Expression, purification, and crystallography of the conserved methionine-rich domain of human signal recognition particle 54 kDa protein

被引:7
作者
Gowda, K
Clemons, WM
Zwieb, C
Black, SD
机构
[1] Univ Texas, Hlth Sci Ctr, Dept Biol Mol, Tyler, TX 75710 USA
[2] Univ Utah, Sch Med, Dept Biochem, Salt Lake City, UT 84132 USA
关键词
protein crystallography; protein secretion; RNA-protein interactions; signal peptide;
D O I
10.1110/ps.8.5.1144
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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; M-r = 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 Angstrom, b = 63.693 Angstrom, and c = 129.601 Angstrom, in space group P2(1)2(1)2(1), with reflections extending to at least 2.0 Angstrom.
引用
收藏
页码:1144 / 1151
页数:8
相关论文
共 32 条
[1]   MOLECULAR EVOLUTION OF SRP CYCLE COMPONENTS - FUNCTIONAL IMPLICATIONS [J].
ALTHOFF, S ;
SELINGER, D ;
WISE, JA .
NUCLEIC ACIDS RESEARCH, 1994, 22 (11) :1933-1947
[2]   EVIDENCE FOR AN EXTENDED 7SL RNA STRUCTURE IN THE SIGNAL RECOGNITION PARTICLE [J].
ANDREWS, DW ;
WALTER, P ;
OTTENSMEYER, FP .
EMBO JOURNAL, 1987, 6 (11) :3471-3477
[3]   MODEL FOR SIGNAL SEQUENCE RECOGNITION FROM AMINO-ACID-SEQUENCE OF 54K SUBUNIT OF SIGNAL RECOGNITION PARTICLE [J].
BERNSTEIN, HD ;
PORITZ, MA ;
STRUB, K ;
HOBEN, PJ ;
BRENNER, S ;
WALTER, P .
NATURE, 1989, 340 (6233) :482-486
[4]   FUNCTIONAL SUBSTITUTION OF THE SIGNAL RECOGNITION PARTICLE 54-KDA SUBUNIT BY ITS ESCHERICHIA-COLI HOMOLOG [J].
BERNSTEIN, HD ;
ZOPF, D ;
FREYMANN, DM ;
WALTER, P .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1993, 90 (11) :5229-5233
[5]   Identification of an RNA-binding-loop in the N-terminal region of signal-recognition-particle protein SRP19 [J].
Black, SD ;
Gowda, K ;
Chittenden, K ;
Walker, KP ;
Zwieb, C .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1997, 245 (03) :564-572
[6]   A STRUCTURE FOR THE SIGNAL SEQUENCE BINDING-PROTEIN SRP54 - 3D RECONSTRUCTION FROM STEM IMAGES OF SINGLE MOLECULES [J].
CZARNOTA, GJ ;
ANDREWS, DW ;
FARROW, NA ;
OTTENSMEYER, FP .
JOURNAL OF STRUCTURAL BIOLOGY, 1994, 113 (01) :35-46
[7]   Structure of the conserved GTPase domain of the signal recognition particle [J].
Freymann, DM ;
Keenan, RJ ;
Stroud, RM ;
Walter, P .
NATURE, 1997, 385 (6614) :361-364
[8]   Protein SRP54 of human signal recognition particle: cloning, expression, and comparative analysis of functional sites [J].
Gowda, K ;
Black, SD ;
Moller, I ;
Sakakibara, Y ;
Liu, MC ;
Zwieb, C .
GENE, 1998, 207 (02) :197-207
[9]   Binding site of the M-domain of human protein SRP54 determined by systematic site-directed mutagenesis of signal recognition particle RNA [J].
Gowda, K ;
Chittenden, K ;
Zwieb, C .
NUCLEIC ACIDS RESEARCH, 1997, 25 (02) :388-394
[10]   Determinants of a protein-induced RNA switch in the large domain of signal recognition particle identified by systematic site directed mutagenesis [J].
Gowda, K ;
Zwieb, C .
NUCLEIC ACIDS RESEARCH, 1997, 25 (14) :2835-2840