Two distinct Raf domains mediate interaction with Ras.

A key event for Ras transformation involves the direct physical association between Ras and the Raf-1 kinase. This interaction promotes both Raf translocation to the plasma membrane and activation of Raf kinase activity. Although substantial experimental evidence has demonstrated that Raf residues 51-131 alone are sufficient for Ras binding, conflicting observations have suggested that the Raf cysteine-rich domain (residues 139-184) may also be important for interaction with Ras.

Biological and structural characterization of a Ras transforming mutation at the phenylalanine-156 residue, which is conserved in all members of the Ras superfamily.

Although Ras residue phenylalanine-156 (F156) is strictly conserved in all members of the Ras superfamily of proteins, it is located outside of the consensus GDP/GTP-binding pocket. Its location within the hydrophobic core of Ras suggests that its strict conservation reflects a crucial role in structural stability. However, mutation of the equivalent residue (F157L) in the Drosophila Ras-related protein Rap results in a gain-of-function phenotype, suggesting an alternative role for this residue.

Biomolecular applications of heteronuclear multidimensional NMR.

Recent advances in stable-isotope enrichment and heteronuclear multidimensional NMR techniques have transformed NMR into a more powerful and versatile method for the structural and dynamic characterization of biomolecules. Current efforts still focus on improving the methodology to increase the number of systems amenable to NMR analysis, to generate better structures and to investigate biomolecular motions in solution.

Isolation and refolding of H-ras from inclusion bodies of Escherichia coli: refold procedure and comparison of refolded and soluble H-ras.

A refolding and purification method for economically producing large quantities of H-ras isolated from Escherichia coli inclusion bodies is described. Experiments were performed to optimize the yield of refolded H-ras for structural analysis by NMR spectroscopy. Protein concentration, temperature, and the presence of 10% glycerol during refolding were varied.

Solution structure and dynamics of ras p21.GDP determined by heteronuclear three- and four-dimensional NMR spectroscopy.

A high-resolution solution structure of the GDP form of a truncated version of the ras p21 protein (residues 1-166) has been determined using NMR spectroscopy. Ras p21 is the product of the human ras protooncogene and a member of a ubiquitous eukaryotic gene family which is highly conserved in evolution. A virtually complete assignment (13C, 15N, and 1H), including stereospecific assignments of 54 C beta methylene protons and 10 C gamma methyl protons of valine residues, was obtained by analysis of three- and four-dimensional (3D and 4D) heteronuclear NMR spectra using a newly developed 3D/4D version of the ANSIG software.

Identification of residues critical for Ras(17N) growth-inhibitory phenotype and for Ras interaction with guanine nucleotide exchange factors.

The Ras(17N) dominant negative antagonizes endogenous Ras function by forming stable, inactive complexes with Ras guanine nucleotide exchange factors (GEFs; e.g., SOS1).

Secondary structure and signal assignments of human-immunodeficiency-virus-1 protease complexed to a novel, structure-based inhibitor.

We report comprehensive NMR studies in solution of the human-immunodeficiency-virus (HIV)-1 protease. Stable solutions of the protease were obtained by complexing the protein to a designed cyclic urea inhibitor DMP 323. A variety of triple-resonance experiments provided essentially complete 1H, 13C and 15N NMR signal assignments of the protease.

Sequential assignment of the backbone nuclei (1H, 15N and 13C) of c-H-ras p21 (1-166).GDP using a novel 4D NMR strategy.

The c-H-ras p21 protein is the product of the human ras proto-oncogene, a member of a ubiquitous eukaryotic gene family which is highly conserved in evolution. These proteins play an important role in the control of cellular growth. We report here the sequential assignment of the backbone nuclei in a truncated form of the 21-kD gene product, using our recently proposed 4D NMR strategy (Boucher et al.

Structural and dynamic differences between normal and transforming N-ras gene products: a 31P and isotope-edited 1H NMR study.

[15N]Glycine was biosynthetically incorporated into normal cellular N-ras p21 and a position 12 transforming mutant, in order to produce p21 proteins containing several site-specific NMR probes at or near activating positions in the guanine nucleotide binding domain. We have previously assigned all five glycine resonances located in loops directly involved in binding of guanosine diphosphate in the wild-type p21 protein [Campbell-Burk, S., Papastavros, M.

31P NMR saturation-transfer measurements in Saccharomyces cerevisiae: characterization of phosphate exchange reactions by iodoacetate and antimycin A inhibition.

31P nuclear magnetic resonance (NMR) saturation-transfer (ST) techniques have been used to measure steady-state flows through phosphate-adenosine 5'-triphosphate (ATP) exchange reactions in glucose-grown derepressed yeast. Our results have revealed that the reactions catalyzed by glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK) and by the mitochondrial ATPase contribute to the observed ST. Contributions from these reactions were evaluated by performing ST studies under various metabolic conditions in the presence and absence of either iodoacetate, a specific inhibitor of GAPDH, or the respiratory chain inhibitor antimycin A.

31P NMR saturation-transfer and 13C NMR kinetic studies of glycolytic regulation during anaerobic and aerobic glycolysis.

31P NMR saturation-transfer techniques have been employed in glucose-grown derepressed yeast to determine unidirectional fluxes in the upper part of the Embden-Meyerhof-Parnas pathway. The experiments were performed during anaerobic and aerobic glycolysis by saturating the ATP gamma resonances and monitoring changes in the phosphomonoester signals from glucose 6-phosphate and fructose 1,6-bis-phosphate. These experiments were supplemented with 13C NMR measurements of glucose utilization rates and 13C NMR label distribution studies.

High-resolution NMR studies of Saccharomyces cerevisiae.

High-resolution NMR studies of yeast cells have contributed to our understanding of metabolism and energetics. The above studies of glycolytic control, enzyme kinetics, and metabolism during dormancy have shown how the strengths of NMR investigations can build upon existing knowledge to create a qualitatively different understanding of the processes in yeast.