This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Several new pulse sequences for distance measurements were developed over the last few years at ACERT and by other groups. The demands of structural biology require their further development in order to realize the potential of ESR in this field. This is essential in the study of RNA, which can tax the method due to its large size, and for large water-soluble proteins and their complexes. Since long relaxation times are necessary to determine distances exceeding 40?, the reporter spin labels are placed on the outer molecular surfaces where they are fully exposed to the aqueous solvent medium. In this case the use of D2O increases T2's but there are still relaxation processes caused by residual protons in the system, in particular those of the protein, and by a number of spectral diffusion mechanisms, which result in relaxation with a quadratic to cubic law in time. Suppression of this type of diffusion requires multiple refocusing. To increase the upper range of distances in the DQC method, we modified the way the signal in the 6-pulse sequence was acquired. The method that we call double-quantum filtered refocused primary echo (DQF-RPE) is based on standard 6-pulse DQC pulse sequence, but the signal is acquired differently. This results in better suppression of quadratic relaxation and permits DQC signal acquisition on a time-scale of 10 ?s. This made it possible to measure a distance of 70? in RNA. Longer distances or protonated solvents require a larger number of refocusing pulses, which is a formidable task for pulses of finite width. Other developments include implementation of pulse sequences with small phase steps for multiple-quantum coherence separation and for increasing the effectiveness of phase cycling and better suppression of unwanted coherence pathways. Multiple-pulse sequence capability is being developed in the context of new methods of 2D-ELDOR and ESR-microscopy. The instrumentation development is described in related projects. A short communication describing application of DQF-RPE to 26 b.p. RNA is in press in J. Am. Chem. Soc.
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