Purification, crystallization, and preliminary X-ray diffraction analysis of an M.HhaI-AdoMet complex

Kumar, S., Cheng, X., Pflugrath, J. W., Roberts, R. J. (1992) Purification, crystallization, and preliminary X-ray diffraction analysis of an M.HhaI-AdoMet complex. Biochemistry, 31 (36). pp. 8648-53. ISSN 0006-2960 (Print)0006-2960 (Linking)

URL: http://www.ncbi.nlm.nih.gov/pubmed/1390649
DOI: 10.1021/bi00151a035

Abstract

The type-II DNA-(cytosine-5)-methyltransferase M.HhaI was overexpressed in Escherichia coli and purified to apparent homogeneity. The purification scheme exploits a unique high salt back-extraction step to solubilize M.HhaI selectively, followed by FPLC chromatography. The yield of purified protein was 0.75-1.0 mg per gram of bacterial paste. M.HhaI could be isolated in two forms: bound with its cofactor S-adenosylmethionine (AdoMet) or devoid of the cofactor. The AdoMet-bound form was capable of methylating DNA in vitro in the absence of exogenous AdoMet. From kinetic studies of the purified enzyme, values for KmAdoMet (60 nM), KiAdoHye (0.4 nM), and Kcat (0.22 s-1) were determined. The purified enzyme bound with its cofactor was crystallized by the hanging drop vapor diffusion technique. Crystals were of monoclinic space group P2(1) and had unit-cell dimensions of a = 55.3 A, b = 72.7 A, c = 91.0 A, and beta = 102.5 degrees, with two molecules of M.HhaI in each of the two asymmetric units. The crystals diffract beyond 2.5 A and are suitable for structure determination.

Item Type: Paper
Uncontrolled Keywords: Crystallization DNA-Cytosine Methylases/biosynthesis/*chemistry/isolation & purification Escherichia coli/genetics Macromolecular Substances Protein Conformation Recombinant Proteins/biosynthesis/chemistry/isolation & purification S-Adenosylmethionine/*chemistry X-Ray Diffraction
Subjects: Investigative techniques and equipment > X-Ray Diffraction
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > methyltransferase
Investigative techniques and equipment > x ray crystallography
CSHL Authors:
Communities: CSHL labs > Roberts lab
Depositing User: Matt Covey
Date Deposited: 01 Oct 2015 19:08
Last Modified: 01 Oct 2015 19:08
Related URLs:
URI: http://repository.cshl.edu/id/eprint/31774

Actions (login required)

Administrator's edit/view item Administrator's edit/view item
CSHL HomeAbout CSHLResearchEducationNews & FeaturesCampus & Public EventsCareersGiving