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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.

The binding of spermine (SPM), N¹,N12-bismethylspermine (BMS) and N¹,N12-bisethylspermine (BES) to tRNA Phe was studied using ¹H-NMR at 750 MHz. The polyamines were enriched in 13C at the 5-CH2 and 8-CH2 residues and the nuclear Overhauser enhancement (NOE) cross peaks connecting the ¹H-NMR resonances of the13C-methylenes and several base paired imino protons of tRNA Phe were obtained using 1D13C-half filteredspectra. It was found that while SPM and BMS bind to the N(3)-H of base pairs T54-m¹A58, U50-A64 and U52-A62, BES binds only to T54-m¹A58 and U50-A64. This regioselectivity in the binding of the three polyamines to tRNA was correlated with their biological effects on cell growth. Using human melanoma cancer cells (MALME-3M), we found that SPM and BMS were without effect and cytostatic, respectively, while BES was distinctly cytotoxic. The latter also affected cell cycling and, at variance with SPM and BMS, lead to a distinctG1/S cell cycle arrest.

Isotope-based methods are commonly used for metabolic flux analysis and metabolite quantification in biological extracts. Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical tool for these studies because NMR can unambiguously identify compounds and accurately measure [sup.13]C enrichment. We have developed a new pulse sequence, isotope-edited total correlation spectroscopy (ITOCSY), that filters two-dimensional [sup.1]H-[sup.1]H NMR spectra from [sup.12]C- and [sup.13]C-containing molecules into separate, quantitatively equivalent spectra. The ITOCSY spectra of labeled and unlabeled molecules are directly comparable and can be assigned using existing bioinformatics tools. In this study, we evaluate ITOCSY using synthetic mixtures of standards and extracts from Escherichia coli. We show that ITOCSY has low technical error (6.6% for metabolites ranging from 0.34 to 6.2 mM) and can detect molecules at concentrations less than 10 [micro]M. We propose ITOCSY as a practical NMR strategy for metabolic flux analysis, isotope dilution experiments, and other methods that rely on carbon-13 labeling. 10.1021/ac100565b

One-dimensional (1D) [sup.1]H nuclear magnetic resonance (NMR) spectroscopy is used extensively for high-throughput analysis of metabolites in biological fluids and tissue extracts. Typically, such spectra are treated as multivariate statistical objects rather than as collections of quantifiable metabolites. We report here a two-dimensional (2D) [sup.1]H-[sup.13]C NMR strategy (fast metabolite quantification, FMQ, by NMR) for identifying and quantifying the ~40 most abundant metabolites in biological samples. To validate this technique, we prepared mixtures of synthetic compounds and extracts from Arabidopsis thaliana, Saccharomyees cerevisiae, and Medicago sativa. We show that accurate (technical error 2.7%) molar concentrations can be determined in 12 min using our quantitative 2D [sup.1]H-[sup.13]C NMR strategy. In contrast, traditional 1D [sup.1]H NMR analysis resulted in 16.2% technical error under nearly ideal conditions. We propose FMQ by NMR as a practical alternative to 1D [sup.1]H NMR for metabolomics studies in which 50-mg (extract dry weight) samples can be obtained.

The rubredoxin from Clostridium pasteurianum (CpRd) provides an excellent system for investigating how the protein sequence modulates the reduction potential of the active site in an iron-sulfur protein, [sup.15]N NMR spectroscopy has allowed us to determine with unprecedented accuracy the strengths of all six key hydrogen bonds between protein backbone amides and the sulfur atoms of the four cysteine residues that ligate the iron in the oxidized ([Fe.sup.III]) and reduced ([Fe.sup.III]) forms of wild-type CpRd and nine mutants (V44G, V44A, V44I, V44L, V8G, V8A, V8I, V8L, and V8G/V44G). The length (or strength) of each hydrogen bond was inferred from the magnitude of electron spin delocalized across the hydrogen bond from the iron atom onto the nitrogen. The aggregate lengths of these six hydrogen bonds are shorter in both oxidation states in variants with higher reduction potential than in those with lower reduction potential. Differences in aggregate hydrogen bonding upon reduction correlate linearly with the published reduction potentials for the 10 CpRd variants, which span 126 mV. Sequence effects on the reduction potential can be explained fully by their influence on hydrogen-bond strengths. iron-sulfur protein | reduction potential tuning | [sup.15]N NMR | paramagnetic NMR

Hybrid density functional quantum mechanical calculations were used to study the strength of the hydrogen bond between His(super 57)N(super delta1) and Asp(super 102)O(super delta1) in chymotrypsin and how it changes along the reaction coordinate. The results confirm the hypothesis that the strengthened hydrogen bond plays an important role in lowering the energy of the transition state and hence, in the catalytic efficiency of the enzyme.

Research was conducted to examine the redox-dependent magnetic alignment of Clostridium pasteurianum rubredoxin (Rdx). One bond 1H(super N) - 15N and 1H(super alpha) - 13C(super alpha) couplings in oxidized and reduced Rdx at multiple magnetic field strengths were measured which enabled the decomposition of the couplings into their scalar and dipolar components. Results reveal that the redox-dependent chemical shift differences can be explained adequately by electron-nuclear dipolar effects without any need to postulate conformational differences.

Research was conducted to compare and predict the contribution of unpaired electrons to the relaxation times for 15N nuclei in oxidized Clostridium pasteurianum rubredoxin using high-level, all-electron, density functional measurements in conjunction with high-resolution X-ray structural data. A 104-atom model for the iron center that included all atoms shown to have strong electronic interactions with the unpaired iron electrons was constructed. Using effective distances greatly improves the correlation for a plot of experimental relaxation rates versus resonances in rubredoxin.

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.

Fermi contact spin densities in the Clostridium pasteurianum rubredoxin can be computed from a structural model derived from a protein x-ray structure by means of a hybrid density functional treatment. Calculations using this method made strong distinctions between cysteine nitrogens shifted to higher and to lower frequency. The spin densities showed linear correlation with isotropic hyperfine 1H, 2H, 13C, and 15N nuclear magnetic resonance chemical shifts determined for Fe(III). Possible sources for errors in calculation include incomplete assignments and use of oversimplified basis sets.

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.

Individual protein domains and two domains in combination were prepared by enzymatic and chemical cleavage of turkey ovomucoid followed by isolation and purification by size-exclusion and ion-exchange chromatography. Silica bonded-phase HPLC columns were made from either whole or isolated domains of turkey ovomucoid. The protein columns were tested for chiral recognition by their abilities to resolve enantiomers among a wide range of racemates. The columns made from whole turkey ovomucoid displayed chiral activity toward many racemates, where as a combination of the first and second domain resolved only a selected number of aromatic weak bases. The first and second domains independently gave no appreciable chiral activity. The turkey ovomucoid third domain exhibited enantioselective protein binding for fused-ring aromatic weak acids. Glycosylation of the third domain did not affect chiral recognition. Titration of the third domain with model compounds in conjunction with NMR measurements enabled the identification of the amino acids responsible for binding. Molecular modeling of the ligand-protein complexation provided insights into the ability of a protein surface to discriminate enantiomers on the basis of multiple intermolecular interactions.

Study on one dimensional 1H, 2H and 15H nuclear magnetic spectroscopy indicates the hyperfine-shifted resonances of all the alpha and beta hydrogens and N atoms for four cysteinyl ligands of rubredoxin from Clostridium pasteurianum in its oxidized and reduced states. For the beta hydrogen, the isotropic shift is larger than the alpha hydrogen. The paramagnetic iron center affects the nearby nuclei, resulting in large isotropic shifts.

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 distribution of rotamer probability about a dihedral angle was studied by Continuous Probability Distribution (CUPID). An equation was presented involving vicinal nuclear spin-spin coupling constants and dihedral angles. The rotationally averaged nuclear Overhauser enhancement was expressed as anintegral which can be solved by expanding the exponential terms in a Fourier series. Angular probability distribution, linear regression and Gaussian curves were some of the techniques used in CUPID.

The Continuous Probability Distribution (CUPID) method using NMR spin-spin coupling constants and nuclear Overhauser enhancement data was applied to analyze the rotamer populations and angles about the Calpha-Cbeta bond in the anion and cation of L-leucine based from six coupling constants. The assumed initial distributions and CUPID calculated distributions of fast internal rotation and reconstruction for slow internal motions were presented.