Christopher J. Halkides

The frozen solution structure of p21 ras determined by ESEEM spectroscopy reveals weak coordination of Thr35 to the active site metal ion

BACKGROUND The G protein p21 ras is a molecular switch in the signal transduction pathway for cellular growth and differentiation. Hydrolysis of tightly bound GTP alters the conformation of p21, terminating the signal. The coordination of the p21 residue Thr35 to Mg2+ in its active site, which has been observed in the crystal structure of p21 in complex with a GTP-analog GMPPNP but not with GDP, has been proposed to drive the conformational change accompanying nucleotide substitution and may have a role in the GTP hydrolysis reaction itself. However, previous electron spin-echo envelope modulation (ESEEM) studies of selectively 2H beta-threonine and 15N-threonine labeled p21.Mn2+ GMPPNP suggest that Thr35 only weakly coordinates the metal ion in the growth-active GTP-bound state of p21. RESULTS A 13C beta-Thr35 to Mn2+ distance of 4.3 +/- 0.2 A and a 15N epsilon-Lys16 to Mn2+ distance of 5.3 +/- 0.2 A were determined from ESEEM spectra of the selectively 13C beta-Thr and 15N epsilon-Lys labeled p21.Mn2+ GMPPNP frozen solution structure. The 13C beta-Thr35 to Mn2+ distance is greater than that (3.16 A) observed in the crystal structure. In contrast, the 15N epsilon-Lys16 to Mn2+ distance is in good agreement with the 5.1 A crystal structure distance. CONCLUSIONS The 13C beta of Thr35 is more distant from the active site Mn2+ in the frozen solution structure than in the crystal structure of p21.Mg2+ GMPPNP, indicating that Thr35 only weakly coordinates the metal ion in frozen solution. Thr35 coordination of the metal ion is therefore unlikely to drive the conformational change between GTP- and GDP-bound states of p21. Thr35 may be essential for GTPase-activating protein (GAP)-stimulated GTP hydrolysis and/or signal transduction for other reasons.

A Mixed Quantum-Classical Approach for Computing Effects of Intramolecular Motion on the Low-Barrier Hydrogen Bond

The fractionation factor is defined as the equilibrium constant for the reaction: R − H + DOH ↔ R − D + HOH. Of interest are values of fractionation factors for reactions where reactants and/or products form intramolecular low-barrier hydrogen bonds. Experimentally measured isotopic fractionation factors are usually interpreted via a one-dimensional potential energy surface along the intrinsic proton hydrogen bond coordinate. Such a one-dimensional picture cannot be completely correct. Intramolecular motions, such as vibrations and librations, can modulate the underlying potential energy surface along the hydrogen bond coordinate and thus affect the isotopic fractionation factor. We have recently generated a picture of the motion of the proton in a low-barrier hydrogen bond as taking place in an effective single-dimensional potential, which we term the potential of mean force (PMF). In this paper, we compute the PMF for a molecule with an intramolecular hydrogen bond in order to quantify the effect of intramolecular motions on the fractionation factor. The PMF and isotopic fractionation factor are computed with a combination of high-level density functional theory and molecular dynamics simulations.

The structures of T87I phosphono-CheY and T87I/Y106W phosphono-CheY help to explain their binding affinities to the FliM and CheZ peptides

CheY is a response regulator in bacterial chemotaxis. Escherichia coli CheY mutants T87I and T87I/Y106W CheY are phosphorylatable on Asp57 but unable to generate clockwise rotation of the flagella. To understand this phenotype in terms of structure, stable analogs of the two CheY-P mutants were synthesized: T87I phosphono-CheY and T87I phosphono-CheY. Dissociation constants for peptides derived from flagellar motor protein FliM and phosphatase CheZ were determined for phosphono-CheY and the two mutants. The peptides bind phosphono-CheY almost as strongly as CheY-P; however, they do not bind T87I phosphono-CheY or T87I/Y106W phosphono-CheY, implying that the mutant proteins cannot bind FliM or CheZ tightly in vivo. The structures of T87I phosphono-CheY and T87I/Y106W phosphono-CheY were solved to resolutions of 1.8 and 2.4A, respectively. The increased bulk of I87 forces the side-chain of Y106 or W106, into a more solvent-accessible conformation, which occludes the peptide-binding site.

Synthesis of a Stable Analog of the Phosphorylated Form of CheY: Phosphono-CheY

The chemical modification of a cysteinyl residue of D57C CheY by the addition of a phosphonomethyl group, (HO)(2)P(O)-CH(2)-, is described. This modification produces a nonlabile analog of an aspartyl phosphate residue in the active form of CheY. The chemically modified protein, phosphono-CheY, is suitable for structural and functional studies. An extensive discussion of the synthetic methodology and purification strategy is presented. A detailed protocol is given.

Synthesis of Benzyl Diisopropyl 5-Phosphonopentanoate and 5-Phosphonopentanoic Acid: An Analog of Succinyl Phosphate

Benzyl 5-(diisopropoxyphosphoryl)pentanoate (benzyl diisopropyl 5-phosphonopentanoate) was synthesized from 5-bromopentanoic acid in three steps. The first step esterified 5-bromopentanoic acid with benzyl alcohol, the second step was a Finkelstein halogen exchange with sodium iodide, and the third step was a Michaelis–Arbuzov reaction. This compound was characterized by NMR and high-resolution mass spectrometry. This compound was hydrolyzed to produce 5-phosphonopentanoic acid, which is an analog of succinyl phosphate and a possible enzyme inhibitor.

On the Possibility of Detecting Low Barrier Hydrogen Bonds with Kinetic Measurements

Recent experimental evidence has pointed to the possible presence of a short, strong hydrogen bond in the enzyme-substrate transition states in some biochemical reactions. To date, most experimental measures of these short, strong hydrogen bonds have monitored their equilibrium properties. In this work we show that kinetic measurements can also be used to detect the presence of short, strong hydrogen bonds. In particular, we find nontrivial differences among rate constant ratios of protonated to deuterated hydrogen bonds between strong and weak hydrogen bonds for proton transfer between donor-acceptor sites. We quantify this kinetic isotope effect by performing dynamical calculations of these rate constants by computing reactive flux through a dividing surface. This reactive flux is computed by evolving trajectories on an effective quantum mechanical potential energy surface.

Production, characterization, and assessment of a stable analog of the response regulator CheY‐phosphate from Thermotoga maritima

Phosphorylation of CheY promotes association with the flagellar motor and ultimately controls the directional bias of the motor. However, biochemical studies of activated CheY‐phosphate have been challenging due to the rapid hydrolysis of the aspartyl‐phosphate in vitro. An inert analog of Tm CheY‐phosphate, phosphono‐CheY, was synthesized by chemical modification and purified by cation‐exchange chromatography. Changes in HPLC retention times, chemical assays for phosphate and free thiol, and mass spectrometry experiments demonstrate modification of Cys54 with a phosphonomethyl group. Additionally, a crystal structure showed electron density for the phosphonomethyl group at Cys54, consistent with a modification at that position. Subsequent biochemical experiments confirmed that protein crystals were phosphono‐CheY. Isothermal titration calorimetry and fluorescence polarization binding assays demonstrated that phosphono‐CheY bound a peptide derived from FliM, a native partner of CheY‐phosphate, with a dissociation constant of ∼29 µM, at least sixfold more tightly than unmodified CheY. Taken together these results suggest that Tm phosphono‐CheY is a useful and unique analog of Tm CheY‐phosphate.