BUILDING UP THE FACILITY FOR MEMBRANE PROTEIN MANIPULATION AND SPIN LABELING

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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 major activities at ACERT is aimed at developing technologies for increasing the throughput of biomedical research. This important activity is poised to strengthen ACERT core research and collaborations, but also to benefit its service and dissemination activates by means of developing more sensitive and thereby considerably more productive pulse ESR spectrometers. Given that increasing sensitivity carries the highest priority, this task should not be considered in isolation from the other side of PDS spectroscopy in the part of sample preparation, since sample good quality is also essential for achieving high throughput. To address this goal, we are setting up a small but well-equipped local facility, suited for variety of biochemical manipulation with proteins. The emphasis will be given to the research on membrane proteins and everyday activities will be addressing efficient protein spin labeling; reconstitutions into liposomes, nanodisks or bicelles, dialysis, sample concentration, etc. We expect to improve the quality of our work by having been able to prepare and study fresh membrane protein samples, since they often are very delicate and do not tolerate excessive manipulations, for example freezing/thawing cycles. It will enable developing the most efficient protocols for spin-labeling, depending on the particular protein, thus helping our collaborators who have limited or lacking capacity to assay spin-labeling and optimize samples for PDS study. ACERT user support and training in sample preparation for ESR spectroscopy, in particular for pulsed dipolar ESR will be provided by ACERT staff. We are confident that such facility will contribute to the flexibility and merits of our biomedical research and will back the efficiency of the center. Additionally, such a facility will enable ACERT scientists to apply routinely the methods complementary to the ESR studies by providing on site access to standard techniques, such as fluorescent spectroscopy and circular dichroism. This will strengthen our existing research, and enable us to quickly implement the new ideas, which will be beneficial for collaborative projects, core research, and service activities.