The Critical Role of N- and C-Terminal Contact in Protein Stability and Folding of a Family 10 Xylanase under Extreme Conditions

Background: Stabilization strategies adopted by proteins under extreme conditions are very complex and involve various kinds of interactions. Recent studies have shown that a large proportion of proteins have their N- and C-terminal elements in close contact and suggested they play a role in protein folding and stability. However, the biological significance of this contact remains elusive. Methodology: In the present study, we investigate the role of N- and C-terminal residue interaction using a family 10 xylanase (BSX) with a TIM-barrel structure that shows stability under high temperature,alkali pH, and protease and SDS treatment. Based on crystal structure,an aromatic cluster was identified that involves Phe4, Trp6 and Tyr343 holding the Nand C-terminus together; this is a unique and important feature of this protein that might be crucial for folding and stabilityunder poly-extreme conditions. Conclusion: A series of mutants was created to disrupt this aromatic cluster formation and study the loss of stability and function under given conditions. While the deletions of Phe4 resulted in loss of stability, removal of Trp6 and Tyr343 affected in vivo folding and activity. Alanine substitution with Phe4, Trp6 and Tyr343 drastically decreased stability under all parameters studied. Importantly,substitution of Phe4 with Trp increased stability in SDS treatment.Mass spectrometry results of limited proteolysis further demonstrated that the Arg344 residue is highly susceptible to trypsin digestion in sensitive mutants such as DF4, W6A and Y343A, suggesting again that disruption of the Phe4-Trp6-Tyr343 (F-W-Y) cluster destabilizes the N-and C-terminal interaction. Our results underscore the importance of N- and C-terminal contact through aromatic interactions in protein folding and stability under extreme conditions, and these results may be useful to improve the stability of other proteins under suboptimal conditions.

A binderless, bulk modified, renewable surface amperometric sensor for NADH and ethanol

Graphite particles are exfoliated and subsequently functionalized with toluidine blue. The resulting covalently modified graphite particles are restacked without any binder to form a surface-renewable, bulk-modified electrode. Electrocatalytic oxidation of NADH and its application in the amperometric biosensing of ethanol using alcohol dehydrogenase enzyme have been demonstrated with this material.

A modified least squares algorithm to treat associations in liquid alloys

The associated model for binary systems has been modified to include volume effects and excess entropy arising from preferential interactions between the associate and the free atoms or between the free atoms. Equations for thermodynamic mixing functions have been derived. An optimization procedure using a modified conjugate gradient method has been used to evaluate the enthalpy and entropy interaction energies, the free energy of dissociation of the complex, its temperature dependance and the size of the associate. An expression for the concentration—concentration structure factor [Scc (0)] has been deduced from the modified associated solution model. The analysis has been applied to the thermodynamic mixing functions of liquid Ga-Te alloys at 1120 K, believed to contain Ga2Te3 associates. It is observed that the modified associated solution model incorporating volume effects and terms for the temperature dependance of interaction energies, describes the thermodynamic properties of Ga-Te system satisfactorily.

Quantitation of serological cross-reactivity between two geographical isolates of Oryctes baculovirus by a modified ELISA

An assay was developed for quantitation of the antigenic relationship between viruses, by modification of the indirect ELISA. The principle of this method is to estimate the epitopes not shared between the related viruses, after titration of the antibodies specific to the common epitopes as in a blocking ELISA. In practice, varying concentrations of purified virus are preincubated with a fixed dilution of heterologous or homologous antiserum and the unbound antibodies present in the mixture are back titrated with virus particles bound to microtitre plates. The antigenic relationship is described in terms of differentiation index (DI) and total antigenic reactivity (TAR). This method has been used to quantitate cross-reactivity between two geographically different isolates of Oryctes baculovirus.

Bi2−xPbx(Ca, Sr)n+1CunO2n+4+δ (n = 1, 2, 3, and 4) family of superconductors

Superconducting oxides of the Bi1.5Pb0.5(Ca, Sr)n+1CunO2n+4+δ series with n = 1, 2, 3 and 4 have been characterized. The superconducting transition temperature increases markedly with n up to n = 3, but the Tc of the n = 4 member is not much higher than that of the n = 3 member. The Tc does not change significantly in Bi2−xPbxCaSr2Cu2O8+δ with x (0.1 < x ≤ 0.5).

Modified linear prediction method for directions of arrival estimation of narrow-band plane waves

A modified linear prediction (MLP) method is proposed in which the reference sensor is optimally located on the extended line of the array. The criterion of optimality is the minimization of the prediction error power, where the prediction error is defined as the difference between the reference sensor and the weighted array outputs. It is shown that the L2-norm of the least-squares array weights attains a minimum value for the optimum spacing of the reference sensor, subject to some soft constraint on signal-to-noise ratio (SNR). How this minimum norm property can be used for finding the optimum spacing of the reference sensor is described. The performance of the MLP method is studied and compared with that of the linear prediction (LP) method using resolution, detection bias, and variance as the performance measures. The study reveals that the MLP method performs much better than the LP technique.

An improved representation of vehicle incompatibility in frontal NCAP tests using a modified rigid barrier

With the objective of better understanding the significance of New Car Assessment Program (NCAP) tests conducted by the National Highway Traffic Safety Administration (NHTSA), head-on collisions between two identical cars of different sizes and between cars and a pickup truck are studied in the present paper using LS-DYNA models. Available finite element models of a compact car (Dodge Neon), midsize car (Dodge Intrepid), and pickup truck (Chevrolet C1500) are first improved and validated by comparing theanalysis-based vehicle deceleration pulses against corresponding NCAP crash test histories reported by NHTSA. In confirmation of prevalent perception, simulation-bascd results indicate that an NCAP test against a rigid barrier is a good representation of a collision between two similar cars approaching each other at a speed of 56.3 kmph (35 mph) both in terms of peak deceleration and intrusions. However, analyses carried out for collisions between two incompatible vehicles, such as an Intrepid or Neon against a C1500, point to the inability of the NCAP tests in representing the substantially higher intrusions in the front upper regions experienced by the cars, although peak decelerations in cars arc comparable to those observed in NCAP tests. In an attempt to improve the capability of a front NCAP test to better represent real-world crashes between incompatible vehicles, i.e., ones with contrasting ride height and lower body stiffness, two modified rigid barriers are studied. One of these barriers, which is of stepped geometry with a curved front face, leads to significantly improved correlation of intrusions in the upper regions of cars with respect to those yielded in the simulation of collisions between incompatible vehicles, together with the yielding of similar vehicle peak decelerations obtained in NCAP tests.

Computing a matrix symmetrizer exactly using modified multiple modulus residue arithmetic

A symmetric solution X satisfying the matrix equation XA = AtX is called a symmetrizer of the matrix A. A general algorithm to compute a matrix symmetrizer is obtained. A new multiple-modulus residue arithmetic called floating-point modular arithmetic is described and implemented on the algorithm to compute an error-free matrix symmetrizer.

High temperature deformation processing maps for boron modified Ti-6Al-4V alloys

The alloy, Ti-6Al-4V is an alpha + beta Ti alloy that has large prior beta grain size (similar to 2 mm) in the as cast state. Minor addition of B (about 0.1 wt.%) to it refines the grain size significantly as well as produces in-situ TiB needles. The role played by these microstructural modifications on high temperature deformation processing maps of B-modified Ti64 alloys is examined in this paper.Power dissipation efficiency and instability maps have been generated within the temperature range of 750-1000 degrees C and strain rate range of 10(-3)-10(+1) s(-1). Various deformation mechanisms, which operate in different temperature-strain rate regimes, were identified with the aid of the maps and complementary microstructural analysis of the deformed specimens. Results indicate four distinct deformation domains within the range of experimental conditions examined, with the combination of 900-1000 degrees C and 10(-3)-10(-2) s(-1) being the optimum for hot working. In that zone, dynamic globularization of alpha laths is the principle deformation mechanism. The marked reduction in the prior beta grain size, achieved with the addition of B, does not appear to alter this domain markedly. The other domains, with negative values of instability parameter, show undesirable microstructural features such as extensive kinking/bending of alpha laths and breaking of beta laths for Ti64-0.0B as well as generation of voids and cracks in the matrix and TiB needles in the B-modified alloys. (C) 2010 Elsevier B.V. All rights reserved.

Modified governing equations for gas-particle nozzle flows

A modified set of governing equations for gas-particle flows in nozzles is suggested to include the inertial forces acting on the particle phase. The problem of gas-particle flow through a nozzle is solved using a first order finite difference scheme. A suitable stability condition for the numerical scheme for gas-particle flows is defined. Results obtained from the present set of equations are compared with those of the previous set of equations. It is also found that present set of equations give results which are in good agreement with the experimental observation.

Super-plasticized natural rubber latex modified concretes

Polymeric admixtures to concrete ingredients modify the properties of the processed concrete. Ductility is one such property modification. This investigation deals with the development of a method of incorporating natural rubber latex into concrete ingredients with only marginal effects on the compressive strength of base plain concrete. This retention of the strength has been effected by reducing the water/cement ratio with the aid of a superplasticizer. The quantity of natural rubber latex is expressed as the dry rubber content by percentage of volume of concrete. The compressive and tensile strengths, as well as post peak ductile behaviour have been the basis for comparison with those of unmodified concrete.

Syntheses and structural studies of new bicyclic 1,3-diphenyl-1,3,2.lambda.3,4.lambda.3-diazadiphosphetidines

Reactions of cis-[(C6H5N)PC1]z(1 ) with the difunctional reagents HO(CH2)20H,H (CH3)N(CHz)zN(CH3)HH, (CH3)N(CH& OH, and HO(CHz)30Hi n the presence of triethylamine yield the new bicyclic 1,3,2X3,4h3-diazadiphosphetidines[( C6H5- N)PIZ[-O(CHZ)Zo-l (2), [(C6H5N)PlZ[-(CH3)N(CHZ)ZN(CH3)-l (319 [(C6H~N)PlZ~-(CH3)N(cHZ)20 (4), and [(C6H5 N)P],[-Q(CH2),0-] (5), respectively. The products have been characterized by elemental analyses and IR and NMR spectroscopic data. The structures of 4 and 5 have been determined by single-crystal X-ray analysis. Crystal data for 4: monoclinic, P2,/c, a = 9.823 (2) A, b = 8.608 (1) A, c = 18.423 (3) A, i3 = 90.55 (1)O, Z = 4. Crystal data for 5 monoclinic, P2,/c, a = 9.727 (2) A, b = 8.064 (2) A, c = 19.702 (4) A, @ =I 91.31 (l)', 2 = 4. The structures have been solved by direct methods and refined to R = 0.028 for 4 and R = 0.050 for 5. Compound 4 is the first example of an aminoalkoxy-l,3,2X3,4X3-diazadiphosphetidine. The PzNz ring is slightly puckered in both 4 and 5 and the puckering occurs in a manner opposite to that observed for cis-[(RN)PX],structures.