NMR Research Today is a free monthly online journal that collates and summarizes the latest research about NMR, including details on nuclear magnetic resonance, structural determination, techniques. | ||||||||
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Computer-aided NMR assay for detecting natively folded structural domains.Hondoh T, Kato A, Yokoyama S, Kuroda Y Protein Research Group, RIKEN Genomic Sciences Center, Tsurumi, Yokohama 230-0045, Japan. 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. Published 7 April 2006 in Protein Sci, 15(4): 871-83.
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