For the first time, an arbitrary pulse shaping module will be integrated into an Electron Paramagnetic Resonance (EPR) spectrometer that will lead to wide ranging and fundamentally important advances, including the realization of Fourier Transform EPR with high spectral resolution and time-resolved EPR spectroscopy. A Dynamic Nuclear Polarization module built into the pulsed EPR instrument will capitalize on this new pulse shaping capability to enable the next generation of quantitative and time resolved measurements of diffusive dynamics of hydration water that is lubricating the exterior and interior of proteins and membrane systems, with site-specific resolution. These are all entirely new experimental capabilities. For the broadest possible dissemination of these novel technologies to the biomedical user community, a commercial spectrometer will provide the core of the system. Despite the variety of software-customizable configurations offered by state of the art EPR instruments, they offer no ability to shape individual pulses, as is done routinely in nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI). Although shaped pulses have been implemented in NMR for over 25 years and are routinely implemented in all clinical MRI scanners, the application of arbitrary pulse shaping has, in fact, never been reported before for any EPR instrument at GHz frequencies and higher. The proposed development will capitalize on a state of the art technique that employs integrated circuit components to generate digital waveforms at ~10 GHz, whose amplitude and phase can be specified with 0.25-1 ns resolution. The expected merits of this technology are broad and significant, and include advancing the study of structure, dynamics and function of proteins, nucleic acids, assemblies or lipid membrane systems. The sensitivity for nanometer scale distance measurements of membrane proteins, including G protein-coupled receptors (GPCR), will be significantly enhanced. Critical conformation changes upon ligand-activation of GPCRs can be probed with improved temporal and spectral resolution. Conformational dynamics on the ms timescale-critical for enzyme function-can be quantified and compared between the active and inactive state, while probing the conformational substates. Equipped with these capabilities, drug effects can be effectively mapped out by directly probing the enzyme activity, through the modulation of protein conformation and hydration dynamics. The new instruments will also enable the study of early aggregation events of proteins implicated in neurodegenerative diseases, e.g. tau and amyloid-b in Alzheimer's or a-synnuclein in Parkinson's disease. One critical early event is the (mis)folding of the protein monomer that is thought to template aggregation. Soluble protein oligomers formed in the early stages of aggregation has been found to bear critical neurotoxic effects, likely more than the fibrous aggregates. The new tools will be capable of characterizing the dynamic structure of these oligomers, and quantifying the kinetics of protein folding, oligomer formation and fiber maturation with site-specificity and high time resolution. Thus, the effects of potential drugs, inhibitors or mutations can be probed on the formation or disappearance of these critical species that usually escape the detection of existing tools, and their effect on the rate limiting step of aggregation. These advances address several critical barriers in biomedical research.

Public Health Relevance

A set of new instrumental capabilities will be developed that will significantly advance the sensitivity, selectivity and time resolution of currently prominent structural biology tools based on electron paramagnetic resonance spectroscopy, and address several critical barriers to progress in biomedical research This will significantly advance the study of membrane protein structures that present the majority of current drug targets, facilitate drug screening for activating or deactivating important enzymes or help unravel the diseases effects of protein aggregation mechanisms implicated in neurodegenerative diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRR1-BT-7 (01))
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Friedman, Fred K
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University of California Santa Barbara
Schools of Arts and Sciences
Santa Barbara
United States
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Rudraraju, A V; Amoyaw, P N A; Hubin, T J et al. (2014) Determination of log P values of new cyclen based antimalarial drug leads using RP-HPLC. Pharmazie 69:655-62