AMINO ACID LABELING FOR LARGER PROTEINS METHYL GROUP LABELING

SIR提供3,4,6二氯- 5,15纳米嘧啶；20毫克

The SIR provided 3,4,6 dichloro-5 15 Namino-pyrimidine; 20mg 6,2'deoxy[9J5N]adenosine; 20mg I a tellurocystine; 20mg 93-94% "Selenium; 87mg Selenomethionine containing proteins analyzed by multiwavelength anomalous diffraction (MAD) provide a facile means of addressing the phase problem, whose solution is necessary to determine protein structures by X-ray crystallography [Hendrickson, W.A. (1991) Science, 254, 51-58]. Since this method requires synchrotron radiation, we sought to incorporate a true heavy atom into protein, allowing the solution of the phase problem by more traditional methods of data collection. Media containing Te-Met alone or Te-Met with low levels of Met failed to sustain growth of a methionine auxotroph of Escherichia coli carrying the dihydrofolate reductase expression vector. Growth of the organism to stationary phase and incorporation of Te-Met was observed when the culture was initiated in media containing minimal Met levels and Te-Met was added after induction with IPTG. The purified enzyme exhibited properties similar to those of the native enzyme. Atomic absorption spectroscopy and amino acid analysis indicated that 40% of the methionines were replaced with Te-Met. Sequence analysis did not indicate significant levels of replacement in the first three sites (1, 16, and 20), suggesting that Te-Met was present only in the last two sites (42 and 92).Crystals of this enzyme were grown in the presence of methotrexate and were isomorphous with crystals of wild-type DHFR. Difference Fourier maps and restrained least-squares refinement showed no substitution at the first three methionines, while incorporation was seen at positions 42 and 92.