Lac repressor N-terminal DNA binding domain: cloning, isolation and preliminary /sup 15/N NMR observations

Conference · Fri May 01 04:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6173094

Khoury, A; Leighton, P; Lu, P

E. coli Lac repressor N-terminal DNA binding domain (headpiece) has previously been isolated by cleavage with clostripain, chymotrypsin, and trypsin to yield N-terminal fragments of 51, 56, and 59 amino acids respectively. This method is both inefficient and limited by the specificity of available proteases. To circumvent these difficulties, the authors are cloning the DNA sequence encoding various lengths of the lac1 gene into a plasmid containing the P/sub L/ promoter from bacteriophage ..gamma.. as well as an improved ribosome binding site. In the authors initial efforts they cloned a 56 amino acid headpiece (pAK3-4); they describe here the isolation and characterization of this headpiece. It has been shown that /sup 15/N NMR can be extremely useful in investigating the structure and dynamics of the bacteriophage ..gamma.. cro repressor protein with its binding site O/sub R/3. Uniformly labelled lac headpiece (56 aa) has been prepared by growth of E. coli transformed with pAK3-4 in a minimal medium containing /sup 15/N ammonium sulfates as the sole nitrogen source. NMR experiments were used to measure /sup 15/N-//sup 1/H/ NOEs which are sensitive to mobility on a nanosecond timescale. They are thus able to observe changes in dynamics of individual amino acid side chains and backbone nitrogen of the peptide upon binding operator DNA.

• Research Organization: Univ. of Pennsylvania, Philadelphia
• OSTI ID: 6173094
• Report Number(s): CONF-870644-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 46:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
BACTERIA
BACTERIOPHAGES
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CLONING
DNA
DNA-CLONING
ENZYMES
ESCHERICHIA COLI
GENES
HYDROLASES
ISOTOPES
LIGHT NUCLEI
MICROORGANISMS
MOLECULAR STRUCTURE
NITROGEN 15
NITROGEN ISOTOPES
NMR SPECTRA
NUCLEI
NUCLEIC ACIDS
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANOIDS
PARASITES
PEPTIDE HYDROLASES
PEPTIDES
PROTEINS
RIBOSOMES
SPECTRA
STABLE ISOTOPES
VIRUSES