Glutamate64 to glycine substitution in G1 beta-bulge of ubiquitin impairs function and stabilizes structure of the protein.
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 beta-bulge. Glutamate at 64 is the first residue of this beta-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.
Mishra
Pradeep
P
Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390002, India.
Volety
Srinivas
S
Rao
Ch Mohan
ChM
Prabha
C Ratna
CR
D019943:Amino Acid Substitution; D017124:Conserved Sequence; D018698:Glutamic Acid / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D009154:Mutation; D011487:Protein Conformation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013499:Surface Properties; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D025801:Ubiquitin; D018698:Glutamic Acid; D005998:Glycine