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Research Article

Computer-aided NMR assay for detecting natively folded structural domains

¹ Protein Research Group, RIKEN Genomic Sciences Center, Tsurumi, Yokohama 230-0045, Japan
² Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Koganei-shi, Tokyo 184-8588, Japan
³ Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan

(RECEIVED September 29, 2005; FINAL REVISION December 9, 2005; ACCEPTED December 13, 2005)

Structural genomics projects require strategies for rapidly recognizing protein sequences appropriate for routine structure determination. For large proteins, this strategy includes the dissection of proteins into structural domains that form stable native structures. However, protein dissection essentially remains an empirical and often a tedious process. Here, we describe a simple strategy for rapidly identifying structural domains and assessing their structures. This approach combines the computational prediction of sequence regions corresponding to putative domains with an experimental assessment of their structures and stabilities by NMR and biochemical methods. We tested this approach with nine putative domains predicted from a set of 108 Thermus thermophilus HB8 sequences using PASS, a domain prediction program we previously reported. To facilitate the experimental assessment of the domain structures, we developed a generic 6-hour His-tag-based purification protocol, which enables the sample quality evaluation of a putative structural domain in a single day. As a result, we observed that half of the predicted structural domains were indeed natively folded, as judged by their HSQC spectra. Furthermore, two of the natively folded domains were novel, without related sequences classified in the Pfam and SMART databases, which is a significant result with regard to the ability of structural genomics projects to uniformly cover the protein fold space.

Keywords: structural genomics; protein domain; domain prediction; NMR; native structure

This paper is dedicated to the memory of Dr. Zheng-yu Peng (University of Connecticut Health Center).

Reprint requests to: Y. Kuroda, Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi, Tokyo 184-8588, Japan; e-mail ykuroda{at}cc.tuat.ac.jp; fax: +81-42-388-7794; or S. Yokoyama, Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; e-mail yokoyama{at}biochem.s.u-tokyo.ac.jp; fax: +81-3-5841-8057.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051880406

Supplementary material: see www.proteinscience.org

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