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. One of the limiting factors in analysis and interpretation of multidimensional NMR data for protein structural analysis is the assignment of resonances. Standard strategies for resonance assignment are laborious and expensive. Recent progress has been made in the use of sparse labeling strategies for multidimensional NMR, where only a limited number of residues are labeled and analyzed. In order to take advantage of the simpler spectra resulting from sparse labeling NMR, alternative technology for resonance assignment are being pursued. One technology studied was the use of amide deuterium exchange kinetics to assign resonances. Amide exchange kinetics can be followed readily by NMR;however, the assignment of the resonances cannot be determined solely by NMR. This subproject attempts to utilize rapid pepsin digestion followed by non-ergodic electron capture dissociation (ECD) mass spectrometry in order to measure rates of amide exchange for different amino acid residues. Once the rate of amide exchange for each of the labeled residues can be measured, resonances in sparse labeling NMR can be assigned based on the rate of amide exchange measured in the NMR spectrum.
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