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

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

Varieties of living things: Life at the intersection of lineage and metabolism

publication-status: Publishedtypes: Articl

Principles of genetic circuit design

Cells navigate environments, communicate and build complex patterns by initiating gene expression in response to specific signals. Engineers seek to harness this capability to program cells to perform tasks or create chemicals and materials that match the complexity seen in nature. This Review describes new tools that aid the construction of genetic circuits. Circuit dynamics can be influenced by the choice of regulators and changed with expression 'tuning knobs'. We collate the failure modes encountered when assembling circuits, quantify their impact on performance and review mitigation efforts. Finally, we discuss the constraints that arise from circuits having to operate within a living cell. Collectively, better tools, well-characterized parts and a comprehensive understanding of how to compose circuits are leading to a breakthrough in the ability to program living cells for advanced applications, from living therapeutics to the atomic manufacturing of functional materials.National Institute of General Medical Sciences (U.S.) (Grant P50 GM098792)National Institute of General Medical Sciences (U.S.) (Grant R01 GM095765)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (EEC0540879)Life Technologies, Inc. (A114510)National Science Foundation (U.S.). Graduate Research FellowshipUnited States. Office of Naval Research. Multidisciplinary University Research Initiative (Grant 4500000552

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