4. Project Summary/Abstract
Proton-transfer in protein is a fundamental process underlying protein functions including
signaling/regulation, bio-energetics, and bio-catalysis. Understanding what triggers proton-transfer and
how proton-transfer drives subsequent functionally important structural transformations are of significant
importance to understand the structure-function relations of many proteins. Such research is significantly
hampered by the paucity of widely accessible structural techniques for probing dynamic changes in proton
positions during protein function. The long-term goal of this project is to overcome this barrier by developing
a time-resolved infrared vibrational spectroscopy-based structural technology.
Protein infrared encode rich structural information and are particularly sensitive to cleavage or
formation of X-H bonds during proton-transfer. Time-resolved infrared technology consists of three steps:
i) detecting time-resolved infrared signals that capture protein structural dynamics; ii) identifying which
amino acids contribute to specific infrared signals; iii) reliably translating infrared signals into proton
positions in proteins. In this project, we will focus on detecting dynamic changes in proton positions in
histidine side chains during protein function, using a bacterial blue-light photoreceptor (PYP) as a model
system. The three protonation states of histidine are His+ (two protons, on N¿ & N¿), His0D (a sole proton
on N¿), and His0E (a sole proton on N¿). Aim 1 is to detect pH-induced protonation of buried His108 and
solvent-exposed His3 in static PYP; Aim 2 is to detect time-resolved dynamic changes in protonation states
of His108 in the signaling state upon light activation; and Aim 3 is to detect chemically activated time-
resolved proton transfer in PYP. We will use high-precision FT-IR (Aim 1), time-resolved rapid-scan FT-IR
(Aim 2), and integrating a microfluidic rapid-mixing device with an FT-IR microscope for collecting 16,000
FT-IR spectra at once using a focal plane array detector (Aim 3). We will assign infrared signals using
specific isotope-editing combined with site-specific mutations. Histidine protonation states will be derived
using vibrational structural marker bands that we recently developed.
The PI has extensive experience in advanced infrared technologies and is the director of Oklahoma
Center for Advanced Infrared Biology. The key collaborator is an expert in PYP, molecular biology and
isotope-editing of PYP. An NSF MRI funded state-of-the-art FT-IR system will be used for this project. Six
undergraduate students will carry out the FT-IR experiments, prepare various PYP samples, perform FT-
IR data analysis, contribute to FT-IR data interpretation, and present results at professional conferences.
Women, native American students, and other minority students will be encouraged to join the project via
University’s three programs 1) Oklahoma Louis Stokes Alliance for Minority Participation, 2) the Center for
Sovereign Nations, and 3) the Freshman Research Scholar program.