Structural Basis of Drug Resistance by Genetic Variants of HIV Type 1 Clade C Protease from India

Using computer modeling of three-dimensional structures and structural information available on the crystal structures of HIV-1 protease, we investigated the structural effects of mutations, in treatment-naive and treatment-exposed individuals from India and postulated mechanisms of resistance in clade C variants. A large number of models (14) have been generated by computational mutation of the available crystal structures of drug bound proteases. Localized energy minimization was carried out in and around the sites of mutation in order to optimize the geometry of interactions present. Most of the mutations result in structural differences at the flap that favors the semiopen state of the enzyme. Some of the mutations were also found to confer resistance by affecting the geometry of the active site. The E35D mutation affects the flap structure in clade B strains and E35N and E35K mutation, seen in our modeled strains, have a more profound effect. Common polymorphisms at positions 36 and 63 in clade C also affected flap structure. Apart from a few other residues Gln-58, Asn-83, Asn-88, and Gln-92 and their interactions are important for the transition from the closed to the open state. Development of protease inhibitors by structure-based design requires investigation of mechanisms operative for clade C to improve the efficacy of therapy.

Studies on the variants of the protein toxins ricin and abrin

his study elucidates some structural and biological features of galactose-binding variants of the cytotoxic proteins ricin and abrin. An isolation procedure is reported for ricin variants from Ricinus communis seeds by using lactamyl-Sepharose affinity matrix, similar to that reported previously for variants of abrin from Abrus precatorius seeds [Hegde, R., Maiti, T. K. & Podder, S. K. (1991) Anal. Biochem. 194, 101–109]. Ricin variants, subfractionated on carboxymethyl-Sepharose CL-6B ion-exchange chromatography, were characterized further by SDS/PAGE, IEF and a binding assay. Based on the immunological cross-reactivity of antibody raised against a single variant of each of ricin and abrin, it was established that all the variants of the corresponding type are immunologically indistinguishable. Analysis of protein titration curves on an immobilized pH gradient indicated that variants of abrin I differ from other abrin variants, mainly in their acidic groups and that variance in ricin is a cause of charge substitution. Detection of subunit variants of proteins by two-dimensional gel electrophoresis showed that there are twice as many subunit variants as there are variants of holoproteins, suggesting that each variant has a set of subunit variants, which, although homologous, are not identical to the subunits of any other variant with respect to pI. Seeds obtained from polymorphic species of R. communis showed no difference in the profile of toxin variants, as analyzed by isoelectric focussing. Toxin variants obtained from red and white varieties of A. precatorius, however, showed some difference in the number of variants as well as in their relative intensities. Furthermore, variants analyzed from several single seeds of A. precatorius red type revealed a controlled distribution of lectin variants in three specific groups, indicating an involvement of at least three genes in the production of Abrus lectins. The complete absence or presence of variants in each group suggested a post-translational differential proteolytic processing, a secondary event in the production of abrin variants.

Structure and Function of Hemoglobin Confined Inside Silica Nanotubes

Investigations on the structure and function of hemoglobin (Hb) confined inside sol-gel template synthesized silica nanotubes (SNTs) have been discussed here. Immobilization of hemoglobin inside SNTs resulted in the enhancement of direct electron transfer during an electrochemical reaction. Extent of influence of nanoconfinement on protein activity is further probed via ligand binding and thermal stability studies. Electrochemical investigations show reversible binding of n-donor liquid ligands, such as pyridine and its derivatives, and predictive variation in their redox potentials suggests an absence of any adverse effect on the structure and function of Hb confined inside nanometer-sized channels of SNTs. Immobilization also resulted in enhanced thermal stability of Hb. The melting or denaturation temperature of Hb immobilized inside SNTs increase by approximately 4 degrees C as compared with that of free Hb in solution.

Functional diversity of human protein kinase splice variants marks significant expansion of human kinome

Background: Protein kinases are involved in diverse spectrum of cellular processes. Availability of draft version of the human genomic data in the year 2001 enabled recognition of repertoire of protein kinases. However, over the years the human genomic data is being refined and the current release of human genomic data has helped us to recognize a larger repertoire of over 900 human protein kinases represented mainly by splice variants. Results: Many of these identified protein kinases are alternatively spliced products. Interestingly, some of the human kinase splice variants appear to be significantly diverged in terms of their functional properties as represented by incorporation or absence of one or more domains. Many sets of protein kinase splice variants have substantially different domain organization and in a few sets of splice variants kinase domains belong to different subfamilies of kinases suggesting potential participation in different signal transduction pathways. Conclusions: Addition or deletion of a domain between splice variants of multi-domain kinases appears to be a means of generating differences in the functional features of otherwise similar kinases. It is intriguing that marked sequence diversity within the catalytic regions of some of the splice variant kinases result in kinases belonging to different subfamilies. These human kinase splice variants with different functions might contribute to diversity of eukaryotic cellular signaling.

Action of N-bromosuccinimide on human hemoglobin and its possible bearing on heme-globin linkage

A reaction of N-bromosuccinimide with the heme groups of hemoglobin has been studied spectrophotometrically. The reaction brings about the disappearance of characteristic absorption peaks of hemoglobin and is accompanied by the release of inorganic iron from the heme groups. Urea is obligatory for the reaction to take place at pH 4.0, while it can occur in the absence of urea at pH 7.0. The spectrum of hemoglobin which does not show any peak in the Soret region at pH 4.0 is “normalized” in the presence of urea or sucrose at the same pH. The effect of “normalization” in 8 M urea is apparent over the pH range 3.0–4.5. From the obligatory requirement of urea and sucrose for “normalization” of spectrum and the dependence of the release of inorganic iron on the concentration of urea, it is suggested that heme groups are “buried” within the globin at pH 4.0 and not dissociated from globin as supposed before.

X-ray Crystallographic Analysis of the Complexes of Enoyl Acyl Carrier Protein Reductase of Plasmodium falciparum with Triclosan Variants to Elucidate the Importance of Different Functional Groups in Enzyme Inhibition

Triclosan, a well-known inhibitor of Enoyl Acyl Carrier Protein Reductase (ENR) from several pathogenic organisms, is a promising lead compound to design effective drugs. We have solved the X-ray crystal structures of Plasmodium falciparum ENR in complex with triclosan variants having different substituted and unsubstituted groups at different key functional locations. The structures revealed that 4 and 2' substituted compounds have more interactions with the protein, cofactor, and solvents when compared with triclosan. New water molecules were found to interact with some of these inhibitors. Substitution at the 2' position of triclosan caused the relocation of a conserved water molecule, leading to an additional hydrogen bond with the inhibitor. This observation can help in conserved water-based inhibitor design. 2' and 4' unsubstituted compounds showed a movement away from the hydrophobic pocket to compensate for the interactions made by the halogen groups of triclosan. This compound also makes additional interactions with the protein and cofactor which compensate for the lost interactions due to the unsubstitution at 2' and 4'. In cell culture, this inhibitor shows less potency, which indicates that the chlorines at 2' and 4' positions increase the ability of the inhibitor to cross multilayered membranes. This knowledge helps us to modify the different functional groups of triclosan to get more potent inhibitors. (C) 2010 IUBMB IUBMB Life, 62(6): 467-476.

Self accomodation morphology of martensite variants in Zr---2.5wt%Nb alloy

The role of self accomodation of the different mertensite variants controlling the morphologies of the Zr---2.5wt%Nb alloy martensite has been examined. Three distinct types of grouping of martensite variants have been found to be predominantly present. Crystallographic descriptions of these groups have been provided and the degrees of self accomodation for these have been estimated and compared with those corresponding to other possible variant groupings around the symmetry axes of the parent phase. The frequently observed 3-variant group, which shows an “indentation mark” morphology when viewed along left angle bracket111right-pointing angle bracketβ directions in the transmission electron microscope, has been seen to have the highest degree of self accomodation amongst the cases considered. Based on the observations made, a growth sequence leading to the formation of the final martensitic structure has been proposed.

The variants of the protein toxins abrin and ricin A useful guide to understanding the processing events in the toxin transport

Kinetic data on inhibition of protein synthesis in thymocyte by three abrins and ricin have been obtained. The intrinsic efficiencies of A chains of four toxins to inactivate ribosomes, as analyzed by k1-versus-concentration plots were abrin II, III > ricin > abrin I. The lag times were 90, 66, 75 and 105 min at a 0.0744 nM concentration of each of abrin I, II, III and ricin, respectively. To account for the observed differences in the dose-dependent lag time, functional and structural variables of toxins such as binding efficiency of B chains to receptors and low-pH-induced structural alterations have been analyzed. The association constants obtained by stopped flow studies showed that abrin-I (4.13 × 105 M−1 s−1) association with putative receptor (4-methylumbelliferyl-α-D-galactoside) is nearly two times more often than abrin III (2.6 × 105 M−1 s−1) at 20°C. Equillibrium binding constants of abrin I and II to thymocyte at 37°C were 2.26 × 107 M−1 and 2.8 × 107 M−1 respectively. pH-induced structural alterations as studied by a parallel enhancement in 8-anilino-L-naphthalene sulfonate fluorescence revealed a high degree of qualitative similarity. These results taken with a nearly identical concentration-independent lag time (minimum lag of 41–42 min) indicated that the binding efficiencies and internalization efficiencies of these toxins are the same and that the observed difference in the dose-dependent lag time is causally related to the proposed processing event. The rates of reduction of inter-subunit disulfide bond, an obligatory step in the intoxication process, have been measured and compared under a variety of conditions. Intersubunit disulfide reduction of abrin I is fourfold faster than that of abrin II at pH 7.2. The rate of disulfide reduction in abrin I could be decreased 1 I-fold by adding lactose, compared to that without lactose. The observed differences in the efficiencies of A chains, the dose-dependent lag period, the modulating effect of lactose on the rates of disulfide reduction and similarity in binding properties make the variants a valuable tool to probe the processing events in toxin transport in detail.

Structural variants of hyperbranched polyesters

Hyperbranched polyesters based on 3,5-dihydroxybenzoic acid and its derivatives were prepared by self-condensation of the corresponding ester under standard trans-esterification conditions. The spacer segment length that connects the branching points was systematically varied by starting from the appropriate ethyl 3,5-bis(omega-hydroxyoligo(ethyleneoxy))benzoate. The thermal properties of the hyperbranched polyesters were studied using DSC, and they have been compared with those of the linear analogues prepared from the corresponding p-hydroxybenzoic acid derivatives and also with the molecularly ''kinked'' analogues prepared from the meta isomers. These hyperbranched polyesters were also terminally functionalized by using a potentially mesogenic 4-butoxybiphenylcarboxylic acid derivative in an attempt to prepare novel hyperbranched liquid crystalline polyesters. This was achieved by copolymerization of the AB(2) monomer with the mesogenic A-type capping unit. These polymers were found to be amorphous and did not exhibit any liquid crystalline phases, probably due to the random distribution of the mesogenic segments on the polymer framework, making it difficult to both crystallize and form mesophases.

Variants of Miyachi�s Theorem for Nilpotent lie Groups

We formulate and prove two versions of Miyachi�s theorem for connected, simply connected nilpotent Lie groups. This allows us to prove the sharpness of the constant 1/4 in the theorems of Hardy and of Cowling and Price for any nilpotent Lie group. These theorems are proved using a variant of Miyachi�s theorem for the group Fourier transform.

Variants Of Miyachi’S Theorem For Nilpotent Lie Groups

We formulate and prove two versions of Miyachi’s theorem for connected, simply connected nilpotent Lie groups. This allows us to prove the sharpness of the constant 1/4 in the theorems of Hardy and of Cowling and Price for any nilpotent Lie group. These theorems are proved using a variant of Miyachi’s theorem for the group Fourier transform.

Bufferless lysis of erythrocytes for isolation of hemoglobin using modified cellulose acetate membranes

This paper presents a modified cellulose acetate membrane prepared using a dry casting technique that can be used to perform lysis of erythrocytes and isolation of hemoglobin. Isolation of hemoglobin is thus achieved without the use of lysis buffers. Cellulose acetate (CA) membranes are embedded with ammonium chloride (NH4Cl) and potassium bicarbonate (KHCO3), which act as lysing agents. The presence of embedded salts is confirmed using EDS analysis. The pores in the CA membrane act as filters. The average pore size in these membranes is designed to be 1.5 mu M, as characterized by SEM analysis, so that they allow hemoglobin to pass through and block all other cells and unlysed erythrocytes present in blood. When a drop of blood is added to the membrane, the NH4Cl and KHCO3 embedded in the membrane dissolve in plasma and lyse the erythrocytes. The filtered hemoglobin is characterized using UV-Vis Spectroscopy. The results indicate extraction of higher concentration of hemoglobin compared with conventional methods.

Enhanced J-protein interaction and compromised protein stability of mtHsp70 variants lead to mitochondrial dysfunction in Parkinsons disease

Parkinsons disease (PD) is the second most prevalent progressive neurological disorder commonly associated with impaired mitochondrial function in dopaminergic neurons. Although familial PD is multifactorial in nature, a recent genetic screen involving PD patients identified two mitochondrial Hsp70 variants (P509S and R126W) that are suggested in PD pathogenesis. However, molecular mechanisms underlying how mtHsp70 PD variants are centrally involved in PD progression is totally elusive. In this article, we provide mechanistic insights into the mitochondrial dysfunction associated with human mtHsp70 PD variants. Biochemically, the R126W variant showed severely compromised protein stability and was found highly susceptible to aggregation at physiological conditions. Strikingly, on the other hand, the P509S variant exhibits significantly enhanced interaction with J-protein cochaperones involved in folding and import machinery, thus altering the overall regulation of chaperone-mediated folding cycle and protein homeostasis. To assess the impact of mtHsp70 PD mutations at the cellular level, we developed yeast as a model system by making analogous mutations in Ssc1 ortholog. Interestingly, PD mutations in yeast (R103W and P486S) exhibit multiple in vivo phenotypes, which are associated with omitochondrial dysfunction', including compromised growth, impairment in protein translocation, reduced functional mitochondrial mass, mitochondrial DNA loss, respiratory incompetency and increased susceptibility to oxidative stress. In addition to that, R103W protein is prone to aggregate in vivo due to reduced stability, whereas P486S showed enhanced interaction with J-proteins, thus remarkably recapitulating the cellular defects that are observed in human PD variants. Taken together, our findings provide evidence in favor of direct involvement of mtHsp70 as a susceptibility factor in PD.

Synchrotron Small-Angle X-ray Scattering Studies of Hemoglobin Nonaggregation Confined inside Polymer Capsules

The effect of confinement on the structure of hemoglobin (Hb) within polymer capsules was investigated here. Hemoglobin transformed from an aggregated state in solution to a nonaggregated state when confined inside the polymer capsules. This was directly confirmed using synchrotron small-angle X-ray scattering (SAXS) studies. The radius of gyration (R-g) and polydispersity (p) of the proteins in the confined state were smaller compared to those in solution. In fact, the R-g value is very similar to theoretical values obtained using protein structures generated from the Protein Databank. In the temperature range (25-85 degrees C, Tm 59 degrees C), the R-g values for the confined Hb remained constant. This observation is in contrary to the increasing R-g values obtained for the bare Hb in solution. This suggested higher thermal stability of Hb when confined inside the polymer capsule than when in solution. Changes in protein configuration were also reflected in the protein function. Confinement resulted in a beneficial enhancement of the electroactivity of Hb. While Hb in solution showed dominance of the cathodic process (Fe3+ -> Fe2+), efficient reversible Fe3+/Fe2+ redox response is observed in the case of the confined Hb. This has important protein functional implications. Confinement allows the electroactive heme to take up positions favorable for various biochemical activities such as sensing of analytes of various sizes from small to macromolecules and controlled delivery of drugs.