A nuclear magnetic resonance study of the DNA-binding affinity of Cro repressor protein stabilized by a disulfide bond.
The structure, dynamics, and DNA-binding characteristics of wild-type and cross-linked Cro repressors are compared by using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The Cro repressor is a small dimeric DNA-binding protein from bacteriophage lambda. Replacement of valine-55 by cysteine in the dimer interaction region of each monomer subunit results in the spontaneous formation of a disulfide cross-link between the subunits. Two-dimensional nuclear Overhauser effect spectroscopy and CD data show the variant has nearly the same conformation as the wild-type protein. However, by monitoring the CD band at 222 nm, the cross-linked protein is shown to have a heat-denaturation midpoint temperature of 67 degrees C, whereas the wild-type protein has a melting temperature of about 47 degrees C. Using 1H-NMR to follow the denaturation by heat, the same melting temperature is observed for wild-type Cro (47 degrees C), but a much lower melting temperature is seen for V55C Cro (58 degrees C). This suggests that between 58 and 67 degrees C the cross-linked protein exists in a molten globule state with the alpha-helices mainly intact, but without the interaction of chemical groups that cause spectral dispersion. Binding parameters for interaction of the proteins with DNA were obtained by observing the NMR spectrum for the imino protons of a 10 base-pair half-operator DNA and titrating in protein. The cross-linked protein binds DNA (Kd = 160 microM) about eight times more weakly than the wild-type protein (Kd = 19 microM). Adjustments in protein structure, necessary to form a tight protein-DNA complex, appear to be hindered by a loss in protein flexibility caused by the intersubunit cross-link.
Baleja
J D
JD
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Sykes
B D
BD
D010582:Bacteriophage lambda / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004220:Disulfides / Q000737:chemistry*; D006358:Hot Temperature; D006860:Hydrogen Bonding; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011487:Protein Conformation; D011489:Protein Denaturation; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D004220:Disulfides; D046911:Macromolecular Substances; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA