Loose Conformation Research Articles

Trapping a Knot into Tight Conformations by Intra-Chain Repulsions.

Knots can occur in biopolymers such as DNA and peptides. In our previous study, we systematically investigated the effects of intra-chain interactions on knots and found that long-range repulsions can surprisingly tighten knots. Here, we use this knowledge to trap a knot into tight conformations in Langevin dynamics simulations. By trapping, we mean that the free energy landscape with respect to the knot size exhibits a potential well around a small knot size in the presence of long-range repulsions, and this potential can well lead to long-lived tight knots when its depth is comparable to or larger than thermal energy. We tune the strength of intra-chain repulsion such that a knot is weakly trapped. Driven by thermal fluctuations, the knot can escape from the trap and is then re-trapped. We find that the knot switches between tight and loose conformations—referred to as “knot breathing”. We use a Yukawa potential to model screened electrostatic interactions to explore the relevance of knot trapping and breathing in charged biopolymers. We determine the minimal screened length and the minimal strength of repulsion for knot trapping. We find that Coulomb-induced knot trapping is possible to occur in single-stranded DNA and peptides for normal ionic strengths.

Use of Spectroscopic Techniques to Reveal the Nature of the Interactions of Two Sialic Acid Specific Lectins with Gold Nanoparticles.

From UV-vis absorption, steady state and time resolved fluorescence measurements coupled with circular dichroism (CD) spectral studies, it was revealed that among the two lectins: Sambucus nigra agglutinin (SNA) and Saraca indica (saracin II), SNA forms stronger binding complex in the ground state with gold nanoparticles (GNPs). From the measurements of Stern-Volmer (SV) constants Ksv, and binding constants K(A) and number of binding sites two important inferences could be drawn. Firstly, the fluorescence quenching is primarily due to static quenching and secondly SNA forms stronger binding with GNPs relative to the other lectin saracin II. Synchronous fluorescence spectral measurements further substantiate this proposition of exhibiting the fully exposed tryptophan residue in case of SNA. It appears that the lectin SNA adopted a relatively looser conformation with the extended polypeptide structures leading to the exposure of the hydrophobic cavities which favoured stronger binding with GNPs. CD measurements demonstrate that gold nanoparticles when interact with the lectins (glycoproteins), no significant distortion in the structural pattern of the later occurs. The unaltered identity in the secondary structural pattern of both SNA and saracin II in presence of gold nanoparticles hints that GNPs may be used as useful drug or drug delivery systems.

Ab initio study of charge transfer between lithium and para-disubstituted benzenes

The present work examines how substituents affect the interaction of lithium with the benzene ring and considers whether two configurations, “loose” and “tight,” can be found for the investigated set of complexes as in the lithium complexes with polyaromatic hydrocarbons. The two conformations were actually found for the electron-donating substituents, whereas for the electron-withdrawing substituents, CF3 and NO2, only tight conformation could be optimized. For complexes in the loose conformation [with the N(CH3)2, NH2, OH, H, Cl, and CH3 substituents], the transfer of electron density was directed from the region occupied by a disubstituted benzene to that occupied by Li. The amount of transferred density was very similar for all the complexes (0.21–0.25 a.u.). By contrast, in the tight complexes, the density transfer direction was opposite and the amount of the transferred density depended on the electronegativity of the substituents on benzene ring, being −0.09, −0.09, −0.13, −0.15, −0.15, −0.20, and −0.30 a.u. for N(CH3)2, NH2, OH, H, Cl, CF3, and NO2, respectively. Geometries of the loose and tight conformations differed notably. The six-membered ring was significantly more warped in the tight conformation than in the loose one: In the latter, the ring was nearly planar, whereas in the former, the ring had the boat structure.

Dimerization of Terminal Domains in Spiders Silk Proteins Is Controlled by Electrostatic Anisotropy and Modulated by Hydrophobic Patches.

The well-tuned spinning technology from spiders has attracted many researchers with the promise of producing high-performance, biocompatible, and yet biodegradable fibers. So far, the intricate chemistry and rheology of spinning have eluded us. A breakthrough was achieved recently, when the 3D structures of the N and C terminal domains of spider dragline silk were resolved and their pH-induced dimerization was revealed. To understand the terminal domains' dimerization mechanisms, we developed a protein model based on the experimental structures that reproduces charge and hydrophobic anisotropy of the complex protein surfaces. Monte Carlo simulations were used to study the thermodynamic dimerization of the N-terminal domain as a function of pH and ionic strength. We show that the hydrophobic and electrostatic anisotropies of the N-terminal domain cooperate constructively in the association process. The dipolar attractions at pH 6 lead to weakly bound dimers by forcing an antiparallel monomer orientation, stabilized by hydrophobic locking at close separations. Elevated salt concentrations reduce the thermodynamic dimerization constant due to screened electrostatic dipolar attraction. Moreover, the mutations on ionizable residues reveal a free energy of binding, proportional to the dipole moment of the mutants. It has previously been shown that dimers, formed at pH 6, completely dissociate at pH 7, which is thought to be due to altered protein charges. In contrast, our study indicates that the pH increase has no influence on the charge distribution of the N-terminal domain. Instead, the pH-induced dissociation is due to an adapted, loose conformation at pH 7, which significantly hampers both electrostatic and hydrophobic attractive interactions.

Comparison of the nature of interactions of two sialic acid specific lectins Saraca indica and Sambucus nigra with N-acetylneuraminic acid by spectroscopic techniques

The present paper deals with the isolation and purification of a new sialic acid binding lectin from the seed integument of Saraca indica (Ashok) and the purified lectin was designated Saracin II. Comparative studies on the interactions of saracin II and another sialic acid specific lectin Sambucus nigra agglutinin (SNA) with N-acetylneuraminic acid (NANA) were made using UV–vis absorption, steady state and time resolved fluorescence along with circular dichroism (CD) spectroscopy to reveal the nature and mechanisms of binding of these two lectins with NANA. The experimental observations obtained from UV–vis, steady state and time resolved fluorescence measurements demonstrated that SNA–NANA system formed relatively stronger ground state complex than saracin II–NANA pair. CD measurements further substantiated the propositions made from steady state and time resolved spectroscopic investigations. It was inferred that during interaction of SNA with NANA, the lectin adopted a relatively looser conformation with the extended polypeptide structures leading to the exposure of the hydrophobic cavities which favoured stronger binding with NANA.

Lipoprotein FtsB in Streptococcus pyogenes Binds Ferrichrome in Two Steps with Residues Tyr137 and Trp204 as Critical Ligands

Lipoprotein FtsB is a component of the FtsABCD transporter that is responsible for ferrichrome binding and uptake in the Gram-positive pathogen Streptococcus pyogenes. In the present study, FtsB was cloned and purified from the bacteria and its Fch binding characteristics were investigated in detail by using various biophysical and biochemical methods. Based on the crystal structures of homogeneous proteins, FtsB was simulated to have bi-lobal structure forming a deep cleft with four residues in the cleft as potential ligands for Fch binding. With the assistance of site-directed mutagenesis, residue Trp204 was confirmed as a key ligand and Tyr137 was identified to be another essential residue for Fch binding. Kinetics experiments demonstrated that Fch binding in FtsB occurred in two steps, corresponding to the bindings to Tyr137 at N-lobe and Trp204 from C-lobe, respectively, and so that closing the protein conformation. Without either residue Tyr137 or Trp204, Fch binding in the protein as mutants Fch-Y137A and Fch-W204A may have a loose conformation, resembling the apo-proteins in proteolysis resistance and migration behaviors in native gel. This study revealed the inconsistence in the key amino acids among Fch-binding proteins from Gram-positive and –negative bacteria, providing interesting findings for understanding the differences between Gram-positive and –negative bacteria in the mechanism of iron uptake via siderophore (Fch) binding and transport.

β-Galactosidase at the membrane–water interface: A case of an active enzyme with non-native conformation

Previously we demonstrated that Escherichia coli beta-galactosidase (β-Gal) binds to zwitterionic lipid membranes improving its catalytic activity. To understand the activation mechanism from the protein perspective, here the thermal dependence of the catalytic activity was evaluated in conjunction with parameters derived from spectroscopy and calorimetry, in the presence and absence of egg-yolk phosphatidylcholine vesicles.In solution, the native state of β-Gal exhibits a loose conformation according to the λmax of fluorescence emission, which is in the upper end of the emission range for most proteins. A non-two state thermal unfolding mechanism was derived from DSC experiments and supported by the sequential unfolding temperatures exhibited by fluorescence (55°C) and CD (60°C) spectroscopies. Quenching of β-Gal's intrinsic fluorescence, provided evidence for a novel and even looser folding for the lipid-bound protein. However, DSC data showed that the thermal unfolding in the presence of lipids occurred with a significant decrease in ΔH compared to what happened in solution, suggesting that only the population of non-bound protein molecules were involved in this process.Concluding, upon binding to a lipid-water interface β-Gal becomes trapped in a partially unfolded state, more active than that of the native protein in solution.

Phenyl Linker-Induced Dense PEG Conformation Improves the Efficacy of C-Terminally MonoPEGylated Staphylokinase

PEGylation can improve the protein efficacy by prolonging serum half-life and reducing proteolytic sensitivity and immunogenicity. However, PEGylation may decrease the bioactivity of a protein by interfering with binding of its substrate or receptors. Here, staphylokinase (SAK), a thrombolysis agent for therapy of myocardial infarction, was mono-PEGylated at the C-terminus of SAK far from its bioactive domain. Phenyl, propyl, and amyl moieties were used as linkers between SAK and polyethylene glycol (PEG), respectively. Flexible propyl and amyl linkers lead to loose conformation. In contrast, rigid and hydrophobic phenyl linker induces dense PEG conformation that can extensively shield most domains adjacent to C-terminus (e.g., the antigen epitopes and proteolytic sites) of SAK and inefficiently shield its bioactive domain. As compared with loose PEG conformation, dense PEG conformation is more efficient to maintain the bioactivity, increase the plasma half-life, and decrease the proteolytic sensitivity and immunogenicity of the PEGylated SAK.

Nitric oxide activates intradomain disulfide bond formation in the kinase loop of Akt1/PKBα after burn injury

Severe burn injury is an acute inflammatory state with massive alterations in gene expression and levels of growth factors, cytokines and free radicals. During the catabolic processes, changes in insulin sensitivity and skeletal muscle wasting (unintended loss of 5–15% of lean body mass) are observed clinically. Here, we reveal a novel molecular mechanism of Akt1/protein kinase Bα (Akt1/PKBα) regulated via cross-talking between dephosphorylation of Thr308 and S-nitrosylation of Cys296 post severe burn injury, which were characterized using nano-LC interfaced with tandem quadrupole time-of-fight mass spectrometry (Q-TOF)micro tandem mass spectrometry in both in vitro and in vivo studies. For the in vitro studies, Akt1/PKBα was S-nitrosylated with S-nitrosoglutathione and derivatized by three methods. The derivatives were isolated by SDS-PAGE, trypsinized and analyzed by the tandem MS. For the in vivo studies, Akt1/PKBα in muscle lysates from burned rats was immuno-precipitated, derivatized with HPDP-Biotin and analyzed as above. The studies demonstrated that the NO free radical reacts with the free thiol of Cys296 to produce a Cys296-SNO intermediate which accelerates interaction with Cys310 to form Cys296-Cys310 in the kinase loop. MS/MS sequence analysis indicated that the dipeptide, linked via Cys296-Cys310, underwent dephosphorylation at Thr308. These effects were not observed in lysates from sham animals. As a result of this dual effect of burn injury, the loose conformation that is slightly stabilized by the Lys297-Thr308 salt bridge may be replaced by a more rigid structure which may block substrate access. Together with the findings of our previous report concerning mild IRS-1 integrity changes post burn, it is reasonable to conclude that the impaired Akt1/PKBα has a major impact on FOXO3 subcellular distribution and activities.

Incorporation of chromophores into dendrigraft polybutadiene: effect of dendrigraft matrix on the fluorescent properties

This paper demonstrated fluorescence behaviour of two novel coumarin-containing dendrigraft polybutadienes (PB-Cou) with linear-comb (LC-PB-Cou) and star-comb (SC-PB-Cou) architectures. LC-PB-Cou and SC-PB-Cou with 12–13 mol% coumarin content were synthesized via the combination of anionic polymerization and “click” chemistry methodology. The photophysical behaviours were investigated based on UV-Vis spectroscopy and fluorescence spectroscopy. The tailor-made LC-PB-Cou and SC-PB-Cou were designed to possess almost identical branching parameters except for architecture. The results of the study suggested that the fluorescent properties of coumarin were improved after being introduced into the dendrigraft polymer. The fluorescence quantum yield (ΦF) of LC-PB-Cou (7.3%) and SC-PB-Cou (5.4%) significantly increased, compared with that of coumarin itself (1.7%). Furthermore, LC-PB-Cou had the advantage over SC-PB-Cou in fluorescent properties because of its looser conformation.

Chitin-binding proteins of Artemia diapause cysts participate in formation of the embryonic cuticle layer of cyst shells

The brine shrimp Artemia reproduces either ovoviviparously, producing free-swimming nauplii, or oviparously, producing encysted embryos (diapause cysts) able to cope with harsh and complex habitats. When the cysts enter diapause they are encased in a complex external shell that protects them from certain extreme environments. The genomic comparison of oviparous and ovoviviparous ovisacs has been described previously. We isolated three significantly up-regulated genes in oviparous oocytes and identified them as Arp-CBP (Artemia parthenogenetica chitin-binding protein) genes. Quantitative real-time PCR indicated that the expression of Arp-CBP genes gradually increases during diapause cyst formation and significant mRNA accumulation occurs during the ovisac stage of oviparous development. Moreover, in situ hybridization results demonstrated that Arp-CBP mRNAs are expressed in the embryo. Interestingly, the results of immune electron microscopy showed that all three Arp-CBPs are distributed throughout the cellular ECL (embryonic cuticle layer) of the cyst shell. Furthermore, knockdown of Arp-CBP by RNA interference resulted in marked changes in the composition of the embryonic cuticular layer. The fibrous layer of the cyst shell adopted a loose conformation and the inner and outer cuticular membranes exhibited marked irregularities when Arp-CBP expression was suppressed. Finally, an invitro recombinant protein-binding assay showed that all three Arp-CBPs have carbohydrate-binding activities. These findings provide significant insight into the mechanisms by which the ECL of Artemia cyst shell is formed, and demonstrate that Arp-CBPs are involved in construction of the fibrous lattice and are required for formation of the ECL of the cyst shell.

Tightening or loosening a pH-sensitive double-lasso molecular machine readily synthesized from an ends-activated [c2]daisy chain

The ready synthesis of a new double-lasso molecular machine from an ends-activated [c2]daisy chain has been achieved. This original architecture consists of a unique double-lasso structure, where the two macrocycles can slide along the threaded pseudo-macrocycle upon variation of pH. The interlocked structure allows for two very different conformations: at low pH, it has a loose conformation with a big cavity. The interlocked molecule thus behaves like a controllable “jump rope”. After deprotonation, the molecule adopts a helical-type tightened conformation with a shrunken cavity. In this conformation, no jump rope movement is possible due to the tightened lasso.

Soft‐Surface DNA Nanotechnology: DNA Constructs Anchored and Aligned to Lipid Membrane

Soft‐Surface DNA Nanotechnology: DNA Constructs Anchored and Aligned to Lipid Membrane

Fibrin Fibers Exhibit Two Distinct Temporal Regimes of Recoil Dynamics

Fibrin fibers form the mechanical backbone of blood clots, stemming the flow of blood at the sights of vascular injury. The heart pumps blood at approximately 1Hz, requiring fibers within the vasculature to recover elastically within a second, or risk clot failure. Recent work on fibrin fiber mechanics showed that fibers are soft in stretching (1-10 MPa modulus), reversibly stretch to up to three times their original length and strain stiffen above 100% strain; however, fibrin fiber dynamics has received less attention. Using a combined AFM/Optical fluorescent microscope we have probed the recoil dynamics of fibrin fibers on the millisecond timescale. Fluorescently labeled fibers were suspended between channels and then stretched from the center with an AFM tip. Fibers were allowed to slip off the tip and fiber relaxation was measured optically at 3-11 ms time intervals. The data indicate two temporal regimes of fibrin dynamics: A fast initial regime (≤1ms) where the fiber snaps back into a loose conformation, and a slow regime on the order of 10's of ms, where the fiber regains a taut conformation. The two timescales of fiber relaxation indicate two different mechanisms. We find that the timescale of the slow regime is directly related to the strain of the fiber at the point of release. The fast timescale is likely governed by the entropic recoil of unstructured polypeptide chains, while the second timescale is likely governed by either protein folding or a macroscopic re-ordering of the fiber substructure. Further experiments will differentiate between these mechanisms, and the implications of these two regimes of fiber recovery will be discussed.

Mutation of conserved tryptophan residues at the dimer interface of Staphylococcus aureus nitric oxide synthase

Nitric oxide synthases (NOSs) share two invariant tryptophan residues within a conserved helical lariat that is part of the pterin-binding site and dimer interface. We mutated Staphylococcus aureus NOS Trp-314 (to alanine, phenylalanine, tyrosine and histidine) and Trp-316 (to alanine, phenylalanine and tyrosine) and characterized the effects of mutation on heme environment, quaternary structure, enzymatic activity, and substrate affinity. With arginine present, all saNOS variants bound heme with native thiolate ligation, formed high spin ferric complexes and were dimeric. All variants catalyze the peroxide-dependent oxidation of N-hydroxy- l-arginine, at rates from 10% to 55% of wild type activity. Arginine-free proteins are dimeric with the exception of W314A. Arginine affinity for all variants decreases with increasing temperature between 15 and 42 °C but is precipitous for position-314 variants. Previous structural and biophysical characterization of NOS oxygenase domains demonstrated that the protein can exist in either a tight or loose conformation, with the former corresponding to the active state of the protein. In the position-314 variants it is likely that the loose conformation is favoured, owing to the loss of a hydrogen bond between the indole side chain and the polypeptide backbone of the helical lariat.

Phase behavior of temperature‐ and pH‐sensitive poly(acrylic acid‐g‐N‐isopropylacrylamide) in dilute aqueous solution

AbstractSeveral different composition temperature‐ and pH‐sensitive poly(acrylic acid‐g‐N‐isopropylacrylamide) (P(AA‐g‐NIPAM)) graft copolymers were synthesized by free‐radical copolymerization utilizing macromonomer technique. The phase behavior and conformation change of P(AA‐g‐NIPAM) in aqueous solutions were investigated by UV–vis transmittance measurements, fluorescence probe, and fluorescence quenching techniques. The results demonstrate that the P(AA‐g‐NIPAM) copolymers have temperature‐ and pH‐sensitivities, and these different composition graft copolymers have different lower critical solution temperature (LCST) and critical phase transition pH values. The LCST of graft copolymer decreases with increasing PNIPAM content, and the critical phase transition pH value increases with increasing Poly(N‐isopropylacrylamide) (PNIPAM) content. At room temperature (20°C), different composition of P(AA‐g‐NIPAM) graft copolymers in dilute aqueous solutions (0.001 wt %) have a loose conformation, and there is no hydrophobic microdomain formation within researching pH range (pH 3 ∼ 10). In addition, for the P(AA‐g‐NIPAM) aqueous solutions, transition from coil to globular is an incomplete reversible process in heating and cooling cycles. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Structure and Dynamics of Single Conjugated Polymer Chromophores by Surface-Enhanced Raman Spectroscopy

Dramatic localization of optical fields by interactions with surface plasmons on nanotextured metal surfaces allows us to record Raman spectra of individual chromophores on single chains of a high-molecular-weight model conjugated polymer, poly(2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene. The data are broadly consistent with two configurations of the chromophore that are characteristic of packed and loose conformations of the backbone. Within these types, fluctuations of the chromophore planarity are observed, and we show how they can be analyzed by studying the temporal correlation of successive spectra. Chromophores exhibit some configurational memory on the time scale of minutes. We also observe substantial spectral fluctuations and associate these mostly with thermally induced motions of the chromophore in the "hot spot". However, we also provide instances of data representing irreversible photochemistry as well as charging and discharging of the chromophore. Finally, we show that single chromophores embedded in the polymer reorganize in response to poor solvents.

Life at low temperatures: is disorder the driving force?

The thermodynamic characterization of various biological systems from psychrophiles points to a larger entropic contribution when compared to the corresponding mesophilic or (hyper) thermophilic counterparts, either at the level of the macromolecules (thermodynamic and kinetic stabilities) or of their function (ligand binding, catalytic activity). It is suggested here that in an environment characterized by a low heat content (enthalpy) and at temperatures that strongly slowdown molecular motions, the cold-adapted biological systems rely on a larger disorder to maintain macromolecular dynamics and function. Such pre-eminent involvement of entropy is observed in the experimental results and, from a macroscopic point of view, is also reflected for instance by the steric hindrances introduced by cis-unsaturated and branched lipids to maintain membrane fluidity, by the loose conformation of psychrophilic proteins or by the local destabilization of tRNA by dihydrouridine in psychrophilic bacteria.

Deletion of Epidermal Growth Factor-like Domains Converts Mammalian Tolloid into a Chordinase and Effective Procollagen C-proteinase

Bone morphogenetic protein (BMP)-1 and mammalian tolloid (mTld) are Ca(2+)-dependent metalloproteinases that result from alternative splicing of the bmp1 gene. They have different proteinase activities, e.g. BMP-1 effectively cleaves procollagen (an extracellular matrix protein) and chordin (a BMP antagonist), whereas mTld is a poor procollagen proteinase and will not cleave chordin in the absence of twisted gastrulation. This is perplexing because mTld (being the longer variant) might be expected to cleave all substrates cleaved by BMP-1. Studies have shown that the minimal structure for procollagen proteinase activity is proteinase-CUB1-CUB2 (BMP-1DeltaEC3) and therefore lacking the epidermal growth factor (EGF)-like domain thought to account for the Ca(2+) dependence of BMP-1. In this study we generated three deletion mutants of mTld that lacked either one or both EGF-like domains (referred to as "mTld-DeltaEGF"). The mutated proteins were poorly but sufficiently secreted from 293-EBNA cells for in vitro assays of procollagen and chordin cleavage. Most surprisingly, the mTld-DeltaEGF mutants required Ca(2+) for proteolytic activity, thereby showing that the EGF-like domains do not account for the Ca(2+) dependence of BMP-1/mTld. Moreover, the mTld-DeltaEGFs are effective procollagen proteinases and cleave chordin. Furthermore, BMP-1DeltaEC3 cleaves chordin and requires Ca(2+) for activity. Studies using nondenaturing gels showed that mTld molecules lacking EGF-like domains have a loose conformation such that in the presence of Ca(2+) binding sites for chordin and procollagen on the "BMP-1-part" of the molecule are exposed. We propose that the EGF-like domains could hold CUB4/5 domains in locations that exclude substrates cleavable by BMP-1.

Theoretical Studies on Mechanisms of Cycloaddition Reaction between Dichlorovinylidene and Formaldehyde: Concerted and Stepwise?

The mechanism of cycloaddition reaction between singlet dichlorovinylidene (R1) and formaldehyde (R2) has been investigated with MP2 and B3LYP /6-31G* methods, including geometry optimization, vibrational analysis, and energy for the involved stationary points on the potential energy surface. Energies from both methods are also further corrected by CCSD(T)/6-31G* single-point calculations. Although the relative energies do differ especially for the loose conformations such as transition states and intermediates, generally the geometries predicted by MP2 and B3LYP are in good agreement. CCSD(T) relative energies for the stationary points predicted by MP2 and B3LYP agree quite well, and they are more comparative to those from B3LYP than those from MP2. The results also show that both three-centered and [2+2] cycloadditions can happen in concerted pathways. The former leads to a stable three-membered ring product (P1), while the two intermediates (INT1c and INT1d) from the latter are not so stable and will rearrange into either P1 or a more stable four-membered ring product (P2). The orbital interactions are also discussed for the leading intermediates and products.