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1 Joint Center for Structural Genomics, Genomics Institute of the Novartis Research Foundation, San Diego, California 92121, USA
2 Department of Medicine and The Biomedical Sciences Graduate Program, University of California at San Diego, La Jolla, California 92093, USA
3 Joint Center for Structural Genomics, The Scripps Research Institute, La Jolla, California 92037, USA
(RECEIVED June 17, 2004; FINAL REVISION August 25, 2004; ACCEPTED August 26, 2004)
The structure of two Thermotoga maritima proteins, a conserved hypothetical protein (TM0160) and a transcriptional regulator (TM1171), have now been determined at 1.9 Å and 2.3 Å resolution, respectively, as part of a large-scale structural genomics project. Our first efforts to crystallize full-length versions of these targets were unsuccessful. However, analysis of the recombinant purified proteins using the technique of enhanced amide hydrogen/deuterium exchange mass spectroscopy (DXMS) revealed substantial regions of rapid amide deuterium hydrogen exchange, consistent with flexible regions of the structures. Based on these exchange data, truncations were designed to selectively remove the disordered C-terminal regions, and the resulting daughter proteins showed greatly enhanced crystallizability. Comparative DXMS analysis of full-length protein versus truncated forms demonstrated complete and exact preservation of the exchange rate profiles in the retained sequence, indicative of conservation of the native folded structure. This study presents the first structures produced with the aid of the DXMS method for salvaging intractable crystallization targets. The structure of TM0160 represents a new fold and highlights the use of this approach where any prior structural knowledge is absent. The structure of TM1171 represents an example where the lack of a substrate/cofactor may impair crystallization. The details of both structures are presented and discussed.
Keywords: crystallization; mass spectrometry; protein structure; novel fold; sequence complexity
Abbreviations: DXMS, deuterium exchange mass spectroscopy
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04939904.
Reprint requests to: Scott A. Lesley, Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA; e-mail: slesley{at}gnf.org; fax: (858) 812-1746; or Virgil L. Woods Jr., Department of Medicine, University of California at San Diego, 9500 Gilman Drive, BSB 5078, La Jolla, CA 92093, USA; e-mail: vwoods{at}ucsd.edu; fax: (858) 534-2180.
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