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Bacterial expression and characterization of the CREB bZip module: Circular dichroism and 2D 1 H‐NMR studies
Author(s) -
SantiagoRivera Zulma I.,
Gorenstein David G.,
Williams John S.,
Andrisani Ourania M.
Publication year - 1993
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560020910
Subject(s) - leucine zipper , creb , circular dichroism , peptide , bzip domain , cysteine , chemistry , recombinant dna , binding site , nuclear magnetic resonance spectroscopy , biochemistry , peptide sequence , stereochemistry , transcription factor , gene , enzyme
In this paper we describe the expression and purification from bacteria of the recombinant basic leucine zipper (bZip) domain of the cAMP response element binding protein, CREB 327 . The bZip peptide, CREB 259–327 , purified to near homogeneity, maintains the sequence‐specific CRE site recognition demonstrated by in vitro competition assays. Alkylation of the three cysteine residues of CREB 259–327 was employed to prevent aggregation of the peptide due to cysteine oxidation. The K d of the purified native and modified CREB 259–327 for the CRE site was determined by gel retardation assays to be on the order of 10 −7 M. We employed CD spectroscopy to study the folding properties of the native and modified CREB 259–327 . The CD analyses of the native/modified CREB 259–327 peptide demonstrated a 20% increase in the α‐helical content upon binding to the cAMP response‐element. Only a 5% increase in the α‐helical content of CREB 259–327 is observed upon binding to the AP‐1 site. This observation contrasts with CREB from the GCN4 protein (Weiss, M.A., et al., 1990, Nature 347 , 575–578). In addition, the two‐dimensional (2D) 1 H‐NMR studies of the bZip CREB peptide further support the distinct features of the CREB protein, in comparison to GCN4. Analysis by CD and 2D NMR of the dimerization domain of CREB suggests that the distinct DNA binding characteristics of CREB reside in the basic portion of the bZip module.