MUTATIONAL APPROACHES TO INVESTIGATION OF PROTEIN STRUCTURE, FUNCTION & FOLDING

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My lab is interested in the structure and function of f-actin binding proteins. We arecurrently determining the structure of the "headpiece" domain of the f-actin bundfing protein villin by NMR and X-ray crystallography. We have expressed a series of mutants of headpiece in E. coli and require mass spectral analysis to insure the product that we purify is the correct sequence and has not been modified during purification. Recently, we have developed conditions where we can reproducibly get crystals of headpiece that diffiract well in the X-ray beam (to beyond 1.8 A resolution). In order to the obtain phase information necessary to solve the high resolution X-ray crystal structure of villin headpiece, we turned to the multiple anomolous dispersion (MAD) method. MAD phasing requires data to be collected at a synchrotron source with headpiece derivatives labeled with selenomethionine. Data collection time at the Brookhaven Synchrotron Source is limited and must be applied for. Therefore, it was essential to have our selenium-labeled crystal prepared and characterized in advance. Is was important for us to insure that our labeling was effective and, in addition, that the selenium was not oxidized or lost during thepurification, before our visit to the synchrotron. The mass spectral analysis performed by the center was critical in insuring our crystal contained the expected selenium and was therefore suitable for synchrotron data collection and MAD phasing. No other method is as effective as mass spectral analysis in providing proof of the presence and percent occupancy of the selenium in our protein crystals.