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) Study holds ProTherm entries: 3506, 3507, 3508, 3509 Extra Details: gene 32 protein; differential scanning calorimetry;,thermal stability; enthalpy change

Submission Details

ID: T37uZsj93

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:21 p.m.

Version: 1

Publication Details
Keating KM;Ghosaini LR;Giedroc DP;Williams KR;Coleman JE;Sturtevant JM,Biochemistry (1988) Thermal denaturation of T4 gene 32 protein: effects of zinc removal and substitution. PMID:3262371
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1GPC 1995-06-01T00:00:00+0000 2.2 CORE GP32, DNA-BINDING PROTEIN
1CB4 1999-02-26T00:00:00+0000 2.3 CRYSTAL STRUCTURE OF COPPER, ZINC SUPEROXIDE DISMUTASE
1CBJ 1999-02-26T00:00:00+0000 1.65 CRYSTAL STRUCTURE OF BOVINE SUPEROXIDE DISMUTASE CRYSTAL.
1COB 1992-02-19T00:00:00+0000 2.0 CRYSTAL STRUCTURE SOLUTION AND REFINEMENT OF THE SEMISYNTHETIC COBALT SUBSTITUTED BOVINE ERYTHROCYTE ENZYME SUPEROXIDE DISMUTASE AT 2.0 ANGSTROMS RESOLUTION
1E9O 2000-10-25T00:00:00+0000 1.85 Crystal structure of bovine SOD - 1 of 3
1E9O 2000-10-25T00:00:00+0000 1.85 Crystal structure of bovine SOD - 1 of 3
1E9P 2000-10-25T00:00:00+0000 1.7 Crystal structure of bovine Cu, Zn SOD to 1.7 Angstrom (3 of 3)
1E9P 2000-10-25T00:00:00+0000 1.7 Crystal structure of bovine Cu, Zn SOD to 1.7 Angstrom (3 of 3)
1E9Q 2000-10-26T00:00:00+0000 1.75 Crystal structure of bovine Cu Zn SOD - (1 of 3)
1E9Q 2000-10-26T00:00:00+0000 1.75 Crystal structure of bovine Cu Zn SOD - (1 of 3)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Single-stranded DNA-binding protein O21959 VHED_BPR69
100.0 Single-stranded DNA-binding protein O21958 VHED_BPR32
100.0 Single-stranded DNA-binding protein O21957 VHED_BPR27
100.0 Single-stranded DNA-binding protein O21950 VHED_BPR03
100.0 Single-stranded DNA-binding protein O21960 VHED_BPS76
100.0 Single-stranded DNA-binding protein O21956 VHED_BPR18
100.0 Single-stranded DNA-binding protein O21955 VHED_BPR15
100.0 Single-stranded DNA-binding protein O21951 VHED_BPR06
100.0 Single-stranded DNA-binding protein O21954 VHED_BPR10
100.0 Single-stranded DNA-binding protein O21952 VHED_BPR08
100.0 Single-stranded DNA-binding protein O21949 VHED_BPPST
100.0 Single-stranded DNA-binding protein O21948 VHED_BPM1
100.0 Single-stranded DNA-binding protein O21947 VHED_BPFSA
100.0 Single-stranded DNA-binding protein O21953 VHED_BPR09
97.8 Single-stranded DNA-binding protein O21946 VHED_BPS14
97.9 Single-stranded DNA-binding protein O21945 VHED_BPR70
98.2 Single-stranded DNA-binding protein P09035 VHED_BPT2
99.1 Single-stranded DNA-binding protein P09797 VHED_BPT6
100.0 Single-stranded DNA-binding protein P03695 VHED_BPT4
99.2 Azurin B3EWN9 AZUR_PSEAI
100.0 Azurin P00282 AZUR_PSEAE
92.3 Alkaline phosphatase P21948 PPB_ESCF3
100.0 Alkaline phosphatase P00634 PPB_ECOLI
94.7 Superoxide dismutase [Cu-Zn] O46412 SODC_CEREL
97.4 Superoxide dismutase [Cu-Zn] P09670 SODC_SHEEP
98.0 Superoxide dismutase [Cu-Zn] Q5FB29 SODC_CAPHI
98.7 Superoxide dismutase [Cu-Zn] Q52RN5 SODC_BOSMU
100.0 Superoxide dismutase [Cu-Zn] P00442 SODC_BOVIN