Gene 32 protein, the single-stranded DNA binding protein from bacteriophage T4, is a zinc metalloprotein.

Abstract

Gene 32 protein (g32P) isolated from bacteriophage T4-infected Escherichia coli and from an overproduction vector derived from the plasmid pKC30 contains 1 mol of tightly incorporated Zn(II) per mol of protein. A linear incorporation of three molar equivalents of p-hydroxymercuriphenylsulfonate (PMPS) results in a linear release of 1.1 mol of Zn(II) from the protein. Reversal of formation of the g32P-PMPS complex with thiol in the presence of EDTA results in a zinc-free apo-g32P. Cd(ll) and Co(ll) can be exchanged with the intrinsic Zn(ll) ion. The Cd(II) protein shows a charge-transfer band at m250 nm. The Co(II) protein shows a set of absorption bands typical of a tetrahedral Co(ll) complex (e. = 660 M-1 cm-1 at 645 nm), and two intense chargetransfer bands are present at 355 nm (e = 2250 M-1 cm-1) and 320 nm (e = 3175 M-1 cm-1). These observations are consistent with three cysteines as ligands to the Zn(ll) ion in g32P. Zn(ll) g32P undergoes precise limited proteolysis by trypsin to produce the small fragments A and B and the core, g32P-(A+B). Under identical conditions, apo-g32P is hydrolyzed rapidly beyond the g32P-(A+B) stage to produce many proteolyzed fragments. Fluorescence quenching experiments show that at low protein concentration apo-g32P has markedly altered binding affinity for poly(dT) relative to native g32P. Three of the four cysteines of g32P are found in a tyrosine-rich sequence corresponding to residues 72-116 and implicated in DNA binding by 1H NMR investigations. Zn(ll) appears to provide a conformational element contributing to DNA binding by coordinating the cysteine and possibly histidine side chains in the sequence -Cys-X3-His-X5-Cys-X2-Cys-, residues 77-90, located in the DNA binding domain of g32P.The product of gene 32 of bacteriophage T4 (g32P) is one of a class of proteins that bind to single-stranded (ss) DNA (or ss RNA) in a stoichiometric fashion and are referred to as helix-destablizing proteins or ss DNA binding proteins (SSBs) (1-4). Other widely studied members of this class include gene S protein from bacteriophage fd and SSB from Escherichia coli (5, 6). g32P is known to play key roles in DNA replication, recombination, and repair (2). g32P is thought to be able to quickly cover those transiently singlestranded regions that arise near the advancing T4 DNA replication forks and in so doing stabilize a particular ss DNA conformation that is most appropriate to serve as a substrate for other catalytic proteins (2). E. coli SSB probably functions in much the same way (6). Gene 5 protein, in contrast, acts to prevent replicative DNA synthesis by covering newly synthesized fd ss DNA until packaging into the phage coat can occur (3, 5).The molecular details of how each of these SSBs interacts with ss DNA has been probed by one-and two-dimensional 1H NMR techniques applied to the oligonucleotide complexes of these proteins (7-11). For both gene 5 protein and the g32P tryptic core, the results support a model involving intercalation of tyrosine and phenylalanine side cha...

Monitoring metal ion flux in reactions of metallothionein and drug‐modified metallothionein by electrospray mass spectrometry

“…We note that the nucleic acid binding activities (Karpel, 1990), autotranslational control (Shamoo et al, 1991;Green et al, 1994), and resistance to proteolytic breakdown (Nielson & Winge, 1983;Giedroc et al, 1986) of gene 32 protein are all very dependent on the presence of the bound metal. The results presented herein raise the possibility that these activities might be regulated in vivo by metal transfer to or from metallothionein or analogous proteins.…”

Molecular cloning and characterization of a cDNA showing negative regulation in v-src-transformed 3Y1 rat fibroblasts.

“…One of these groups has the form of Cys-Xaa2-CYs-Xaa13-CYs-Xaa2-CYS and thus is referred to as the C4 family (36). Because included in the C4 family with the exception that one of the cysteine residues has been replaced by histidine (38). We found no sequence homology between the putative finger domain of the N03 protein and the members of the C4 family (36,39); however, in the finger domain of N03 and the Mel-18 gene product which has been reported to be a zinc-finger protein (40), 11 out of 27 amino acid residues are identical (Fig.…”

FYVE Domain Targets Pib1p Ubiquitin Ligase to Endosome and Vacuolar Membranes

“…Zinc content of recombinant proteins was determined using a colorimetric assay (19) and compared to a standard curve generated using known amounts of ZnCl 2 . The standard curve was generated by determining the absorbance of 4-(2-pyridylazo)resorcinol at 500 nm of 1, 5, 10, 15, and 20 M ZnCl 2 .…”