Serum response factor (SRF) is a MADS transcription factor that binds to the CArG box sequence of the serum response element (SRE). Through its binding to CArG sequences, SRF activates several muscle‐specific genes as well as genes that respond to mitogens. The thermodynamic parameters of the interaction of core‐SRF (the 124–245 fragment of serum response factor) with specific oligonucleotides from c‐fos and desmin promoters, were determined by spectroscopy. The rotational correlation time of core‐SRF labeled with bis‐ANS showed that the protein is monomeric at low concentration (10−7 m). The titration curves for the fluorescence anisotropy of fluorescein‐labeled oligonucleotide revealed that under equilibrium conditions, the core‐SRF monomers were bound sequentially to SRE at very low concentration (10−9 m). Curve‐fitting data showed also major differences between the wild‐type sequence and the oligonucleotide sequences mutated within the CArG box. The fluorescence of the core‐SRF tyrosines was quenched by the SRE oligonucleotide. This quenching indicated that under stoichiometric conditions, core‐SRF was bound as a dimer to the wild‐type oligonucleotide, and as a monomer or a tetramer to the mutant oligonucleotides. Far‐UV CD spectra indicated that the flexibility of core‐SRF changed profoundly upon its binding to its specific target SRE. Lastly, the rotational correlation time of fluorescein‐labeled SRE revealed that formation of the specific complex was accompanied by a change in the SRE internal dynamics. These results indicated that the flexibility of the two partners is crucial for the DNA–protein interaction.
Structural and dynamic constraints produced by the surrounding solvent on the aquometmyoglobin molecule were investigated by means of circular dichroism and Fourier-transform infrared spectroscopies, tritiumhydrogen exchange kinetics and small-angle neutron-scattering experiments. Formamide and ethanol were chosen as cosolvents because they are known to increase and decrease protein activity, respectively. The CD measurements in the Soret region show that no changes occur in the heme molecular structure nor in the protein near the heme. The results of proton-exchange kinetics experiments indicate that the conformational dynamics of aquometmyoglobin is only marginally affected by the cosolvents. However, the small-angle neutron-scattering spectra strongly suggest that these cosolvents induce some distortions of the tertiary conformation. According to the ultraviolet CD and Fourier-transform infrared data, the alteration of the tertiary conformation results from changes in both the number of intrachain hydrogen bonds and the structures of p turns of type I' for formamide and of type I1 for either of the two cosolvents. The use of several techniques allows the present approach to demonstrates that the myoglobin structure is extremely sensitive to its environmental conditions. Keywords. Myoglobin; conformation change ; mixed solvents ; neutron scattering ; Fourier-transform infrared spectroscopy.All we know about the myoglobin structure derives primarily from X-ray crystallography (Kendrew et al., 1960(Kendrew et al., , 1961Takano, 1977; Brayer, 1988, 1990). This structural approach has led to the idea that the three-dimensional structure of proteins basically is identical in crystal and in solution (Stuhrmann, 1973;Makinen and Fink, 1977;Wiithrich, 1989). However, it is difficult to eliminate the possibility that the crystal lattice packing forces may induce significant constraints on the macromolecules (Phillips, 1990). As a matter of fact it has already been demonstrated that small differences exist between both the topology of particular regions and the tertiary conformation of myoglobin in solution and in the crystalline state (Fedorov and Denesyuk, 1978;Bianconi et al., 1985). There are also some instances where the native protein structure in solution is radically different from the crystalline one (Timchenko et al., 1978;Heidorn and Trewhella, 1988;Trewhella et al., 1988). Other studies have also suggested that the dynamic properties of proteins could be related to those of the bulk solvent (Beece et al., 1980;Zentz et al., 1991).A better knowledge of mixed-solvent effects on a monomeric protein such as myoglobin could help us to understand why protein structures and dynamics can be different in crystal and in solution. However our understanding of protein-solvent interactions is far from being complete. Two classes of neutral cosolvents, amides and alcohols, are known to alter the water structure and to affect the protein-ligand equilibria and enzymic Correspondence to P. Calmettes, Laboratoire LCon Brillou...
The effects of the solvent conditions (buffer pH 9, 8, or 7 or buffer pH 6.5 alone or mixed with 3.2% ethanol or 6.2% formamide) on the protein dynamics of horse apomyoglobin were investigated through tryptophan fluorescence quenching, spectra, and decay properties. Raising the pH (which induces discontinuous protein conformation changes) increases the structural fluctuations inside the hydrophobic A, G, and H helix core. Mixed solutions containing either 3.2% ethanol or 6.2% formamide (which redistribute water molecules on the protein surface) produce protein dynamics changes in the vicinity of the two Trp residues, without inducing particular constraints on these very residues. Formamide increases, in the same way, the polarity and the protein flexibility while ethanol reduces both. The present fluorescence work also shows that, whatever the outside solvent, the two Trp residues W7 and W14, embedded in the A, G, and H helix core, are equally and statistically reached by small molecules diffusing inside the protein matrix. Hydrogen-tritium exchange measurements on the protein in mixed solvents reveal that the dynamics of the A, G, and H helix cluster and of the B and E helixes are greatly influenced by the nature of the outside medium. A small amount of formamide in the buffer increases the protein fluctuations while an ethanol-water mixture reduces them. We suggest that the hydratation state of the protein surface could be the relevant parameter of the protein dynamics.
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