Petr P. Borbat

Multiple-quantum ESR and distance measurements

Abstract It is shown that allowed double-quantum coherences (DQC) can now be routinely generated in disordered and oriented solids containing nitroxide biradicals and random distributions of stable radicals. The Pake doublets obtained from DQC pathways can be effectively used to determine long (∼30 A) distances in the former case, and concentrations in the latter. The DQC signals are strong and often comparable to standard single-quantum signals. In the limit of non-selective pulses their interpretation becomes independent of complicating features which affect other ESR distance methods.

Effect of freezing conditions on distances and their distributions derived from Double Electron Electron Resonance (DEER): A study of doubly-spin-labeled T4 lysozyme

Pulsed dipolar ESR spectroscopy, DEER and DQC, require frozen samples. An important issue in the biological application of this technique is how the freezing rate and concentration of cryoprotectant could possibly affect the conformation of biomacromolecule and/or spin-label. We studied in detail the effect of these experimental variables on the distance distributions obtained by DEER from a series of doubly spin-labeled T4 lysozyme mutants. We found that the rate of sample freezing affects mainly the ensemble of spin-label rotamers, but the distance maxima remain essentially unchanged. This suggests that proteins frozen in a regular manner in liquid nitrogen faithfully maintain the distance-dependent structural properties in solution. We compared the results from rapidly freeze-quenched (≤100 μs) samples to those from commonly shock-frozen (slow freeze, 1 s or longer) samples. For all the mutants studied we obtained inter-spin distance distributions, which were broader for rapidly frozen samples than for slowly frozen ones. We infer that rapid freezing trapped a larger ensemble of spin label rotamers; whereas, on the time-scale of slower freezing the protein and spin-label achieve a population showing fewer low-energy conformers. We used glycerol as a cryoprotectant in concentrations of 10% and 30% by weight. With 10% glycerol and slow freezing, we observed an increased slope of background signals, which in DEER is related to increased local spin concentration, in this case due to insufficient solvent vitrification, and therefore protein aggregation. This effect was considerably suppressed in slowly frozen samples containing 30% glycerol and rapidly frozen samples containing 10% glycerol. The assignment of bimodal distributions to tether rotamers as opposed to protein conformations is aided by comparing results using MTSL and 4-Bromo MTSL spin-labels. The latter usually produce narrower distance distributions.

Double-Quantum ESR and Distance Measurements

“Allowed” double quantum ccherences (DQC) can now be routinely generated in disordered and oriented solids containing nitroxide biradicals and random distributions of stable radicals. The Pake doublets obtained from DQC pathways can be effectively used to determine a broad range of distances in the former case whereas decay constants yield concentrations in the latter. The DQC signals are strong and often comparable to standard single quantum signals. They are free of any large undesirable signals, so the DQ experiment is easy to perform. Their strong intensity permits the study of low concentrations of spins in samples typical of those ordinarily met in the case of doubly-labeled macromolecules such as proteins and polypeptides. The upper range of distances for systems labeled with nitroxides is estimated to be ca. 80 A. In the limit of non-selective pulses the interpretation of DQC signals becomes independent of complicating geometric features which affect other ESR distance methods. The method is compared to other existing pulse distance measurement techniques and future improvements are also discussed.

Two‐Pulse Electron Spin Echo Study of Deuterium‐Helium Solids

We measured electron spin echoes from deuterium atoms within deuterium‐helium solids with an X‐band pulse ESR spectrometer. Our two‐pulse electron spin echo envelope modulation (ESEEM) measurements are well described by a model that places 50% – 60% of the deuterium atoms at the interface between the molecular deuterium nanoclusters and the superfluid liquid helium. The remainder of the atoms lie in substitutional sites within the nanoclusters. We also report the spin‐lattice relaxation times T1 for these atoms.

Pulse Electron Spin Resonance Method For Investigation Of Atoms In Impurity‐Helium Solids

We have constructed an X‐band pulse electron spin resonance spectrometer for the investigation of hydrogen, deuterium, and nitrogen impurity‐helium solids at low temperatures. The spectrometer is of a conventional homodyne detection design incorporating a sampling oscilloscope but uses a modified CW ESR resonant cavity.