No abstract

The catalytic oxidation of beta-D-glucose by the enzyme glucose oxidase involves a redox change of the flavin coenzyme. The structure and the dynamics of the two extreme glucose oxidase forms were studied by using infrared absorption spectroscopy of the amide I'band, tryptophan fluorescence quenching and hydrogen isotopic exchange. The conversion of FAD to FADH2 does not change the amount of alpha-helix present in the protein outer shell, but reorganizes a fraction of random coil to beta-sheet structure. The dynamics of the protein interior vary with the redox states of the flavin without affecting the motions of the structural elements near the protein surface. From the structure of glucose oxidase given by X-ray crystallography, these results suggest that the dynamics of the interface between the two monomers are involved in the catalytic mechanism.

No abstract

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.