CALORIMETRIC STUDIES OF MAT A2 HOMEODOMAIN
CALORIMETRIC STUDIES OF MAT A2 HOMEODOMAIN
批准号:
6122033
负责人:
JOHN H CARRA
金额:
$0.0万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
1997
资助国家:
美国
项目状态:
已结题
起止时间:
1997-08-05 至 1998-08-04
中文摘要
同源结构域是一类dna结合蛋白结构域
英文摘要
Homeodomains are a class of DNA-binding protein domains which have
important roles in control of transcription and development in
eukaryotes, and in some cases are involved in human oncogenesis. We
are using the MAT a2 homeodomain of yeast (Li et al., 1995, Science
270, 262-269) as a model system to characterize the thermodynamics of
protein folding and sequence-specific DNA binding by this protein
motif. Using differential scanning and isothermal titration
calorimetry, we measured the enthalpy, entropy, heat capacity, and
Gibbs free energy changes of these processes for the wild-type
sequence of a2 homeodomain (Carra & Privalov, 1997,Biochemistry 36,
526-535). The protein-DNA interaction is enthalpically driven at
physiological temperatures. Protein folding and DNA binding are
linked, as for several other DNA binding proteins. A comparison of
the circular dichroism spectra of the free and DNA-bound protein
species revealed that formation of protein structure is induced by DNA
binding. The energies measured for association therefore include a
component due to folding. We are currently extending these studies to
mutant versions of the a2 homeodomain. Site-directed mutagenesis was
used to create versions of the homeodomain that contain alanine
substitutions in the DNA recognition helix, removing important
contacts to DNA. Changes in the core packing of the protein were also
made to assay the importance of certain conserved residues to folding,
and the ability of the protein core to accept compensating
substitutions. Deletion of the N-terminal arm of the protein, which
wraps around the back of the DNA in the complex, results in a drastic
loss of DNA binding affinity. The MATa2 homeodomain can also bind DNA
cooperatively with the MATa1 homeodomain, as a heterodimer. This
protein-protein interaction, and DNA binding by the heterodimer, will
be studied using the isolated
homeodomains and a genetically produced fusion of the two.
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