Advanced Infrared Biology of Protein Structure & Dynamics

4. 项目总结/文摘

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.