Chimeras of Escherichia coli and Mycobacterium tuberculosis Single-Stranded DNA Binding Proteins: Characterization and Function in Escherichia coli

Single stranded DNA binding proteins (SSBs) are vital for the survival of organisms. Studies on SSBs from the prototype, Escherichia coli (EcoSSB) and, an important human pathogen, Mycobacterium tuberculosis (MtuSSB) had shown that despite significant variations in their quaternary structures, the DNA binding and oligomerization properties of the two are similar. Here, we used the X-ray crystal structure data of the two SSBs to design a series of chimeric proteins (m beta 1, m beta 1'beta 2, m beta 1-beta 5, m beta 1-beta 6 and m beta 4-beta 5) by transplanting beta 1, beta 1'beta 2, beta 1-beta 5, beta 1-beta 6 and beta 4-beta 5 regions, respectively of the N-terminal (DNA binding) domain of MtuSSB for the corresponding sequences in EcoSSB. In addition, m beta 1'beta 2(ESWR) SSB was generated by mutating the MtuSSB specific `PRIY' sequence in the beta 2 strand of m beta 1'beta 2 SSB to EcoSSB specific `ESWR' sequence. Biochemical characterization revealed that except for m beta 1 SSB, all chimeras and a control construct lacking the C-terminal domain (Delta C SSB) bound DNA in modes corresponding to limited and unlimited modes of binding. However, the DNA on MtuSSB may follow a different path than the EcoSSB. Structural probing by protease digestion revealed that unlike other SSBs used, m beta 1 SSB was also hypersensitive to chymotrypsin treatment. Further, to check for their biological activities, we developed a sensitive assay, and observed that m beta 1-beta 6, MtuSSB, m beta 1'beta 2 and m beta 1-beta 5 SSBs complemented E. coli Delta ssb in a dose dependent manner. Complementation by the m beta 1-beta 5 SSB was poor. In contrast, m beta 1'beta 2(ESWR) SSB complemented E. coli as well as EcoSSB. The inefficiently functioning SSBs resulted in an elongated cell/filamentation phenotype of E. coli. Taken together, our observations suggest that specific interactions within the DNA binding domain of the homotetrameric SSBs are crucial for their biological function.

Distributed Function Computation over Fields and Rings via Linear Compression of Sources

The setting considered in this paper is one of distributed function computation. More specifically, there is a collection of N sources possessing correlated information and a destination that would like to acquire a specific linear combination of the N sources. We address both the case when the common alphabet of the sources is a finite field and the case when it is a finite, commutative principal ideal ring with identity. The goal is to minimize the total amount of information needed to be transmitted by the N sources while enabling reliable recovery at the destination of the linear combination sought. One means of achieving this goal is for each of the sources to compress all the information it possesses and transmit this to the receiver. The Slepian-Wolf theorem of information theory governs the minimum rate at which each source must transmit while enabling all data to be reliably recovered at the receiver. However, recovering all the data at the destination is often wasteful of resources since the destination is only interested in computing a specific linear combination. An alternative explored here is one in which each source is compressed using a common linear mapping and then transmitted to the destination which then proceeds to use linearity to directly recover the needed linear combination. The article is part review and presents in part, new results. The portion of the paper that deals with finite fields is previously known material, while that dealing with rings is mostly new.Attempting to find the best linear map that will enable function computation forces us to consider the linear compression of source. While in the finite field case, it is known that a source can be linearly compressed down to its entropy, it turns out that the same does not hold in the case of rings. An explanation for this curious interplay between algebra and information theory is also provided in this paper.

Abstract Characteristic Function

The characteristic function for a contraction is a classical complete unitary invariant devised by Sz.-Nagy and Foias. Just as a contraction is related to the Szego kernel k(S)(z, w) = ( 1 - z(w)over bar)- 1 for |z|, |w| < 1, by means of (1/k(S))( T, T *) = 0, we consider an arbitrary open connected domain Omega in C(n), a kernel k on Omega so that 1/k is a polynomial and a tuple T = (T(1), T(2), ... , T(n)) of commuting bounded operators on a complex separable Hilbert spaceHsuch that (1/k)( T, T *) >= 0. Under some standard assumptions on k, it turns out that whether a characteristic function can be associated with T or not depends not only on T, but also on the kernel k. We give a necessary and sufficient condition. When this condition is satisfied, a functional model can be constructed. Moreover, the characteristic function then is a complete unitary invariant for a suitable class of tuples T.

Structure-function relationship in serine hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT), a pyridoxal-5V-phosphate (PLP)-dependent enzyme catalyzes thetetrahydrofolate (H4-folate)- dependent retro-aldol cleavage of serine to form 5,10-methylene H4-folate and glycine. The structure–function relationship of SHMT wasstudied in our laboratory initially by mutation of residues that are conserved in all SHMTs and later by structure-based mutagenesis of residues located in the active site. The analysis of mutants showed that K71, Y72, R80, D89, W110, S202, C203, H304, H306 and H356 residues are involved in maintenance of the oligomeric structure. The mutation of D227, a residue involved in charge relay system, led to the formation of inactive dimers, indicating that this residue has a role in maintaining the tetrameric structure and catalysis. E74, a residue appropriately positioned in the structure of the enzyme to carry out proton abstraction, was shown by characterization of E74Q and E74K mutants to be involved in conversion of the enzyme from an ‘open’ to ‘closed’ conformation rather than proton abstraction from the hydroxylgroup of serine. K256, the residue involved in the formation of Schiffs base with PLP, also plays a crucial role in the maintenance of the tetrameric structure. Mutation of R262 residue established the importance of distal interactions in facilitating catalysis and Y82 is not involved in the formaldehyde transfer via the postulated hemiacetal intermediate but plays a role in stabilizing the quinonoid intermediate.The mutational analysis of scSHMT along with the structure of recombinant Bacillus stearothermophilus SHMT and its substrate(s)complexes was used to provide evidence for a direct transfer mechanism rather than retro-aldol cleavage for the reaction catalyzed by SHMT.

From anomalies in neat liquid to structure, dynamics and function in the biological world

Water brings its remarkable thermodynamic and dynamic anomalies in the pure liquid state to biological world where water molecules face a multitude of additional interactions that frustrate its hydrogen bond network. Yet the water molecules participate and control enormous number of biological processes in manners which are yet to be understood at a molecular level. We discuss thermodynamics, structure, dynamics and properties of water around proteins and DNA, along with those in reverse micelles. We discuss the roles of water in enzyme kinetics, in drug-DNA intercalation and in kinetic-proof reading ( the theory of lack of errors in biosynthesis). We also discuss how water may play an important role in the natural selection of biomolecules. (C) 2011 Elsevier B. V. All rights reserved.

Learning polyhedral classifiers using logistic function

In this paper we propose a new algorithm for learning polyhedral classifiers. In contrast to existing methods for learning polyhedral classifier which solve a constrained optimization problem, our method solves an unconstrained optimization problem. Our method is based on a logistic function based model for the posterior probability function. We propose an alternating optimization algorithm, namely, SPLA1 (Single Polyhedral Learning Algorithm1) which maximizes the loglikelihood of the training data to learn the parameters. We also extend our method to make it independent of any user specified parameter (e.g., number of hyperplanes required to form a polyhedral set) in SPLA2. We show the effectiveness of our approach with experiments on various synthetic and real world datasets and compare our approach with a standard decision tree method (OC1) and a constrained optimization based method for learning polyhedral sets.

Construction of equivalent circuit of a transformer winding from driving-point impedance function - analytical approach

Analytical solution is presented to convert a given driving-point impedance function (in s-domain) into a physically realisable ladder network with inductive coupling between any two sections and losses considered. The number of sections in the ladder network can vary, but its topology is assumed fixed. A study of the coefficients of the numerator and denominator polynomials of the driving-point impedance function of the ladder network, for increasing number of sections, led to the identification of certain coefficients, which exhibit very special properties. Generalised expressions for these specific coefficients have also been derived. Exploiting their properties, it is demonstrated that the synthesis method essentially turns out to be an exercise of solving a set of linear, simultaneous, algebraic equations, whose solution directly yields the ladder network elements. The proposed solution is novel, simple and guarantees a unique network. Presently, the formulation can synthesise a unique ladder network up to six sections.

Insights into differential modulation of receptor function by hinge region using novel agonistic lutropin receptor and inverse agonistic thyrotropin receptor antibodies

We report two antibodies, scFv 13B1 and MAb PD1.37, against the hinge regions of LHR and TSHR, respectively, which have similar epitopes but different effects on receptor function. While neither of them affected hormone binding, with marginal effects on hormone response, scFv 13B1 stimulated LHR in a dose-dependent manner, whereas MAb PD1.37 acted as an inverse agonist of TSHR. Moreover, PD1.37 could decrease the basal activity of hinge region CAMs, but had varied effects on those present in ECLs, whereas 13B1 was refractory to any CAMs in LHR. Using truncation mutants and peptide phage display, we compared the differential roles of the hinge region cysteine box-2/3 as well as the exoloops in the activation of these two homologus receptors. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Partition function for hindered, one-and multi-dimensional rotors

Writing the hindered rotor (hr) partition function as the trace of (rho) over cap = e(-beta(H) over cap hr), we approximate it by the sum of contributions from a set of points in position space. The contribution of the density matrix from each point is approximated by performing a local harmonic expansion around it. The highlight of this method is that it can be easily extended to multidimensional systems. Local harmonic expansion leads to a breakdown of the method a low temperatures. In order to calculate the partition function at low temperatures, we suggest a matrix multiplication procedure. The results obtained using these methods closely agree with the exact partition function at all temperature ranges. Our method bypasses the evaluation of eigenvalues and eigenfunctions and evaluates the density matrix for internal rotation directly. We also suggest a procedure to account for the antisymmetry of the total wavefunction in the same. (C) 2012 Elsevier B.V. All rights reserved.

An Online Actor-Critic Algorithm with Function Approximation for Constrained Markov Decision Processes

We develop an online actor-critic reinforcement learning algorithm with function approximation for a problem of control under inequality constraints. We consider the long-run average cost Markov decision process (MDP) framework in which both the objective and the constraint functions are suitable policy-dependent long-run averages of certain sample path functions. The Lagrange multiplier method is used to handle the inequality constraints. We prove the asymptotic almost sure convergence of our algorithm to a locally optimal solution. We also provide the results of numerical experiments on a problem of routing in a multi-stage queueing network with constraints on long-run average queue lengths. We observe that our algorithm exhibits good performance on this setting and converges to a feasible point.

The force distribution function of an oscillator model of polymer stretching at constant velocity

Recent simulations of the stretching of tethered biopolymers at a constant speed v (Ponmurugan and Vemparala, 2011 Phys. Rev. E 84 060101(R)) have suggested that for any time t, the distribution of the fluctuating forces f responsible for chain deformation is governed by a relation of the form P(+ f)/ P(- f) = expgamma f], gamma being a coefficient that is solely a function of v and the temperature T. This result, which is reminiscent of the fluctuation theorems applicable to stochastic trajectories involving thermodynamic variables, is derived in this paper from an analytical calculation based on a generalization of Mazonka and Jarzynski's classic model of dragged particle dynamics Mazonka and Jarzynski, 1999 arXiv:cond-\textbackslashmat/9912121v1]. However, the analytical calculations suggest that the result holds only if t >> 1 and the force fluctuations are driven by white rather than colored noise; they further suggest that the coefficient gamma in the purported theorem varies not as v(0.15)T-(0.7), as indicated by the simulations, but as vT(-1).

MS_RHII-RSD, a Dual-Function RNase HII-(p)ppGpp Synthetase from Mycobacterium smegmatis

In the noninfectious soil saprophyte Mycobacterium smegmatis, intracellular levels of the stress alarmones guanosine tetraphosphate and guanosine pentaphosphate, together termed (p)ppGpp, are regulated by the enzyme Rel(Msm). This enzyme consists of a single, bifunctional polypeptide chain that is capable of both synthesizing and hydrolyzing (p)ppGpp. The rel(Msm), knockout strain of M. smegmatis (Delta rel(Msm)) is expected to show a (p)ppGpp null (p)ppGpp(0)] phenotype. Contrary to this expectation, the strain is capable of synthesizing (p)ppGpp in vivo. In this study, we identify and functionally characterize the open reading frame (ORF), MSMEG_5849, that encodes a second functional (p)ppGpp synthetase in M. smegmatis. In addition to (p)ppGpp synthesis, the 567-amino-acid-long protein encoded by this gene is capable of hydrolyzing RNA(.)DNA hybrids and bears similarity to the conventional RNase HII enzymes. We have classified this protein as actRel(Msm) in accordance with the recent nomenclature proposed and have named it MS_RHII-RSD, indicating the two enzymatic activities present RHII, RNase HII domain, originally identified as (d) under bar omain of (u) under bar nknown (f) under bar unction 429 (DUF429), and RSD, RelA_SpoT nucleotidyl transferase domain, the SYNTH domain responsible for (p)ppGpp synthesis activity]. MS_RHII-RSD is expressed and is constitutively active in vivo and behaves like a monofunctional (p)ppGpp synthetase in vitro. The occurrence of the RNase HII and (p)ppGpp synthetase domains together on the same polypeptide chain is suggestive of an in vivo role for this novel protein as a link connecting the essential life processes of DNA replication, repair, and transcription to the highly conserved stress survival pathway, the stringent response.

PocketAnnotate: towards site-based function annotation

A computational pipeline PocketAnnotate for functional annotation of proteins at the level of binding sites has been proposed in this study. The pipeline integrates three in-house algorithms for site-based function annotation: PocketDepth, for prediction of binding sites in protein structures; PocketMatch, for rapid comparison of binding sites and PocketAlign, to obtain detailed alignment between pair of binding sites. A novel scheme has been developed to rapidly generate a database of non-redundant binding sites. For a given input protein structure, putative ligand-binding sites are identified, matched in real time against the database and the query substructure aligned with the promising hits, to obtain a set of possible ligands that the given protein could bind to. The input can be either whole protein structures or merely the substructures corresponding to possible binding sites. Structure-based function annotation at the level of binding sites thus achieved could prove very useful for cases where no obvious functional inference can be obtained based purely on sequence or fold-level analyses. An attempt has also been made to analyse proteins of no known function from Protein Data Bank. PocketAnnotate would be a valuable tool for the scientific community and contribute towards structure-based functional inference. The web server can be freely accessed at http://proline.biochem.iisc.ernet.in/pocketannotate/.