Light-induced geometric isomerization of 1,2-diphenylcyclopropanes included within Y zeolites: Role of cation-guest binding

Through a systematic study of several diphenylcyclopropane derivatives, we have inferred that the cations present within a zeolite control the excited-state chemistry of these systems. In the parent 1,2-diphenylcylopropane, the cation binds to the two phenyl rings in a sandwich-type arrangement, and such a mode of binding prevents cis-to-trans isomerization. Once an ester or amide group is introduced into the system (derivatives of 2beta,3beta-diphenylcyclopropane-1alpha-carboxylic acid), the cation binds to the carbonyl group present in these chromophores and such a binding has no influence on the cis-trans isomerization process. Cation-reactant structures computed at density functional theory level have been very valuable in rationalizing the observed photochemical behavior of diphenylcyclopropane derivatives included in zeolites. While the parent system, 1,2-diphenyleylopropane, has been extensively investigated in the context of chiral induction in solution, owing to its failure to isomerize from cis to trans, the same could not be investigated in zeolites. However, esters of 2beta,3beta-diphenylcyclopropane-1alpha-carboxylic acid could be studied within zeolites in the context of chiral induction. Chiral induction as high 20% ee and 55% de has been obtained with selected systems. These numbers, although low, are much higher than what has been obtained in solution with the same system or with the parent system by other investigators (maximum similar to10% ee).

On the importance of backbone loop and peptide flip in the Walker sequence in F-1-ATPase action

The Walker sequence, GXXXXGKT, present in all the six subunits of F-1-ATPase exists in a folded form, known as phosphate-binding loop (P-loop). Analysis of the Ramachandran angles showed only small RMS deviation between the nucleotide-bound and nucleotide-free forms. This indicated a good overlap of the backbone loops. The catalytic beta-subunits (chains D, E and F) showed significant changes in the Ramachandran angles and the side chain torsion angles, but not the structural alpha-subunits (chains A, B and C). Most striking among these are the changes associated with Val160 and Gly161 corresponding to a flip in the peptide unit between them when a nucleotide is bound (chains D or F compared to nucleotide-free chain E). The conformational analysis further revealed a hitherto unnoticed hydrogen bond between amide-N of the flipped Gly161 and terminal phosphate-O of the nucleotide. This assigns a role for this conserved amino acid, otherwise ignored, of making an unusual direct interaction between the peptide backbone of the enzyme protein and the incoming nucleotide substrate. Significance of this interaction is enhanced, as it is limited only to the catalytic subunits, and also likely to involve a mechanical rotation of bonds of the peptide unit. Hopefully this is part of the overall events that link the chemical hydrolysis of ATP with the mechanical rotation of this molecule, now famous as tiny molecular motor.

The repertoire of protein kinases encoded in the draft version of the human genome: atypical variations and uncommon domain combinations

Background: Phosphorylation by protein kinases is central to cellular signal transduction. Abnormal functioning of kinases has been implicated in developmental disorders and malignancies. Their activity is regulated by second messengers and by the binding of associated domains, which are also influential in translocating the catalytic component to their substrate sites, in mediating interaction with other proteins and carrying out their biological roles. Results: Using sensitive profile-search methods and manual analysis, the human genome has been surveyed for protein kinases. A set of 448 sequences, which show significant similarity to protein kinases and contain the critical residues essential for kinase function, have been selected for an analysis of domain combinations after classifying the kinase domains into subfamilies. The unusual domain combinations in particular kinases suggest their involvement in ubiquitination pathways and alternative modes of regulation for mitogen-activated protein kinase kinases (MAPKKs) and cyclin-dependent kinase (CDK)-like kinases. Previously unexplored kinases have been implicated in osteoblast differentiation and embryonic development on the basis of homology with kinases of known functions from other organisms. Kinases potentially unique to vertebrates are involved in highly evolved processes such as apoptosis, protein translation and tyrosine kinase signaling. In addition to coevolution with the kinase domain, duplication and recruitment of non-catalytic domains is apparent in signaling domains such as the PH, DAG-PE, SH2 and SH3 domains. Conclusions: Expansion of the functional repertoire and possible existence of alternative modes of regulation of certain kinases is suggested by their uncommon domain combinations. Experimental verification of the predicted implications of these kinases could enhance our understanding of their biological roles.

Crystallization of SrCO3 on a self-assembled monolayer substrate: an in-situ synchrotron X-ray study

Self-assembled monolayers (SAMs) of alkanethiols on gold surfaces show great promise in controlling the nucleation and growth of inorganic minerals from solution. In doing so, they mimic the role of some biogenic macromolecules in natural biomineralisation processes. Crystallization on SAM surfaces is usually monitored ex-situ; by allowing the process to commence and to evolve for some time, removing the substrate from the mother solution, and then examining it using microscopy, diffraction etc. We present here for the first time, the use of high energy monochromatic synchrotron X-radiation in conjunction with a two dimensional detector to monitor in situ, in a time resolved fashion, the growth of SrCO3 (strontianite) crystals on a SAM substrate.

Synthesis and Microstructure of Laser Surface Alloyed Al–Sn–Si Layer on Commercial Aluminum Substrate

A new type of bearing alloy containing ultrafine sized tin and silicon dispersions in aluminum was designed using laser surface alloying and laser remelting techniques. The microstructures of these non-equilibrium processed alloys were studied in detail using scanning and transmission electron microscopy. The microstructures revealed three distinct morphologies of tin particles namely elongated particles co-existing with silicon, globular particles, and very fine particles. Our detailed analyses using cellular growth theories showed that the formation of these globular tin particles was due to the pinching off of the tin rich liquid in the inter-cellular space by the growth of aluminum secondary dendrite arms. Evidence of fine recrystallized aluminum grains at the top layer due to constrained solidification was shown. Thermal analyses suggested that melting of the spherical shaped tin particles was controlled by the binary aluminum-tin eutectic reaction, whereas non-spherical tin particles melted via the tin-silicon eutectic reaction.

Ellipsometry as a Sensitive Technique to Probe film -Substrate Interfaces: Al2O3 on Si(100)

An attempt has been made to study the film-substrate interface by using a sensitive, non- conventional tool. Because of the prospective use of gate oxide in MOSFET devices, we have chosen to study alumina films grown on silicon. Film-substrate interface of alumina grown by MOCVD on Si(100) was studied systematically using spectroscopic ellipsometry in the range 1.5-5.0 eV, supported by cross-sectional SEM, and SIMS. The (ε1,ε2) versus energy data obtained for films grown at 600°C, 700°C, and 750°C were modeled to fit a substrate/interface/film “sandwich”. The experimental results reveal (as may be expected) that the nature of the substrate -film interface depends strongly on the growth temperature. The simulated (ε1,ε2) patterns are in excellent agreement with observed ellipsometric data. The MOCVD precursors results the presence of carbon in the films. Theoretical simulation was able to account for the ellipsometry data by invoking the presence of “free” carbon in the alumina films.

Denaturant mediated unfolding of both native and molten globule states of maltose binding protein are accompanied by large [Delta]Cp's

Maltose binding protein (MBP) is a large, monomeric two domain protein containing 370 amino acids. In the absence of denaturant at neutral pH, the protein is in the native state, while at pH 3.0 it forms a molten globule. The molten globule lacks a tertiary circular dichroism signal but has secondary structure similar to that of the native state. The molten globule binds 8-anilino-1-naphthalene sulfonate (ANS). The unfolding thermodynamics of MBP at both pHs were measured by carrying out a series of isothermal urea melts at temperatures ranging from 274–329 K. At 298 K, values of [Delta]G°, [Delta]Cp, and Cm were 3.1 ± 0.2 kcal mol−1, 5.9 ± 0.8 kcal mol−1 K−1 (15.9 cal (mol-residue)−1 K−1), and 0.8 M, respectively, at pH 3.0 and 14.5 ± 0.4 kcal mol−1, 8.3 ± 0.7 kcal mol−1 K−1 (22.4 kcal (mol-residue)−1 K−1), and 3.3 M, respectively, at pH 7.1. Guanidine hydrochloride denaturation at pH 7.1 gave values of [Delta]G° and [Delta]Cp similar to those obtained with urea. The m values for denaturation are strongly temperature dependent, in contrast to what has been previously observed for small globular proteins. The value of [Delta]Cp per mol-residue for the molten globule is comparable to corresponding values of [Delta]Cp for the unfolding of typical globular proteins and suggests that it is a highly ordered structure, unlike molten globules of many small proteins. The value of [Delta]Cp per mol-residue for the unfolding of the native state is among the highest currently known for any protein.

PocketAlign A Novel Algorithm for Aligning Binding Sites in Protein Structures

A fundamental task in bioinformatics involves a transfer of knowledge from one protein molecule onto another by way of recognizing similarities. Such similarities are obtained at different levels, that of sequence, whole fold, or important substructures. Comparison of binding sites is important to understand functional similarities among the proteins and also to understand drug cross-reactivities. Current methods in literature have their own merits and demerits, warranting exploration of newer concepts and algorithms, especially for large-scale comparisons and for obtaining accurate residue-wise mappings. Here, we report the development of a new algorithm, PocketAlign, for obtaining structural superpositions of binding sites. The software is available as a web-service at http://proline.physicslisc.emetin/pocketalign/. The algorithm encodes shape descriptors in the form of geometric perspectives, supplemented by chemical group classification. The shape descriptor considers several perspectives with each residue as the focus and captures relative distribution of residues around it in a given site. Residue-wise pairings are computed by comparing the set of perspectives of the first site with that of the second, followed by a greedy approach that incrementally combines residue pairings into a mapping. The mappings in different frames are then evaluated by different metrics encoding the extent of alignment of individual geometric perspectives. Different initial seed alignments are computed, each subsequently extended by detecting consequential atomic alignments in a three-dimensional grid, and the best 500 stored in a database. Alignments are then ranked, and the top scoring alignments reported, which are then streamed into Pymol for visualization and analyses. The method is validated for accuracy and sensitivity and benchmarked against existing methods. An advantage of PocketAlign, as compared to some of the existing tools available for binding site comparison in literature, is that it explores different schemes for identifying an alignment thus has a better potential to capture similarities in ligand recognition abilities. PocketAlign, by finding a detailed alignment of a pair of sites, provides insights as to why two sites are similar and which set of residues and atoms contribute to the similarity.

Insulin Growth Factor-2 Binding Protein 3 (IGF2BP3) Is a Glioblastoma-specific Marker That Activates Phosphatidylinositol 3-Kinase/Mitogen-activated Protein Kinase (PI3K/MAPK) Pathways by Modulating IGF-2

Glioblastoma is the most common and malignant form of primary astrocytoma. Upon investigation of the insulin-like growth factor (IGF) pathway, we found the IGF2BP3/IMP3 transcript and protein to be up-regulated in GBMs but not in lower grade astrocytomas (p<0.0001). IMP3 is an RNA binding protein known to bind to the 5'-untranslated region of IGF-2 mRNA, thereby activating its translation. Overexpression-and knockdown-based studies establish a role for IMP3 in promoting proliferation, anchorage-independent growth, invasion, and chemoresistance. IMP3 overexpressing B16F10 cells also showed increased tumor growth, angiogenesis, and metastasis, resulting in poor survival in a mouse model. Additionally, the infiltrating front, perivascular, and subpial regions in a majority of the GBMs stained positive for IMP3. Furthermore, two different murine glioma models were used to substantiate the above findings. In agreement with the translation activation functions of IMP3, we also found increased IGF-2 protein in the GBM tumor samples without a corresponding increase in its transcript levels. Also, in vitro IMP3 overexpression/knockdown modulated the IGF-2 protein levels without altering its transcript levels. Additionally, IGF-2 neutralization and supplementation studies established that the proproliferative effects of IMP3 were indeed mediated through IGF-2. Concordantly, PI3K and MAPK, the downstream effectors of IGF-2, are activated by IMP3 and are found to be essential for IMP3-induced cell proliferation. Thus, we have identified IMP3 as a GBM-specific proproliferative and proinvasive marker acting through IGF-2 resulting in the activation of oncogenic PI3K and MAPK pathways.

Ligand-modulated Parallel Mechanical Unfolding Pathways of Maltose-binding Proteins

Protein folding and unfolding are complex phenomena, and it is accepted that multidomain proteins generally follow multiple pathways. Maltose-binding protein (MBP) is a large (a two-domain, 370-amino acid residue) bacterial periplasmic protein involved in maltose uptake. Despite the large size, it has been shown to exhibit an apparent two-state equilibrium unfolding in bulk experiments. Single-molecule studies can uncover rare events that are masked by averaging in bulk studies. Here, we use single-molecule force spectroscopy to study the mechanical unfolding pathways of MBP and its precursor protein (preMBP) in the presence and absence of ligands. Our results show that MBP exhibits kinetic partitioning on mechanical stretching and unfolds via two parallel pathways: one of them involves a mechanically stable intermediate (path I) whereas the other is devoid of it (path II). The apoMBP unfolds via path I in 62% of the mechanical unfolding events, and the remaining 38% follow path II. In the case of maltose-bound MBP, the protein unfolds via the intermediate in 79% of the cases, the remaining 21% via path II. Similarly, on binding to maltotriose, a ligand whose binding strength with the polyprotein is similar to that of maltose, the occurrence of the intermediate is comparable (82% via path I) with that of maltose. The precursor protein preMBP also shows a similar behavior upon mechanical unfolding. The percentages of molecules unfolding via path I are 53% in the apo form and 68% and 72% upon binding to maltose and maltotriose, respectively, for preMBP. These observations demonstrate that ligand binding can modulate the mechanical unfolding pathways of proteins by a kinetic partitioning mechanism. This could be a general mechanism in the unfolding of other large two-domain ligand-binding proteins of the bacterial periplasmic space.

Synthesis and DNA binding studies of Ni(II), Co(II), Cu(II) and Zn(II) metal complexes of N(1),N(5)-bis[pyridine-2-methylene]-thiocarbohydrazone Schiff-base ligand

The thiocarbohydrazone Schiff-base ligand with a nitrogen and sulphur donor was synthesized through condensation of pyridine-2-carbaldehyde and thiocarbohydrazide. Schiff-base ligands have the ability to conjugate with metal salts. A series of metal complexes with a general formula [MCl(2)(H(2)L)]center dot nH(2)O (M=Ni, Co, Cu and Zn) were synthesized by forming complexes of the N(1),N5-bis[pyridine-2-methylene]thiocarbohydrazone (H2L) Schiff-base ligand. These metal complexes and ligand were characterized by using ultraviolet-visible (UV-Vis), Fourier Transform Infrared (FT-IR), (1)H and (13)C NMR spectroscopy and mass spectroscopy, physicochemical characterization, CHNS and conductivity. The biological activity of the synthesized ligand was investigated by using Escherichia coli DNA as target. The DNA interaction of the synthesized ligand and complexes on E. coli plasmid DNA was investigated in the aqueous medium by UV-Vis spectroscopy and the binding constant (K(b)) was calculated. The DNA binding studies showed that the metal complexes had an improved interaction due to trans-geometrical isomers of the complexes than ligand isomers in cis-positions. (C) 2011 Elsevier B.V. All rights reserved.

Conformational basis for substrate recognition and regulation of catalytic activity in Staphylococcus aureus nucleoside di-phosphate kinase

Nucleoside diphosphate kinases (NDK) are characterized by high catalytic turnover rates and diverse substrate specificity. These features make this enzyme an effective activator of a pro-drug an application that has been actively pursued for a variety of therapeutic strategies. The catalytic mechanism of this enzyme is governed by a conserved histidine that coordinates a magnesium ion at the active site. Despite substantial structural and biochemical information on NDK, the mechanistic feature of the phospho-transfer that leads to auto-phosphorylation remains unclear. While the role of the histidine residue is well documented, the other active site residues, in particular the conserved serine remains poorly characterized. Studies on some homologues suggest no role for the serine residue at the active site, while others suggest a crucial role for this serine in the regulation and quaternary association of this enzyme in some species. Here we report the biochemical features of the Staphylococcus aureus NDK and the mutant enzymes. We also describe the crystal structures of the apo-NDK, as a transition state mimic with vanadate and in complex with different nucleotide substrates. These structures formed the basis for molecular dynamics simulations to understand the broad substrate specificity of this enzyme and the role of active site residues in the phospho-transfer mechanism and oligomerization. Put together, these data suggest that concerted changes in the conformation of specific residues facilitate the stabilization of nucleotide complexes thereby enabling the steps involved in the ping-pong reaction mechanism without large changes to the overall structure of this enzyme. (C) 2011 Elsevier B.V. All rights reserved.

Synergy between the N-terminal and C-terminal domains of Mycobacterium tuberculosis HupB is essential for high-affinity binding, DNA supercoiling and inhibition of RecA-promoted strand exchange

The occurrence of DNA architectural proteins containing two functional domains derived from two different architectural proteins is an interesting emerging research theme in the field of nucleoid structure and function. Mycobacterium tuberculosis HupB, unlike Escherichia coli HU, is a two-domain protein that, in the N-terminal region, shows broad sequence homology with bacterial HU. The long C-terminal extension, on the other hand, contains seven PAKK/KAAK motifs, which are characteristic of the histone H1/H5 family of proteins. In this article, we describe several aspects of HupB function, in comparison with its truncated derivatives lacking either the C-terminus or N-terminus. We found that HupB binds a variety of DNA repair and replication intermediates with K(d) values in the nanomolar range. By contrast, the N-terminal fragment of M. tuberculosis HupB (HupB(MtbN)) showed diminished DNA-binding activity, with K(d) values in the micromolar range, and the C-terminal domain was completely devoid of DNA-binding activity. Unlike HupB(MtbN), HupB was able to constrain DNA in negative supercoils and introduce negative superhelical turns into relaxed DNA. Similarly, HupB exerted a robust inhibitory effect on DNA strand exchange promoted by cognate and noncognate RecA proteins, whereas HupB(MtbN), even at a 50-fold molar excess, had no inhibitory effect. Considered together, these results suggest that synergy between the N-terminal and C-terminal domains of HupB is essential for its DNA-binding ability, and to modulate the topological features of DNA, which has implications for processes such as DNA compaction, gene regulation, homologous recombination, and DNA repair.