Journal of Biochemistry Advance Access originally published online on July 15, 2009
Journal of Biochemistry 2009 146(4):563-569; doi:10.1093/jb/mvp106
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Glutamate64 to Glycine Substitution in G1 β-bulge of Ubiquitin Impairs Function and Stabilizes Structure of the Protein
1Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390002;and 2Centre for Cellular and Molecular Biology, Habsiguda, Uppal Road, Hyderabad 500007, India
*To whom correspondence should be addressed. Tel: +91 265 2795594/+91 932 7201 349, Fax: +91 265 2795 569; E-mail: chivukula_r{at}yahoo.com
Received June 5, 2009; Accepted June 20, 2009
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Abstract |
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Ubiquitin is a globular protein with a highly conserved sequence. Sequence conservation and compact structure make it an ideal protein for structure–function studies. One of the atypical secondary structural features found in ubiquitin is a parallel G1 β-bulge. Glutamate at 64 is the first residue of this β-bulge and the third residue in a type II turn. However, glycine is seen in these positions in several proteins. To understand the effects of substitution of glutamate64 by glycine on the structure, stability and function of ubiquitin, mutant UbE64G has been constructed and characterized in Saccharomyces cerevisiae. The secondary and tertiary structures of UbE64G mutant protein are only marginally different from wild-type protein (UbWt) and fluorescent form of ubiquitin (UbF45W). The earlier studies have shown that the structure and stability of UbWt and UbF45W were similar. However, UbE64G has less surface hydrophobicity than UbWt. UbE64G is found to be more stable compared with UbF45W towards guanidinium chloride induced denaturation. In vivo, complementation shows substrate proteins with Pro as the N-terminal residue, which undergo ubiquitination, have extended half-lives with UbE64G. This altered preference for Pro as opposed to Met might be related to natural preference of glutamate at 64th position in ubiquitin.
Key Words: G1 β-bulge of ubiquitin, mutations in ubiquitin, ubiquitin, ubiquitin function, ubiquitin structure
Abbreviations: UbWt, wild-type ubiquitin; UbE64G, ubiquitin with Glu64 replaced by Gly; UbF45W, Ubiquitin with Phe45 replaced by Trp; UBI4, Polyubiquitin gene; Ub-X-β-gal, Ubiquitin-β-galactosidase fusion, where X is the first residue of β-galactosidase and it can be any of the 20 amino acids; DTT, Dithiothreitol; CD, Circular dichroism; ANS, 1-Anilino 8-naphthalene sulphonic acid; gdmCl, guanidinium chloride; TLTGK, Thr-Leu-Thr-Gly-Lys; NPDG, Asn-Pro-Asp-Gly