A fluorescence anisotropy study of tetramer-dimer equilibrium of λ repressor and its implication for function

Tetramer-dimer equilibrium of λ repressor has been studied by fluorescence anisotropy techniques. We have chosen 1-dimethylamino naphthalene-5-sulfonyl chloride (dansyl chloride)-labeled repressor to study the dissociation-association equilibrium, because of relatively long life-time of the probe (> 10 ns). Polarization of the dansyl-labeled repressor decreases with decreasing protein concentrations in the range of 20 to 0.2 μM. The decrease of anisotropy was shown to be due to reversible dissociation of the protein. Size exclusion high-performance liquid chromatography studies and polyacrylamide gel electrophoresis under native conditions (Ferguson plot) confirmed that at around 20 μM concentrations the repressor exists in predominantly tetrameric form, whereas in lower concentrations it exists in predominantly dimer form. A dissociation constant of 2.3 ± 0.9 μM was estimated in 0.1 M potassium phosphate, pH 8.0, at 25° C. A stoichiometric amount of isolated single operator shifted the tetramer-dimer equilibrium toward the dimer. Increased ionic strength had only a modest effect on the dissociation constant. The thermodynamic constants for the dissociation reaction calculated from the Van't Hoff plot was +26.6 kcal/mol for ΔH and +64.7 e.u. for ΔS. The rotational correlation times derived from isothermal Perrin plot indicated elongated dimers and tetramers

Effect of Cultivars and Season on Grafting Success in Sapota under Paschim Midnapur Conditions of West Bengal

Two sets of experiments were carried out during 2007-08 to assess incompatibility of sapota cultivars to softwood grafting, and to find out the best time for softwood grafting, in a private orchard at Jhargram of Paschim Midnapore, West Bengal. Considerable variation in success of softwood grafting among sapota cultivars was observed. Among ten cultivars studied, CO-2 showed highest compatibility with Khirnee rootstock to softwood grafting, followed by Cricket Ball and DSH-2. There was a total failure in graft-take in cultivars CO-1, DSH-1 and Guthi. Softwood grafting success was highest in sapota when carried out on 1stJuly (72%) followed by 15th August (70%), 5th June (62%) and 15th June (56%)

Theory of Adiabatic fluctuations : third-order noise

We consider the response of a dynamical system driven by external adiabatic fluctuations. Based on the `adiabatic following approximation' we have made a systematic separation of time-scales to carry out an expansion in $\alpha |\mu|^{-1}$, where $\alpha$ is the strength of fluctuations and $|\mu|$ is the damping rate. We show that probability distribution functions obey the differential equations of motion which contain third order terms (beyond the usual Fokker-Planck terms) leading to non-Gaussian noise. The problem of adiabatic fluctuations in velocity space which is the counterpart of Brownian motion for fast fluctuations, has been solved exactly. The characteristic function and the associated probability distribution function are shown to be of stable form. The linear dissipation leads to a steady state which is stable and the variances and higher moments are shown to be finite.Comment: Plain Latex, no figures, 28 pages; to appear in J. Phys.

An operator-induced conformational change in the C-terminal domain of the λ repressor

4,4'-bis(1-anilino-8-naphthalenesulfonic acid (Bis-ANS), an environment-sensitive fluorescent probe for hydrophobic region of proteins, binds specifically to the C-terminal domain of λ repressor. The binding is characterized by positive cooperativity, the magnitude of which is dependent on protein concentration in the concentration range where dimeric repressor aggregates to a tetramer. In this range, positive cooperativity becomes more pronounced at higher protein concentrations. This suggests a preferential binding of Bis-ANS to the dimeric form of the repressor. Binding of single operator OR1 to the N-terminal domain of the repressor causes enhancement of fluorescence of the C-terminal domain bound Bis-ANS. The binding of single operator OR1 also leads to quenching of fluorescence of tryptophan residues, all of which are located in the hinge or the C-terminal domain. Thus two different fluorescent probes indicate an operator-induced conformational change which affects the C-terminal domain. The significance of this conformational change with respect to the function of λ repressor has been discussed

Lamb Wave Modes in Coal-Tar-Coated Steel Plates

In order to study the feasibility of using ultrasonic Lamb wave modes for detection and sizing of corrosion-related flaws in buried steel pipelines, we have calculated Lamb wave modes and performed numerous experiments on steel plates coated on one side with coal-tar enamel. The purpose of this paper is to discuss these theoretical and experimental results

Generalization of escape rate from a metastable state driven by external cross-correlated noise processes

We propose generalization of escape rate from a metastable state for externally driven correlated noise processes in one dimension. In addition to the internal non-Markovian thermal fluctuations, the external correlated noise processes we consider are Gaussian, stationary in nature and are of Ornstein-Uhlenbeck type. Based on a Fokker-Planck description of the effective noise processes with finite memory we derive the generalized escape rate from a metastable state in the moderate to large damping limit and investigate the effect of degree of correlation on the resulting rate. Comparison of the theoretical expression with numerical simulation gives a satisfactory agreement and shows that by increasing the degree of external noise correlation one can enhance the escape rate through the dressed effective noise strength.Comment: 9 pages, 1 figur

Linear systems with adiabatic fluctuations

We consider a dynamical system subjected to weak but adiabatically slow fluctuations of external origin. Based on the ``adiabatic following'' approximation we carry out an expansion in \alpha/|\mu|, where \alpha is the strength of fluctuations and 1/|\mu| refers to the time scale of evolution of the unperturbed system to obtain a linear differential equation for the average solution. The theory is applied to the problems of a damped harmonic oscillator and diffusion in a turbulent fluid. The result is the realization of `renormalized' diffusion constant or damping constant for the respective problems. The applicability of the method has been critically analyzed.Comment: Plain Latex, no figure, 21 page

Master equation approach to DNA-breathing in heteropolymer DNA

After crossing an initial barrier to break the first base-pair (bp) in double-stranded DNA, the disruption of further bps is characterized by free energies between less than one to a few kT. This causes the opening of intermittent single-stranded bubbles. Their unzipping and zipping dynamics can be monitored by single molecule fluorescence or NMR methods. We here establish a dynamic description of this DNA-breathing in a heteropolymer DNA in terms of a master equation that governs the time evolution of the joint probability distribution for the bubble size and position along the sequence. The transfer coefficients are based on the Poland-Scheraga free energy model. We derive the autocorrelation function for the bubble dynamics and the associated relaxation time spectrum. In particular, we show how one can obtain the probability densities of individual bubble lifetimes and of the waiting times between successive bubble events from the master equation. A comparison to results of a stochastic Gillespie simulation shows excellent agreement.Comment: 12 pages, 8 figure