CaltechAUTHORS
  A Caltech Library Service

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

Gowda, Krishne and Clemons, William M., Jr. and Zwieb, Christian and Black, Shaun D. (1999) Expression, purification, and crystallography of the conserved methionine-rich domain of human signal recognition particle 54 kda protein. Protein Science, 8 (5). pp. 1144-1151. ISSN 0961-8368. PMCID PMC2144335. doi:10.1110/ps.8.5.1144. https://resolver.caltech.edu/CaltechAUTHORS:20181030-105013706

Full text is not posted in this repository. Consult Related URLs below.

Use this Persistent URL to link to this item: https://resolver.caltech.edu/CaltechAUTHORS:20181030-105013706

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; 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 Å, b = 63.693 Å, and c = 129.601 Å, in space group P2_12_12_1, with reflections extending to at least 2.0 Å.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1110/ps.8.5.1144DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2144335/PubMed CentralArticle
ORCID:
AuthorORCID
Clemons, William M., Jr.0000-0002-0021-889X
Additional Information:© 1999 The Protein Society. (Received December 15, 1998; Accepted February 19, 1999) This work was supported by National Institutes of Health Grant GM-49034 to C.Z.
Funders:
Funding AgencyGrant Number
NIHGM49034
Subject Keywords:protein crystallography; protein secretion; RNA–protein interactions; signal peptide
Issue or Number:5
PubMed Central ID:PMC2144335
DOI:10.1110/ps.8.5.1144
Record Number:CaltechAUTHORS:20181030-105013706
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20181030-105013706
Official Citation:Gowda, K. , Clemons, W. M., Zwieb, C. and Black, S. D. (1999), Expression, purification, and crystallography of the conserved methionine‐rich domain of human signal recognition particle 54 kda protein. Protein Science, 8: 1144-1151. doi:10.1110/ps.8.5.1144
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:90501
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:30 Oct 2018 20:38
Last Modified:16 Nov 2021 03:33

Repository Staff Only: item control page