Thermal denaturation of T4 gene 32 protein: effects of zinc removal and substitution

Abstract

Gene 32 protein (g32P), the single-stranded (ss) DNA binding protein from bacteriophage T4, is a zinc metalloprotein. The intrinsic zinc is one of the factors required for the protein to bind cooperatively to a ssDNA lattice. We have used differential scanning calorimetry to determine how the thermodynamic parameters characterizing the denaturation of g32P are affected by removal or substitution of the intrinsic zinc. Over a wide concentration range (1-10 mg/mL), the native Zn(II) protein unfolds at a tm of 55 degrees C with an associated mean enthalpy change of 139 kcal mol-1. Under the same conditions, the metal-free apoprotein denatures over a relatively broader temperature range centered at 49 degrees C, with a mean enthalpy change of 84 kcal mol-1. Substitution of Zn(II) in g32P by either Cd(II) or Co(II) does not significantly change the enthalpy of denaturation but does affect the thermal stability of the protein. All metallo forms of g32P when bound to poly(dT) undergo highly cooperative denaturational transitions characterized by asymmetric differential scanning calorimetry peaks with increases in tm of 4-5 degrees C compared to the unliganded metalloprotein. Removal of the metal ion from g32P significantly reduces the cooperativity of binding to poly(dT) [Giedroc, D. P., Keating, K. M., Williams, K. R., & Coleman, J. E. (1987) Biochemistry 26, 5251-5259], and presumably as a consequence of this, apo-g32P shows no change in either the shape or the midpoint of the thermal transition on binding to poly(dT).(ABSTRACT TRUNCATED AT 250 WORDS)

Poly(ADP-ribose)polymerase: a novel finger protein

“…Although the term 'zinc finger' protein was initially restricted to regulatory proteins having finger motifs closely related to TFIIIA (27) this family has now broadened its scope by the recent recruitment of the nuclear receptors (28)(29)(30) and the nucleic acid binding motifs associated with the retroviral gag gene protein (31) and T4 phage gene 32 protein (32,33). On the basis of our results we propose that poly(ADP-ribose)polymerase should also be considered as a member of the zinc-finger protein family.…”

p10 Single-stranded nucleic acid binding protein from murine leukemia virus binds metal ions via the peptide sequence Cys26-X2-Cys29-X4-His34-X4-Cys39

“…In the case of g32P, Zn(I1) appears to organize a subdomain within the core of the protein that is essential along with the aminoterminal domain in allowing cooperative binding of g32P to oligonucleotides such as d(pT)16 that are sufficiently long to permit protein-protein interactions along the DNA lattice. In addition, limited proteolysis, circular dichroism, and differential scanning microcalorimetry all indicate that Zn(I1) makes a substantial contribution to maintaining the overall structure of g32P (Giedroc et al, 1986(Giedroc et al, , 1987Keating et al, 1988).…”

Zinc and an N‐terminal extra stretch of the ferredoxin from a thermoacidophilic archaeon stabilize the molecule at high temperature

“…With such a stabilizing role, the zinc is referred to as`structural zinc', as has been reported for other proteins including alcohol dehydrogenase [23], adenylate kinase [24] and GP32 of bacteriophage T4 [25]. In these cases, Zn is ligated by either four Cys residues or three Cys and one His.…”