W0168

Rapid Screening for Heavy Atom Derivatives Using Mass Spectrometry. G. Bains, T. Niedenzu, D. Roeleke, P. Franke and W. Saenger, Institut fur Kristallographie, and Institut fur Biochemie, Freie Universitat Berlin, Berlin, Germany

Mass Spectrometry (MALDI) is employed to efficiently screen protein solutions and crystals for heavy atom (HA) derivatives. It is observed that the shift in molecular weight of a protein upon formation of a covalent complex with heavy atoms may be detected. Experiments performed with cytochrome C (12226), RNase A (13671), lysozyme (14304), chymotrypsin (25673), RepA (29806), aldolase (39400), catalase (58018), and BSA (66861) systematically reveal the rapid selective incorporation of the compounds o-chloromercuriphenol (CMP) and thimoseral (EMTS) in a manner dependent upon the accessibility and reduction state of the sulfhydryls. Noncovalent heavy atom binding could not be detected with this technique. Addition of some HA compounds resulted in the absence of protein interaction with the matrix used to prepare the samples for the MALDI experiment and consequent loss of signal presumably due to protein precipitation. This effect may allow an efficient additonal screen against strongly destabilizing heavy atom derivatives.

These results have been used to screen solutions of RepA and engineered cysteine mutants for complexes with mercury-containing compounds. Three promising candidates were identified, out of seven tested, as possible candidates for RepA HA derivatives. Diffraction data have been collected from crystals soaked with 2-chloro-mercuri- 4-nitrophenol (CMNP), cocrystallized with CMP, and a mutant soaked with methyl-mercurychloride. Data from the CMNP-soaked crystals result in a Patterson map which may be interpreted in terms of the hexameric structure of RepA.