Chaperonin-Affected Folding of Globular Proteins

We studied the effect of GroEL on the kinetic refolding of-lactalbumin by stopped-flow fluorescence techniques. We usedwild-type GroEL and its ATPase-defficient mutant D398A, and studied thebinding constants between GroEL and the molten globule foldingintermediate at various concentrations of ADP and ATP. The results arecompared with titration of GroEL with the nucleotides, ADP, ATP-analogs(ATP-S and AMP-PNP) and ATP, which have shown that bothADP and the ATP analogs are bound to GroEL in a non-cooperativemanner but that ATP shows a cooperative effect. Similarly, the bindingconstant between GroEL and the folding intermediate decreased in acooperative manner with an increase in ATP concentration although itshowed non-cooperative decrease with respect to ADP concentration. Itis shown that the allosteric control of GroEL by the nucleotides isresponsible for the above behavior of GroEL and that the observeddifference between the ATP- and ADP-induced transitions of GroEL isbrought about by a small difference in an allosteric parameter (the ratio ofthe nucleotide affinities of GroEL in the high-affinity and the low-affinitystates), i.e., 4.1 for ATP and 2.6 for ADP.     

Structural features and molecular evolution of Bowman-Birk protease inhibitors and their potential application

The Bowman-Birk inhibitors (BBIs) are well-studied serine protease inhibitors that are abundant in dicotyledonous and monocotyledonous plants. BBIs from dicots usually have a molecular weight of 8k and are double-headed with two reactive sites, whereas those from monocots can be divided into two classes, one approximately 8 kDa in size with one reactive site (another reactive site was lost) and the other approximately 16 kDa in size with two reactive sites. The reactive site is located at unique exposed surfaces formed by a disulfide-linked β-sheet loop that is highly conserved, rigid and mostly composed of nine residues. The structural features and molecular evolution of inhibitors are described, focusing on the conserved disulfide bridges. The sunflower trypsin inhibitor-1 (SFTI-1), with 14 amino acid residues, is a recently discovered bicyclic inhibitor, and is the most small and potent naturally occurring Bowman-Birk inhibitor.Recently, BBIs have become a hot topic because of their potential applications. BBIs are now used for defense against pathogens and insects in transgenic plants, which has advantages over using toxic and polluting insecticides. BBIs could also be applied in the prevention of cancer, Dengue fever, and inflammatory and allergic disorders, because of their inhibitory activity with respect to the serine proteases that play apivotal role in the development and pathogenesis of these diseases. The canonical nine-residue loop of BBIs/STH-1 provides an ideal template for drug design of specific inhibitors to target their respective proteases.     

Platelets do not express the oxidized or reduced forms of tissue factor

Background

Expression of tissue factor (TF) antigen and activity in platelets is controversial and dependent upon the laboratory and reagents used. Two forms of TF were described: an oxidized functional form and a reduced nonfunctional form that is converted to the active form through the formation of an allosteric disulfide. This study tests the hypothesis that the discrepancies regarding platelet TF expression are due to differential expression of the two forms.

Methods

Specific reagents that recognize both oxidized and reduced TF were used in flow cytometry of unactivated and activated platelets and western blotting of whole platelet lysates. TF-dependent activity measurements were used to confirm the results.

Results

Western blotting analyses of placental TF demonstrated that, in contrast to anti-TF#5, which is directed against the oxidized form of TF, a sheep anti-human TF polyclonal antibody recognizes both the reduced and oxidized forms. Flow cytometric analyses demonstrated that the sheep antibody did not react with the surface of unactivated platelets or platelets activated with thrombin receptor agonist peptide, PAR-1. This observation was confirmed using biotinylated active site-blocked factor (F)VIIa: no binding was observed. Likewise, neither form of TF was detected by western blotting of whole platelet lysates with sheep anti-hTF. Consistent with these observations, no FXa or FIXa generation by FVIIa was detected at the surface of these platelets. Similarly, no TF-related activity was observed in whole blood using thromboelastography.

Conclusion and significance

Platelets from healthy donors do not express either oxidized (functional) or reduced (nonfunctional) forms of TF.     

Signification differentielle des photoperiodes pour l'horloge biologique de Pieris brassicae (Lepidoptera)

Abstract

Photoperiodic insects are able to distinguish between long days and short days. In various models the long day response is classifically considered the “actively” induced state. The short day response is thought to be “passive”, caused by failure of light to coincide with a photosensitive part of the night or failure of coincidence of constituent oscillators. The photoperiodic response curve of Pieris brassicae showed that diapause is induced by short days (4–14 h), and non‐diapause state by several conditions (natural and non‐natural): long days (16 h or more), LL, DD and ultrashort days (0.1 h). By reciprocal transfers of larvae between non‐diapausing determining and diapause determining conditions, it was proved possible to estimate the differential capacity of four non‐diapausing conditions vs. the diapausing action of LD 8: 16 in decreasing sequence: LD 16:8 > LL > DD = LD 0.1: 23.9. DD may be considered a “neutral” condition. In darkness the development seems to be determined by an endogenous program without external influence. LL, although beingan aperiodic signal as DD, has a weak antidiapausingeffect.Thebiological clock of Pieris differentiates between two constant conditions. The four non‐diapausing conditions have the same effect on the development when applied during the entire larval life, but have different effects when only applied during a few days. Both ecological conditions LD 16:8 and LD 8:16 have an action on the development but in an opposite way. There was not a “passive” state caused by failure of another inductive photoperiod. Ultra‐short days, DD and LL are without ecological meaning. Nevertheless, in these experiments, they provided informations in attempts to determine the mechanism of the time measurement. The external coincidence model of Pittendrigh and Minis (1964) was the more adequate to explain theearlier results on the biological clock of Pieris. However, this model has to be modified to account for the differential significance of several non‐diapausing conditions.     

All-Atom Structural Models for Complexes of Insulin-Like Growth Factors IGF1 and IGF2 with Their Cognate Receptor

Type 1 insulin-like growth factor receptor (IGF1R) is a membrane-spanning glycoprotein of the insulin receptor family that has been implicated in a variety of cancers. The key questions related to molecular mechanisms governing ligand recognition by IGF1R remain unanswered, partly due to the lack of testable structural models of apo or ligand-bound receptor complexes. Using a homology model of the IGF1R ectodomain IGF1RΔβ, we present the first experimentally consistent all-atom structural models of IGF1/IGF1RΔβ and IGF2/IGF1RΔβ complexes. Our explicit-solvent molecular dynamics (MD) simulation of apo-IGF1RΔβ shows that it displays asymmetric flexibility mechanisms that result in one of two binding pockets accessible to growth factors IGF1 and IGF2, as demonstrated via an MD-assisted Monte Carlo docking procedure. Our MD-generated ensemble of structures of apo and IGF1-bound IGF1RΔβ agrees reasonably well with published small-angle X-ray scattering data. We observe simultaneous contacts of each growth factor with sites 1 and 2 of IGF1R, suggesting cross-linking of receptor subunits. Our models provide direct evidence in favor of suggested electrostatic complementarity between the C-domain (IGF1) and the cysteine-rich domain (IGF1R). Our IGF1/IGF1RΔβ model provides structural bases for the observation that a single IGF1 molecule binds to IGF1RΔβ at low concentrations in small-angle X-ray scattering studies. We also suggest new possible structural bases for differences in the affinities of insulin, IGF1, and IGF2 for their noncognate receptors.     

A temperature‐dependent conformational change of NADH oxidase from Thermus thermophilus HB8

Using molecular dynamics simulations and steady‐state fluorescence spectroscopy, we have identified a conformational change in the active site of a thermophilic flavoenzyme, NADH oxidase from Thermus thermophilus HB8 (NOX). The enzyme's far‐UV circular dichroism spectrum, intrinsic tryptophan fluorescence, and apparent molecular weight measured by dynamic light scattering varied little between 25 and 75°C. However, the fluorescence of the tightly bound FAD cofactor increased approximately fourfold over this temperature range. This effect appears not to be due to aggregation, unfolding, cofactor dissociation, or changes in quaternary structure. We therefore attribute the change in flavin fluorescence to a temperature‐dependent conformational change involving the NOX active site. Molecular dynamics simulations and the effects of mutating aromatic residues near the flavin suggest that the change in fluorescence results from a decrease in quenching by electron transfer from tyrosine 137 to the flavin. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Retroviral Oligonucleotide Distributions Correlate with Biased Nucleotide Compositions of Retrovirus Sequences,Suggesting a Duplicative Stepwise Molecular Evolution

A computer-assisted analysis was made of 24 complete nucleotide sequences selected from the vertebrate retroviruses to represent the ten viral groups. The conclusions of this analysis extend and strengthen the previously made hypothesis on the Moloney murine leukemia virus: The evolution of the nucleotide sequence appears to have occurred mainly through at least three overlapping levels of duplication: (1) The distributions of overrepresented (3–6)-mers are consistent with the universal rule of a trend toward TG/CT excess and with the persistence of a certain degree of symmetry between the two strands of DNA. This suggests one or several original tandemly repeated sequences and some inverted duplications. (2) The existence of two general core consensuses at the level of these (3–6)-mers supports the hypothesis of a common evolutionary origin of vertebrate retroviruses. Consensuses more specific to certain sequences are compatible with phylogenetic trees established independently. The consensuses could correspond to intermediary evolutionary stages. (3) Most of the (3–6)-mers with a significantly higher than average frequency appear to be internally repeated (with monomeric or oligomeric internal iterations) and seem to be at least partly the cause of the bias observed by other researchers at the level of retroviral nucleotide composition. They suggest a third evolutionary stage by slippage-like stepwise local duplications. Received: 3 January 1996 / Accepted: 27 March 1996     

Microsecond simulations of mdm2 and its complex with p53 yield insight into force field accuracy and conformational dynamics

The p53‐MDM2 complex is both a major target for cancer drug development and a valuable model system for computational predictions of protein‐ligand binding. To investigate the accuracy of molecular simulations of MDM2 and its complex with p53, we performed a number of long (200 ns to 1 µs) explicit‐solvent simulations using a range of force fields. We systematically compared nine popular force fields (AMBER ff03, ff12sb, ff14sb, ff99sb, ff99sb‐ildn, ff99sb‐ildn‐nmr, ff99sb‐ildn‐phi, CHARMM22*, and CHARMM36) against experimental chemical shift data, and found similarly accurate results, with microsecond simulations achieving better agreement compared to 200‐ns trajectories. Although the experimentally determined apo structure has a closed binding cleft, simulations in all force fields suggest the apo state of MDM2 is highly flexible, and able to sample holo‐like conformations, consistent with a conformational selection model. Initial structuring of the MDM2 lid region, known to competitively bind the binding cleft, is also observed in long simulations. Taken together, these results show molecular simulations can accurately sample conformations relevant for ligand binding. We expect this study to inform future computational work on folding and binding of MDM2 ligands. Proteins 2015; 83:1665–1676. © 2015 Wiley Periodicals, Inc.