Use of a distant reporter group as evidence for a conformational change in a sensory receptor

A highly sensitive method for demonstrating ligand-induced conformational changes in protein molecules in solution is described. The method utilizes an environmentally sensitive reporter group that is known to be distant from the active site. In the present application a conformational change is demonstrated in the galactose receptor of Salmonella typhimurium, involved in bacterial sensing and transport, by means of an extrinsic fluorophore, 5-iodoacetamidofluorescein, attached at a single methionine residue, and the intrinsic tryptophan fluorophore. Binding of the ligand galactose perturbs the microenvironment of both the fluorescein and tryptophan, as shown by both spectral and potassium iodide quenching changes. The distance between the two dyes is established by fluorescence energy transfer methods to be 41 ± 10 angstrom. Since only one molecule of galactose binds per molecule of receptor and since the galactose molecule is only about 5 angstrom in length, changes at one of these sites reflect the result of an indirect effect. Hence, there must be a ligand-induced conformational change that is propagated a minimum of 30 angstrom through the receptor molecule

Precise Asymptotics for a Random Walker's Maximum

We consider a discrete time random walk in one dimension. At each time step the walker jumps by a random distance, independent from step to step, drawn from an arbitrary symmetric density function. We show that the expected positive maximum E[M_n] of the walk up to n steps behaves asymptotically for large n as, E[M_n]/\sigma=\sqrt{2n/\pi}+ \gamma +O(n^{-1/2}), where \sigma^2 is the variance of the step lengths. While the leading \sqrt{n} behavior is universal and easy to derive, the leading correction term turns out to be a nontrivial constant \gamma. For the special case of uniform distribution over [-1,1], Coffmann et. al. recently computed \gamma=-0.516068...by exactly enumerating a lengthy double series. Here we present a closed exact formula for \gamma valid for arbitrary symmetric distributions. We also demonstrate how \gamma appears in the thermodynamic limit as the leading behavior of the difference variable E[M_n]-E[|x_n|] where x_n is the position of the walker after n steps. An application of these results to the equilibrium thermodynamics of a Rouse polymer chain is pointed out. We also generalize our results to L\'evy walks.Comment: new references added, typos corrected, published versio

Substrate-Assisted Catalysis Unifies Two Families of Chitinolytic Enzymes

Hen egg-white lysozyme has long been the paradigm for enzymatic glycosyl hydrolysis with retention of configuration, with a protonated carboxylic acid and a deprotonated carboxylate participating in general acid-base catalysis. In marked contrast, the retaining chitin degrading enzymes from glycosyl hydrolase families 18 and 20 all have a single glutamic acid as the catalytic acid but lack a nucleophile on the enzyme. Both families have a catalytic (βα)8-barrel domain in common. X-ray structures of three different chitinolytic enzymes complexed with substrates or inhibitors identify a retaining mechanism involving a protein acid and the carbonyl oxygen atom of the substrate’s C2 N-acetyl group as the nucleophile. These studies unambiguously demonstrate the distortion of the sugar ring toward a sofa conformation, long postulated as being close to that of the transition state in glycosyl hydrolysis.

Area distribution and the average shape of a L\'evy bridge

We consider a one dimensional L\'evy bridge x_B of length n and index 0 < \alpha < 2, i.e. a L\'evy random walk constrained to start and end at the origin after n time steps, x_B(0) = x_B(n)=0. We compute the distribution P_B(A,n) of the area A = \sum_{m=1}^n x_B(m) under such a L\'evy bridge and show that, for large n, it has the scaling form P_B(A,n) \sim n^{-1-1/\alpha} F_\alpha(A/n^{1+1/\alpha}), with the asymptotic behavior F_\alpha(Y) \sim Y^{-2(1+\alpha)} for large Y. For \alpha=1, we obtain an explicit expression of F_1(Y) in terms of elementary functions. We also compute the average profile < \tilde x_B (m) > at time m of a L\'evy bridge with fixed area A. For large n and large m and A, one finds the scaling form = n^{1/\alpha} H_\alpha({m}/{n},{A}/{n^{1+1/\alpha}}), where at variance with Brownian bridge, H_\alpha(X,Y) is a non trivial function of the rescaled time m/n and rescaled area Y = A/n^{1+1/\alpha}. Our analytical results are verified by numerical simulations.Comment: 21 pages, 4 Figure

The kinetics and feedback inhibition of cytidine 5′-triphosphate synthetase in wild-type and mutant Chinese hamster cells

The kinetics and cytidine 5′-triphosphate (CTP) feedback inhibition of CTP synthetase in wild-type and four mutants of Chinese hamster V79 cells have been studied. The enzymes of the wild type and three of the four mutants exhibited positive cooperativity with the substrate uridine 5′-triphosphate (UTP). Three of the mutants had K m app and S 50 valuves distinctly greater than those of the wild type, while the fourth mutant had values similar to those of the wild type. all four mutants exhibited resistance to CTP feedback inhibition, while the wild type was sensitive to such inhibition. It is postulated that a single mutational event in each mutant had caused a concomitant change of the enzyme in its binding both to the substrate UTP and to the end-product CTP.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44151/1/10528_2004_Article_BF00485855.pd

On the conservation of the slow conformational dynamics within the amino acid kinase family: NAGK the paradigm

N-Acetyl-L-Glutamate Kinase (NAGK) is the structural paradigm for examining the catalytic mechanisms and dynamics of amino acid kinase family members. Given that the slow conformational dynamics of the NAGK (at the microseconds time scale or slower) may be rate-limiting, it is of importance to assess the mechanisms of the most cooperative modes of motion intrinsically accessible to this enzyme. Here, we present the results from normal mode analysis using an elastic network model representation, which shows that the conformational mechanisms for substrate binding by NAGK strongly correlate with the intrinsic dynamics of the enzyme in the unbound form. We further analyzed the potential mechanisms of allosteric signalling within NAGK using a Markov model for network communication. Comparative analysis of the dynamics of family members strongly suggests that the low-frequency modes of motion and the associated intramolecular couplings that establish signal transduction are highly conserved among family members, in support of the paradigm sequence→structure→dynamics→function © 2010 Marcos et al

Internal stress as a link between macroscale and mesoscale mechanics

The internal (or residual) stress is among the key notions to describe the state of the systems far from equilibrium. Such stress is invisible on the macroscopic scale where the system is regarded as a blackbox. Yet nonequilibrium macroscopic operations allow to create and observe the internal stress. We present in this lecture some examples of the internal stress and its operations. We describe the memory effect in some detail, the process in which the history of past operations is recalled through the relaxation of internal stress.Comment: 11pages, 2 figures, 7 equations: Refereed notes of the Lectures given at "Morphogenesis through the interplay of nonlinear chemical instabilities and elastic active media" July 2-14 2007. to be published from Springer (NATO series

Telomere disruption results in non-random formation of de novo dicentric chromosomes involving acrocentric human chromosomes

Copyright: © 2010 Stimpson et al.Genome rearrangement often produces chromosomes with two centromeres (dicentrics) that are inherently unstable because of bridge formation and breakage during cell division. However, mammalian dicentrics, and particularly those in humans, can be quite stable, usually because one centromere is functionally silenced. Molecular mechanisms of centromere inactivation are poorly understood since there are few systems to experimentally create dicentric human chromosomes. Here, we describe a human cell culture model that enriches for de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. The induced dicentrics vary in structure near fusion breakpoints and like naturally-occurring dicentrics, exhibit various inter-centromeric distances. Many functional dicentrics persist for months after formation. Even those with distantly spaced centromeres remain functionally dicentric for 20 cell generations. Other dicentrics within the population reflect centromere inactivation. In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the alpha-satellite DNA array associated with CENP-A is reduced compared to the same array before dicentric formation. Extrachromosomal fragments that contained CENP-A often appear in the same cells as dicentrics. Some of these fragments are derived from the same alpha-satellite DNA array as inactivated centromeres. Our results indicate that dicentric human chromosomes undergo alternative fates after formation. Many retain two active centromeres and are stable through multiple cell divisions. Others undergo centromere inactivation. This event occurs within a broad temporal window and can involve deletion of chromatin that marks the locus as a site for CENP-A maintenance/replenishment.This work was supported by the Tumorzentrum Heidelberg/Mannheim grant (D.10026941)and by March of Dimes Research Foundation grant #1-FY06-377 and NIH R01 GM069514

Robustness in Glyoxylate Bypass Regulation

The glyoxylate bypass allows Escherichia coli to grow on carbon sources with only two carbons by bypassing the loss of carbons as CO2 in the tricarboxylic acid cycle. The flux toward this bypass is regulated by the phosphorylation of the enzyme isocitrate dehydrogenase (IDH) by a bifunctional kinase–phosphatase called IDHKP. In this system, IDH activity has been found to be remarkably robust with respect to wide variations in the total IDH protein concentration. Here, we examine possible mechanisms to explain this robustness. Explanations in which IDHKP works simultaneously as a first-order kinase and as a zero-order phosphatase with a single IDH binding site are found to be inconsistent with robustness. Instead, we suggest a robust mechanism where both substrates bind the bifunctional enzyme to form a ternary complex