Gene cloning, expression, and characterization of the Sac7 proteins from the hyperthermophile Sulfolobus acidocaldarius.


Abstract

The genes for two Sac7 DNA-binding proteins, Sac7d and Sac7e, from the extremely thermophilic archaeon Sulfolobus acidocaldarius have been cloned into Escherichia coli and sequenced. The sac7d and sac7e open reading frames encode 66 amino acid (7608 Da) and 65 amino acid (7469 Da) proteins, respectively. Southern blots indicate that these are the only two Sac7 protein genes in S. acidocaldarius, each present as a single copy. Sac7a, b, and c proteins appear to be carboxy-terminal modified Sac7d species. The transcription initiation and termination regions of the sac7d and sac7e genes have been identified along with the promoter elements. Potential ribosome binding sites have been identified downstream of the initiator codons. The sac7d gene has been expressed in E. coli, and various physical properties of the recombinant protein have been compared with those of native Sac7. The UV absorbance spectra and extinction coefficients, the fluorescence excitation and emission spectra, the circular dichroism, and the two-dimensional double-quantum filtered 1H NMR spectra of the native and recombinant species are essentially identical, indicating essentially identical local and global folds. The recombinant and native proteins bind and stabilize double-stranded DNA with a site size of 3.5 base pairs and an intrinsic binding constant of 2 x 10(7) M-1 for poly[dGdC].poly[dGdC] in 0.01 M KH2PO4 at pH 7.0. The availability of the recombinant protein permits a direct comparison of the thermal stabilities of the methylated and unmethylated forms of the protein. Differential scanning calorimetry demonstrates that the native protein is extremely thermostable and unfolds reversibly at pH 6.0 with a Tm of approximately 100 degrees C, while the recombinant protein unfolds at 92.7 degrees C. Study holds ProTherm entries: 5137, 5138 Extra Details: DNA-binding proteins; thermophilic; ribosome binding sites;,local and global folds

Submission Details

ID: KKHW7Zcz

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:28 p.m.

Version: 1

Publication Details
McAfee JG;Edmondson SP;Datta PK;Shriver JW;Gupta R,Biochemistry (1995) Gene cloning, expression, and characterization of the Sac7 proteins from the hyperthermophile Sulfolobus acidocaldarius. PMID:7632679
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
1XX8 2005-02-08 NMR Structure of the W24A Mutant of the Hyperthermophile Sac7d Protein
1SAP 1995-09-15 HYPERTHERMOPHILE PROTEIN, RELAXATION MATRIX REFINEMENT STRUCTURE
1XYI 2005-02-08 1.45 Hyperthermophile chromosomal protein Sac7d double mutant Val26Ala/Met29Ala in complex with DNA GCGATCGC
2XIW 2011-07-13 1.5 Crystal structure of the Sac7d-derived IgG1-binder C3-C24S
4CJ2 2014-05-21 1.5 Crystal structure of HEWL in complex with affitin H4
1WTO 2005-02-22 1.5 Hyperthermophile chromosomal protein SAC7D double mutant V26F/M29F in complex with DNA GCGATCGC
1BF4 1999-11-10 1.6 CHROMOSOMAL DNA-BINDING PROTEIN SSO7D/D(GCGAACGC) COMPLEX
1AZP 1999-01-13 1.6 HYPERTHERMOPHILE CHROMOSOMAL PROTEIN SAC7D BOUND WITH KINKED DNA DUPLEX
1WTV 2005-02-22 1.6 Hyperthermophile chromosomal protein SAC7D single mutant M29A in complex with DNA GTAATTAC
4CJ1 2014-05-21 1.63 Crystal structure of CelD in complex with affitin H3
1WTQ 2005-02-22 1.7 Hyperthermophile chromosomal protein SAC7D single mutant M29F in complex with DNA GTAATTAC
1WTR 2005-02-22 1.8 Hyperthermophile chromosomal protein SAC7D single mutant M29A in complex with DNA GCGATCGC
1WD0 2004-08-03 1.9 Crystal structures of the hyperthermophilic chromosomal protein Sac7d in complex with DNA decamers
1WTP 2005-02-22 1.9 Hyperthermophile chromosomal protein SAC7D single mutant M29F in complex with DNA GCGA(UBr)CGC
1AZQ 1999-01-13 1.94 HYPERTHERMOPHILE CHROMOSOMAL PROTEIN SAC7D BOUND WITH KINKED DNA DUPLEX
1CA6 2000-02-23 2.2 INTERCALATION SITE OF HYPERTHERMOPHILE CHROMOSOMAL PROTEIN SSO7D/SAC7D BOUND TO DNA
1WTX 2005-02-22 2.2 Hyperthermophile chromosomal protein SAC7D single mutant V26A in complex with DNA GTAATTAC
1WD1 2004-08-03 2.2 Crystal structures of the hyperthermophilic chromosomal protein Sac7d in complex with DNA decamers
1WTW 2005-02-22 2.2 Hyperthermophile chromosomal protein SAC7D single mutant V26A in complex with DNA GCGATCGC
1CA5 2000-02-23 2.2 INTERCALATION SITE OF HYPERTHERMOPHILE CHROMOSOMAL PROTEIN SSO7D/SAC7D BOUND TO DNA
1WVL 2005-08-02 2.6 Crystal Structure of Multimeric DNA-binding Protein Sac7d-GCN4 with DNA decamer

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
92.9 DNA-binding protein 7d F4B8X5 DN7C_ACIHW
91.4 DNA-binding protein 7d F4B9I5 DN7B_ACIHW
92.3 DNA-binding protein 7d P13125 DN7E_SULAC
100.0 DNA-binding protein 7d P13123 DN7D_SULAC