title: Expression, purification, and crystallography of the conserved methionine-rich domain of human signal recognition particle 54 kDa protein.
abstract: Protein SRP54 is an essential component of eukaryotic signal recognition particle (SRP). The methionine-rich M-domain (SRP54M or 54M) interacts with the SRP RNA and is also involved in the binding to signal peptides of secretory proteins during their targeting to cellular membranes. To gain insight into the molecular details of SRP-mediated protein targeting, we studied the human 54M polypeptide. The recombinant human protein was expressed successfully in Escherichia coli and was purified to homogeneity. Our studies determined the sites that were susceptible to limited proteolysis, with the goal to design smaller functional mutant derivatives that lacked nonessential amino acid residues from both termini. Of the four polypeptides produced by V8 protease or chymotrypsin, 54MM-2 was the shortest (120 residues; Mr = 13,584.8), but still contained the conserved amino acids suggested to associate with the signal peptide or the SRP RNA. 54MM-2 was cloned, expressed, purified to homogeneity, and was shown to bind human SRP RNA in the presence of protein SRP19, indicating that it was functional. Highly reproducible conditions for the crystallization of 54MM-2 were established. Examination of the crystals by X-ray diffraction showed an orthorhombic unit cell of dimensions a = 29.127 A, b = 63.693 A, and c = 129.601 A, in space group P2(1)2(1)2(1), with reflections extending to at least 2.0 A.
authors.affiliation: Department of Molecular Biology, The University of Texas Health Science Center at Tyler, 75710, USA.
authors.forename: K
authors.initials: K
authors.lastname: Gowda
authors.forename: W M
authors.initials: WM
authors.lastname: Clemons
authors.forename: C
authors.initials: C
authors.lastname: Zwieb
authors.forename: S D
authors.initials: SD
authors.lastname: Black
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D006801:Humans; D008715:Methionine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D018271:Signal Recognition Particle / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D018271:Signal Recognition Particle; D008715:Methionine