Bacterial expression and characterization of the CREB bZip module: circular dichroism and 2D 1H-NMR studies.
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, CREB327. The bZip peptide, CREB259-327, purified to near homogeneity, maintains the sequence-specific CRE site recognition demonstrated by in vitro competition assays. Alkylation of the three cysteine residues of CREB259-327 was employed to prevent aggregation of the peptide due to cysteine oxidation. The Kd of the purified native and modified CREB259-327 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 CREB259-327. The CD analyses of the native/modified CREB259-327 peptide demonstrated a 20% increase in the alpha-helical content upon binding to the cAMP response-element. Only a 5% increase in the alpha-helical content of CREB259-327 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) 1H-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.
Santiago-Rivera
Z I
ZI
Department of Chemistry, School of Science, Purdue University, West Lafayette, Indiana 47907.
Williams
J S
JS
Gorenstein
D G
DG
Andrisani
O M
OM
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D016350:Leucine Zippers; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D014169:Transformation, Bacterial
D017362:Cyclic AMP Response Element-Binding Protein; D010446:Peptide Fragments; D004247:DNA; D003545:Cysteine