Disorderdepleted Mutant Improved Crystallization Efficiency and Produced High Resolution Structure

Despite multiple screening attempts, the TM0160 full-length construct gave only three marginal hits from 2400 individual crystallization tests. In contrast, each of the four TM0160 mutants crystallized well. The TM0160 D3 mutant gave 78 hits

Fig. 12.3 (a) The H/D exchange results of full length TM0160 after 10 s exchange reaction at 0 °C. Each line indicates a peptide fragment analyzed. Red is the region exchanged and blue is the region not exchanged. Long stretches of contiguous sequence (four or more residues) that are rapidly exchanging are indicative of disorder. There are substantial disordered regions at C-terminal of this full-length construct. Four

Fig. 12.3 (a) The H/D exchange results of full length TM0160 after 10 s exchange reaction at 0 °C. Each line indicates a peptide fragment analyzed. Red is the region exchanged and blue is the region not exchanged. Long stretches of contiguous sequence (four or more residues) that are rapidly exchanging are indicative of disorder. There are substantial disordered regions at C-terminal of this full-length construct. Four truncated constructs, D1-D4 were designed by eliminating the C-terminal disordered regions. (b) The repeat H/D exchange results of full-length TM0160 and D3 mutant after 10 s exchange reaction at 0 °C. They showed virtually identical patterns in retained sequence, but are depleted of the disordered regions at the C-terminus of the parental construct.

from 1920 individual tests, including numerous crystals of sufficient size and quality for diffraction studies. The crystallographic structure of TM0160 D3 was subsequently determined at a resolution of 1.9 A [51]. This application of DXMS analysis was further validated by the successful crystallization and structure determination of other proteins in a similar manner [51, 52].

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