1.1. Develop a modern L-band microwave bridge with synthesizer-based microwave sources, high speed A/D, and computer-based data storage and analysis tools.
1.2. Extend NARS to X-band, and both field- and frequency-swept NARS to Q-band.
1.3. Quantify NARS saturability for spin labels at L-, X-, and Q-band as a function of rotational correlation time.
The public health significance of Project 1 lies in development of innovative apparatus and methods for the study of dynamic fluctuations in protein structure as well as macromolecular assemblies that are relevant to biological function.
2.1. Develop an LGR optimized for 15 µl sample at L-band taking into account the goal of high resonator efficiency Ʌ and low resonator quality factor Q for improved detection of FID signals. Extend the technology to X- and Q-band. Develop a bimodal resonator for Specific Aim 2.3.
2.2. Test the hypothesis that 40 Å distances can be measured at L-band using FID methodology for detection of the Pake doublet.
2.2.1. Test the hypothesis that 40 Å spin-label separation can be measured at L-band by improving existing methods.
2.2.2. Test the hypothesis that distance determination at L-band can be enhanced by pulse FID methods.
2.3. Evaluate the proposed L-band FID methodology in measurement of the bimolecular collision rate of the protein-bound marker probe at various positions on the T4L model system with freely diffusing charged and neutral 15N spin-label probes to determine the electrostatic surface potential of the protein.
The public health significance of Project 2 lies in the hypothesis that L-band NARS of spin-labeled biomolecules will lead to dynamic structural information of functional relevance that was not previously available.
3.1. Develop pure absorption and pure dispersion EPR at W-band under non-adiabatic (NARS) conditions.
3.1.1. Extend conventional NARS with triangular magnetic field sweep combined with steps of the main magnetic field across the spectrum to W-band.
3.1.2. Compare microwave frequency ramps under AWG control in combination with steps of the magnetic field across the spectrum with conventional magnetic field sweep NARS.
3.2. Develop the method of segmental adiabatic sweeps of the microwave frequency.
3.2.1. Combine very fast (3 MHz/ns) frequency sweeps (effective π/2) with FID readout.
3.2.2. Combine very fast sweeps of the microwave frequency (effective π/2) with spin echo readout.
3.3. Carry out passage experiments at the interface between adiabatic and non-adiabatic conditions to measure very slow rotational diffusion.
3.3.1. Develop saturation transfer spectroscopy by variation of the microwave frequency sweep rate.
3.3.2. Optimize the signal-to-noise ratio using frequency sweep incipient passage effects.
One aspect of the public health significance is that W-band yields good EPR sensitivity from very small samples. The first aim further enhances sensitivity from these small samples. In addition, W-band is inherently broadband, which allows new experiments based on modern AWGs to achieve microwave frequency agility.
The goal of the second and third aims is development of technology for improved measurement of macromolecular dynamics.