p55-hGRF, a short natural form of the Ras-GDP exchange factor high yield production and characterization.

p55-hGRF, a natural short form of the guanine-nucleotide-releasing factor for p21-Ras from human brain, was expressed at high level in Escherichia coli as well as an engineered truncated form, p39-hGRF. A T7 polymerase expression system was used, resulting in the formation of insoluble cytoplasmic protein aggregates. The recombinant products were resolubilized, renatured and purified to homogeneity. The exchange activity of the refolded hGRF samples on H-Ras was comparable with that published for the soluble catalytic domain of the mouse counterpart, CDC25 Mm. Both p55-hGRF and p39-hGRF form dimers. We established a procedure to prepare and purify the complex with Ras. The results of the characterization study are consistent with a stoichiometry of 1:1 and an equilibrium between dimeric and monomeric forms of the complex.

Dimerization of Cdc25p, the guanine-nucleotide exchange factor for Ras from Saccharomyces cerevisiae, and its interaction with Sdc25p.

The oligomerization state of Cdc25p, the guanine nucleotide exchange factor for ras from yeast, was analyzed using different complementary approaches. The two-hybrid system showed that the C-terminal part of Cdc25p (Cdc25-Ct) can interact with itself but also with Sdc25p-Ct, the corresponding part of Sdc25p, the other guanine exchange factor from yeast. The homotropic interaction of Cdc25p-Ct has been confirmed in yeast using immunoprecipitation experiments with epitope-tagged and beta-galactosidase-fused polypeptides. No other component was required for this interaction, since dimerization was shown to occur with material synthesized in vitro. The size of Cdc25-Ct produced in Escherichia coli has been directly measured on gel filtration columns and corresponds to a dimer. The dimerization domain is localized in the same part of the molecule as the catalytic domain and the portion responsible for membrane localization. The biological relevance of dimerization is still an open question, however by allowing heterodimerization with Sdc25p it could permit a more complex combinatorial regulation of ras in yeast.

Evidence for a dimeric intermediate on the crystallization pathway of ribonuclease A.

Early steps in the crystallization process of pancreatic ribonuclease have been investigated by time-dependent fluorescence anisotropy, using a labeled protein as a fluorescent probe. Previous experiments have shown that steady-state fluorescence anisotropy is sensitive to protein-protein interactions and can be used to find new crystallization conditions. The present work describes an attempt, by means of time-resolved experiments, to detect and characterize species appearing in the early stages of the crystallization pathway. Fluorescence anisotropy decay was measured with synchrotron radiation as a light source under a variety of conditions where it is known that the solutions tend towards crystallization; the decay was analyzed by a maximum-entropy method that calculates a rotational correlation-time distribution. Fluorescence anisotropy originates in the Brownian rotatory motion of macromolecules and the values of the correlation times are related to the size and shape of different species present in the solution. In the presence of high salt concentrations, a bimodal distribution is always observed. Whereas a peak of protein monomer is still present, a second peak appears as a stable intermediate in the crystallization pathway. The correlation time of this new species varies between two and three times the correlation time of the monomer. The second peak is possibly the symmetrical dimer of the ribonuclease molecules commonly observed in all the high-salt crystal forms.

Crystal structure of a fluorescent derivative of RNase A.

The crystal structure of RNase A chemically modified with the fluorescent probe, N-[[(iodoacetyl)-amino]ethyl]-5-naphthylamine-1-sulfonic acid (1,5-IAENS), has been solved and refined to high resolution. It yields information on the mode of binding, the mobility of a probe commonly used in spectroscopic studies, and anion binding sites in RNase A. Trigonal crystals of the fluorescent derivative grown in sodium or cesium chloride and ammonium sulfate, pH 5.1, were nearly isomorphous with those of a semisynthetic RNase [DeMel, et al. (1992) J. Biol. Chem. 267, 247-256]. Refinement starting from semisynthetic RNase led to a model with R = 20% against 1.7-A diffraction data from crystals in ammonium sulfate and another model with R = 17% against 1.9-A data taken in the presence of 3 M NaCl. The second model contains three chloride ions: one is at the active site, and the other two are at molecular interfaces. Otherwise, the two models are very similar. The fluorophore has very little effect on the protein conformation. It is found to be covalently attached to the active site His-12 with the naphthyl group stacked on the imidazole ring of His-119. It remains largely accessible to solvent and in a polar environment on the protein surface, even though the fluorescence emission spectrum is blue shifted as it is in nonpolar solvents.