A novel function for zinc(II) in a nucleic acid-binding protein. Contribution of zinc(II) toward the cooperativity of bacteriophage T4 gene 32 protein binding.
The contribution of Zn(II) toward the binding of bacteriophage T4 gene 32 single-stranded nucleic acid-binding protein (gp32) has been examined by the use of two independent approaches. Studies carried out with successively longer oligonucleotides which have the general structure p(dT)n, where n is equal to 8, 16, 24, or 32 nucleotides, suggest that removal of Zn(II) decreases the cooperativity of binding by as much as 30-fold. Hence, whereas apo-gp32 and native gp32 have similar apparent affinities for the single-site lattice p(dT)8, native gp32 has an approximately 10-fold higher affinity compared to apo-gp32 for a two-site lattice, such as p(dT)16. In contrast to native gp32, where full cooperativity (in terms of the strength of a single gp32-gp32 interaction) is reached with only a two-site lattice, the cooperativity of apo-gp32 binding appears to increase approximately 4-fold upon going from a two- to a four-site lattice such as p(dT)32. The conclusion reached from these oligonucleotide studies agrees well with a series of titrations with polyribo(ethenoadenylic) acid, in 0.275-0.40 M NaCl. These latter studies indicate that the 6-38-fold higher affinity of native gp32 as compared to apo-gp32 for polyribo(ethenoadenylic) acid results primarily from the higher cooperativity of binding of native gp32. By stabilizing a specific subdomain within gp32 that is essential along with the NH2-terminal domain (residues 1-9), Zn(II) contributes from 20 to 50% of the free energy of cooperative gp32-gp32 interactions that occur along a polynucleotide lattice.
Nadler
S G
SG
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510.
Roberts
W J
WJ
Shamoo
Y
Y
Williams
K R
KR
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000378:metabolism; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008667:Metalloproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013604:T-Phages / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D001059:Apoproteins; D004268:DNA-Binding Proteins; D008667:Metalloproteins; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; D015032:Zinc