title: Q2N and S65D substitutions of ubiquitin unravel functional significance of the invariant residues Gln2 and Ser65.
abstract: Ubiquitin is a small, globular protein, structure of which  has been perfected and conserved through evolution to manage diverse functions in the macromolecular metabolism of eukaryotic cells. Several non-homologous proteins interact with ubiquitin through entirely different motifs. Though the roles of lysines in the multifaceted functions of ubiquitin are well documented, very little is known about the contribution of other residues. In the present study, the importance of two invariant residues, Gln2 and Ser65, have been examined by substituting them with Asn and Asp, respectively, generating single residue variants of ubiquitin UbQ2N and UbS65D. Gln2 and Ser65 form part of parallel G1 β-bulge adjacent to Lys63, a residue involved in DNA repair, cell-cycle regulated protein synthesis and imparting resistance to protein synthesis inhibitors. The secondary structure of variants is similar to that of UbF45W, a structural homologue of wild-type ubiquitin (UbWt). However, there are certain functional differences observed in terms of resistance to cycloheximide, while there are no major differences pertaining to growth under normal conditions, adherence to N-end rule and survival under heat stress. Further, expression of UbQ2N impedes protein degradation by ubiquitin fusion degradation (UFD) pathway. Such differential responses with respect to functions of ubiquitin produced by mutations may be due to interference in the interactions of ubiquitin with selected partner proteins, hint at biomedical implications.
authors.affiliation: Department of Biochemistry, The Maharaja Sayajirao University of Baroda, Vadodara 390002, India.
authors.forename: Pradeep
authors.initials: P
authors.lastname: Mishra
authors.forename: C Ratna
authors.initials: CR
authors.lastname: Prabha
authors.forename: Ch Mohan
authors.initials: ChM
authors.lastname: Rao
authors.forename: Srinivas
authors.initials: S
authors.lastname: Volety
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002460:Cell Line; D002942:Circular Dichroism; D003513:Cycloheximide / Q000494:pharmacology; D005973:Glutamine; D018869:Heat-Shock Response; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055550:Protein Stability; D017433:Protein Structure, Secondary; D059748:Proteolysis / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012694:Serine; D013050:Spectrometry, Fluorescence; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D005973:Glutamine; D012694:Serine; D003513:Cycloheximide