Abstract
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.
Highlights
From the $Division of Biophysics, Departmentof Molecular Biology, Universityof Gdansk, 80-822 Gdansk, Poland and the flnstitute of Biochemistry, Medical School, Universityof Ancona, 60100Ancona
Using fluorescence spectroscopy of a single tryptophan located in the N
The which interacts with hydrophobic domains of DnaK hsp70 family of proteins, by binding and releasing to these protein, we wereable to distinguish between two con- protein hydrophobic surfaces, could prevent protein aggregaformational states of DnaK protein
Summary
Deuterium oxide (99.9% deuterium) was purchased from Aldrich. Hepes, 1,4-dithio-~-threito(lDTT),' P-mercaptoethanol, sucrose, ATP, &y-imidoadenosine 5"triphosphate (AMP-PNP),and @,ymethyleneadenosine 5'-triphosphate (AMP-PCP) were obtained from Sigma. l,l'-Bis(4-anilino)naphthalene-5,5'-disulfonicacid (bisANS) was obtained from Molecular Probes. Preparation of Samples for Infrared Measurements-The purified protein solutions were concentrated onCentricon-30 microconcentrators + 50 mM NaCl, 10% sucrose, 6 mM P-mercaptoethanol buffer prepared in DzO, pD7.2 (pD = p H meter reading 0.4) [35,36].After exchange of the medium, the concentrated protein solution was stored for 3 days at room temperature.Prior toFTIR measurements, afinal exchange with 25 mM Hepes, 50 mM NaCl, 3 mM DTT buffer in DzO, pD 7.2 was carried out. The calculations were performed according to the method reported by Blume et al. PCP, &y-methyleneadenosine 5"triphosphate; AMP-PNP, P,y-imidoadenosine 5"triphosphate; FTIR, Fourier transform infrared; bisANS, l,l'-bis(4-anilino)naphthalene-5,5'-disulfoniaccid. The position and the number of amide I components, which were used as an input for the curve-fitting program, were obtained from the second derivative, fourth derivative, and deconvoluted absorption spectra.
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