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The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as "membrane-distal" and estimate to comprise ∼90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.

A study was conducted to investigate the HIV surface glycoprotein gp120-bound conformation of CD4 by determining a CD4 peptide blocking the interaction with gp12 using nuclear magnetic resonance. The Milligen/Perseptive Biosystems 950 continuous flow peptide synthesizer was utilized to obtain peptides. Results suggested that the 36-59 region of CD4 is flexible. Findings also indicated that the region forecasts a gp120-bound conformation not similar with the CD4 crystal formed during the absence of gp120.

The effects of serpin binding on the target proteinase particularly on the stabilization, localized enhanced conformational flexibility, and retained hydrogen bonding at the active site were established. Lower stability of rat trypsin toward thermal denaturation in the free enzyme indicated the presence of extensive protein-protein interactions that stabilize the folding process for the complex.

The formation of the transition state of bovine chymotrypsinogen A is followed by a change in H-bonding strength. Signals from histidine57 (His57) and aspartame102 (Asp102) were detected in the spectra of chymotrypsinogen A during nuclear magnetic resonance studies. The strong H-bonds of the catalytic triad were enhanced by the imidazolium of His. Stabilization of the catalytic triad was affected by energy changes in the network of H-bonds and neighboring protein residues.

The characterization of the green-fluorescent protein chromophore of Aequorea victoria was described. The papain-derived chromophore-containing hexapeptide is formed upon cyclization of the residues Ser-dehydroTyr-Gly within the polypeptide. Amino acid analysis and enzymatic cleavage of the peptide demonstrated that a phenylalanine is present at the amino terminus and that the carboxyl-terminal sequence is Val-Glx.

The flavoenzyme uridine 5'-diphosphate galactopyranose mutase (UGM of Glf) has catalyzed the interconversion of UDP-galactopyranose and UDP-galactofuranose and the mechanistic studies of UGM have shown a novel role for the flavin. The X-ray crystal structures of the substrate-bound enzyme are described in oxidized and reduced forms, respectively.

The catalysis of the interconversion of UDP-galactopyranose and UDP-galactofuranose, a key building block for cell wall construction in Mycobacterium tuberculosis by flavoenzyme uridine 5'-diphosphate galactopyranose mutase is studied. X ray crystallography is employed to investigate the binding of flavin.

A method based on 1H-1H NOE interactions is used to identify the binding site of spermine on tRNA. The carbon-bound methylene protons next to the amino groups are used to follow the interaction with tRNA. These protons resonate between 2.8-3.1 ppm where they overlap with the tRNA sugar resonances. This overlap is resolved by using spermines enriched with 13C in the carbons bonded to the -NH2+- and NH3+ groups. Results show that spermine interacts at the corner of the L-shaped tRNA molecule where the T_C and D loops intersect. These results are useful in the further understanding of the function of polyamines in cell growth and carcinogenesis.

Selective isotope labeling was employed to analyze the origin of the paramagnetically shifed 1H, 2H and 15N resonances of T4MOC, a soluble Rieske ferredoxin component of toluene 4-monooxygenase. The paramagnetic H NMR signals from T4MOC differ from those of the plant-type and vertebrate ferredoxins, and assignments of these signals to residue and atom type in T4MOC question particular 1H NMR assignments made for the Xanthobacter Rieske center. The pattern of H-bonding to the iron-sulfur cluster of T4MOC is discussed.