NMR Spectroscopy of ¹¹³Cd(II)-Substituted Gene 32 Protein

Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol of protein. This intrinsic zinc is retained within the DNA-binding core fragment, g32P-(A+B) (residues 22-253), obtained by limited proteolysis of the intact protein. Ultraviolet circular dichroism provides evidence that Zn(II) binding causes significant changes in the conformation of the peptide chain coupled with alterations in the microenvironments of tryptophan and tyrosine side chains. NMR spectroscopy of the ¹¹³Cd(II) derivative of g32P-(A+B) at both 44.4 and 110.9 MHz shows a single ¹³Cd resonance, δ 637, a chemical shift consistent with coordination to three of the four sulfhydryl groups in the protein. In vitro mutagenesis of Cys¹⁶⁶ to Ser¹⁶⁶ creates a mutant g32P that still contains 1 Zn(II)/molecule. This mutant protein when substituted with ¹¹³Cd(II) shows a ¹¹³Cd signal with a δ and a line width the same as those observed for the wild-type protein. Thus, the S⁻ligands to the metal ion appear to be contributed by Cys⁷⁷, Cys⁸⁷, and Cys⁹⁰. Relaxation data suggest that chemical shift anisotropy is the dominant, but not exclusive, mechanism of relaxation of the ¹¹³Cd nucleus in g32P, since a dipolar modulation from ligandprotons is observed at 44.4 MHz but not at 110.9 MHz. Complexation of core ¹¹³Cd g32P with d(pA)₆ or Co(II) g32P with poly(dT) shows only minor perturbation of the NMR signal or d-d electronic transitions respectively, suggesting that the metal ion in g32P does not add a ligand from the bound DNA. The visible absorption spectra of the Co(II) derivative of g32P and its Ser¹⁶⁶ mutant suggest an approximately tetrahedral ligand field around the Co(II) ion in g32P. ³⁵C1 NMR shows that the Zn(II) site is not accessible to anions from solution, which suggests that a fourth ligand from the protein completes the tetrahedral complex. The best candidate is an imidazole nitrogen from His⁸¹. A simple molecular modeling procedure shows that a series of peptide turns compatible with peptide backbone angles found in proteins can accommodate the formation of a tetrahedral Zn(II) complex with Cys⁷⁷, His⁸¹, Cys⁸⁷, and Cys⁹⁰ as ligands.