Structural and functional relationships in DnaK and DnaK756 heat-shock proteins from Escherichia coli.
The secondary structures of DnaK and the mutant DnaK756 heat-shock proteins from Escherichia coli have been investigated by Fourier transform infrared spectroscopy. The analysis of infrared data showed that DnaK and DnaK756 proteins have different secondary structures that are not affected by the presence of ATP or beta, gamma-methyleneadenosine 5'-triphosphate. The infrared data indicate also that the tertiary structures of DnaK and DnaK756 proteins are different and that DnaK protein undergoes conformational changes in its tertiary structure not only during binding of ATP but also during ATP hydrolysis. Using fluorescence spectroscopy of a single tryptophan located in the N-terminal domain of DnaK protein and fluorescence of 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid, which interacts with hydrophobic domains of DnaK protein, we were able to distinguish between two conformational states of DnaK protein. After binding of triphosphonucleotides, the C-terminal domain of DnaK protein changes in tertiary structure in such a way that fewer hydrophobic segments are exposed on the surface of the protein. After ATP hydrolysis, the number of hydrophobic segments on the surface of the protein is further reduced, and moreover the tertiary structure of the N-terminal domain of the protein changes. These data are discussed in terms of structural and functional relationships of both DnaK and DnaK756 proteins.
Banecki
B
B
Department of Molecular Biology, University of Gdansk, Poland.
Zylicz
M
M
Bertoli
E
E
Tanfani
F
F
D000255:Adenosine Triphosphate / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D005583:Fourier Analysis; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013055:Spectrophotometry, Infrared; D013816:Thermodynamics
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D000255:Adenosine Triphosphate; C063842:dnaK protein, E coli