Pulsed ESR Distance Measurements

Site-directed nitroxide spin labeling opens doors for use of ESR in structure determination and the study of functions of a broad class of biomolecules such as proteins and RNA. One method uses higher order coherences which can be created and manipulated in systems

Double-quantum coherence (DQC) between two electron spins coupled by their dipole-dipole interaction is of particular interest to
us, since this coherence provides the tool for separating weak dipolar couplings from stronger interactions for accurate measurements of
distances over a broad range, 10Å…..40-80Å, depending on the system and conditions.

Let’s consider two interacting spins a and b. Possible coherences are:

Single-quantum in-phase, I_±

Single-quantum antiphase, A_±

Double-quantum, DQ_±

All these coherences can be manipulated by pulses and be refocused. Refocusing of DQ_± is particularly useful, because it singles out the part of the signal that evolves solely due to spin coupling.

Once we have a bilabel rigid molecular structure, the workflow for the 6-pulse DQC sequence will look as follows:

Our overall aim in this area is to fundamentally advance our knowledge of structure and functions of biomolecules and to increase the experimental throughput of protein structure determinations using a small number of longer distance constraints, provided by modern techniques of site-directed spin labeling and pulsed ESR distance measurement techniques, and to further refine these techniques.

Additional:

• The development and implementation of techniques of pulsed ESR distance measurements suitable for a wide range of
  experimental challenges including the determination of distributions in
  the distances. They include methods based upon wide spectral excitation,
  including Double Quantum Coherence (DQC) and Advanced Single Quantum
  Coherence (ASQC); as well as methods based on selective excitation:
  double-electron-electron resonance (DEER);
• The extension of these methods to the higher frequencies of Ka-band (35 GHz) and W-band (95 GHz) to enhance sensitivity to study samples with low concentrations of spin-labeled biomolecules (ca. 1µM);
• The development of methods that lead to enhanced microwave B₁ fields at X, Ku, Ka, and W-bands to improve the accuracy of the DQC and ASQC methods and to increase their sensitivity;
• The utilization of techniques of macroscopic alignment of membrane samples to provide a powerful means of structural
  studies on membrane peptides and proteins;
• The development of methods to study Multiple Quantum Coherences (MQC) by modern pulsed ESR methods and to utilize
  them in the study of aggregates of proteins.

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