Capturing Transient Protein Structures on Multiple Spatial and Temporal Scales

Project: Research project

Project Details

Description

The long term objective of the proposed research is to gain new insight into correlations between metallo active site structures and their functions through high resolution simultaneous structural “snapshots” of the active site during different biological relevant processes, such as electron transfer, ligand binding and protein folding. Although static protein structures have significantly enhanced our understanding of enzymatic reaction mechanisms, they are often insufficient because of the dynamic nature of the active sites while the enzymes are in action. The proposed research aims at building a facility for mapping reaction trajectories through the three-dimensional structures of protein active sites as a function of the fourth dimension, the reaction time. Both transient electronic and nuclear structures of metal centered active sites as a function of the reaction time in a series of metalloproteins will be simultaneously captured using X-ray transient absorption (XTA, or transient X-ray absorption spectroscopy) spectroscopy with time resolutions from 10-13 second (100 fs) to longer, while the protein conformation change along the reaction coordinates can be captured by transient small angle X-ray scattering (TSAXAS) and transient wide angle X-ray scattering (TWAXS). The method uses a laser pulse pump to trigger a biological reaction, which can be direct photodissociation of ligands/inhibitors or photoinduced redox reaction on metal active sites which may trigger the protein conformation change, and an X-ray pulse (from a synchrotron or an X-ray free electron source) to probe the active site structures and the conformations of proteins as a function of the delay time between the laser and X-ray pulses. The proposed research focuses on building a portable and multifunctional sample chamber/detection setup to enable simultaneous detection of the local structures of the metal binding sites and the over protein conformation during enzymatic reactions. The proposed setup will be built upon an existing facility with advanced detectors and laser systems with protein sample specific requirement that the facility currently does not have. Once the facility is built and commissioned, it will be a part of user shared instrumentation as well as a part of portable instrumentation for conducting the experiments in other light source, such as the linear coherence light source (LCLS) with femtosecond pulsed X-rays. Specific testing protein systems are axial ligand dissociation/association from a heme or metalloporphyrin in native or reconstructed hemeproteins. From the case study, we can make correlations of metal center spin state, oxidation state, ligation and geometry with protein conformations and folding pathways in a series of metal substituted cytochrome c. These structural results combined with those of reaction kinetics from optical transient spectroscopy will provide guidance for modulating protein functions via structural modifications around the active sites, which will lead to advances in drug design, enzymatic function enhancement, catalysis, as well as theoretical calculations.
StatusActive
Effective start/end date7/1/156/30/21

Funding

  • National Institute of General Medical Sciences (5R01GM115761-04)

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