PAS domains, is not an essential part in the transmission mechanism of allosteric structural changes during PYP signaling and affects both signaling kinetics and folding cooperativity. (3) We found that structure-function rules for Hhal PYP qualitatively transfer to Rcen PYP, including the role of Glu46 as the electrostatic epicenter for driving conformational changes. The resulting set of Rcen PYP mutants with altered photocycle rate and reduced conformational changes provides a powerful tool for future studies on the photocycle events that are needed for in vivo signaling by PYP.