Thermal denaturation of T4 gene 32 protein: effects of zinc removal and substitution.
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)
Keating
K M
KM
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510.
Ghosaini
L R
LR
Giedroc
D P
DP
Williams
K R
KR
Coleman
J E
JE
Sturtevant
J M
JM
D000465:Algorithms; D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D004277:DNA, Single-Stranded / Q000032:analysis; D011071:Poly T / Q000032:analysis; D011489:Protein Denaturation; D013816:Thermodynamics; D014764:Viral Proteins / Q000032:analysis*; D015032:Zinc / Q000378:metabolism*
D004277:DNA, Single-Stranded; D014764:Viral Proteins; D011071:Poly T; D015032:Zinc