Entropy-driven Mechanism Research Articles

Formation of Nonclassical Ordered Phases of AB-Type Multiarm Block Copolymers.

The formation of ordered phases from block copolymers is driven by a delicate balance between the monomer-monomer interaction and chain configurational entropy. The configurational entropy can be regulated by designed chain architecture, resulting in a new entropy-driven mechanism to control the self-assembly of ordered phases from block copolymers. An effective routine to regulate the configurational entropy is to utilize multiarm architecture, in which the entropic contribution to the free energy could be qualitatively controlled by the fraction of bridging configurations. As an illustration of this mechanism, the phase behavior of two AB-type multiarm block copolymers, B0-(Bi-Ai)m and (B1-Ai-B2)m where the minority A blocks form cylindrical or spherical domains, are examined using the self-consistent field theory (SCFT). The SCFT results demonstrate that the packing symmetry of the cylinders or spheres can be controlled by the length of the bridging B blocks. Several nonclassical ordered phases, including a novel square array cylinder with p4mm symmetry, are predicted to form from the AB-type multiarm block copolymers.

A universal entropy-driven mechanism for thioredoxin–target recognition

Cysteine residues in cytosolic proteins are maintained in their reduced state, but can undergo oxidation owing to posttranslational modification during redox signaling or under conditions of oxidative stress. In large part, the reduction of oxidized protein cysteines is mediated by a small 12-kDa thiol oxidoreductase, thioredoxin (Trx). Trx provides reducing equivalents for central metabolic enzymes and is implicated in redox regulation of a wide number of target proteins, including transcription factors. Despite its importance in cellular redox homeostasis, the precise mechanism by which Trx recognizes target proteins, especially in the absence of any apparent signature binding sequence or motif, remains unknown. Knowledge of the forces associated with the molecular recognition that governs Trx-protein interactions is fundamental to our understanding of target specificity. To gain insight into Trx-target recognition, we have thermodynamically characterized the noncovalent interactions between Trx and target proteins before S-S reduction using isothermal titration calorimetry (ITC). Our findings indicate that Trx recognizes the oxidized form of its target proteins with exquisite selectivity, compared with their reduced counterparts. Furthermore, we show that recognition is dependent on the conformational restriction inherent to oxidized targets. Significantly, the thermodynamic signatures for multiple Trx targets reveal favorable entropic contributions as the major recognition force dictating these protein-protein interactions. Taken together, our data afford significant new insight into the molecular forces responsible for Trx-target recognition and should aid the design of new strategies for thiol oxidoreductase inhibition.

Structure-Based Mechanism for Retroviral Primer Annealing

In order to prime reverse transcription, retroviruses require annealing of a tRNA molecule to the U5-primer binding site (U5-PBS) region of the viral genome. The residues essential for primer annealing are initially locked in intramolecular interactions, and hence, annealing requires the chaperone activity of the retroviral nucleocapsid (NC) protein to facilitate structural rearrangements. Understanding the mechanism of primer annealing has been a challenging problem, both due to the relatively low probability of these domains crystallizing and the complexity of studying them by nuclear magnetic resonance (NMR). In my talk, I will detail the NMR experiments that led to the discovery of the mechanism used by the Moloney murine leukemia virus (MLV) NC protein. I will show that unlike classical chaperones, the MLV-NC uses a unique mechanism, in which it specifically targets multiple structured regions in both the U5-PBS and tRNAPro primer that otherwise sequester residues necessary for annealing. This high-specificity and high-affinity binding by NC consequently liberates these sequestered residues_which are exactly complementary_for intermolecular interactions. Furthermore, I will show that NC utilizes a step-wise, entropy-driven mechanism to trigger both residue-specific destabilization and residue-specific release.

Entropic separations of mixtures of aromatics by selective face-to-face molecular stacking in one-dimensional channels of metal-organic frameworks and zeolites.

Separation of challenging mixtures using metal-organic frameworks can be achieved by an entropy-driven mechanism, where one of the components can arrange into a "face-to-face" stacking, thus reducing its "footprint" and reaching a higher saturation loading.

Piloting the membranolytic activities of peptides with a self-organizing map.

Antimicrobial peptides (AMPs) show remarkable selectivity toward lipid membranes and possess promising antibiotic potential. Their modes of action are diverse and not fully understood, and innovative peptide design strategies are needed to generate AMPs with improved properties. We present a de novo peptide design approach that resulted in new AMPs possessing low-nanomolar membranolytic activities. Thermal analysis revealed an entropy-driven mechanism of action. The study demonstrates sustained potential of advanced computational methods for designing peptides with the desired activity.

A structure-based mechanism for tRNA and retroviral RNA remodelling during primer annealing.

To prime reverse transcription, retroviruses require annealing of a transfer RNA molecule to the U5 primer binding site (U5-PBS) region of the viral genome. The residues essential for primer annealing are initially locked in intramolecular interactions; hence, annealing requires the chaperone activity of the retroviral nucleocapsid (NC) protein to facilitate structural rearrangements. Here we show that, unlike classical chaperones, the Moloney murine leukaemia virus NC uses a unique mechanism for remodelling: it specifically targets multiple structured regions in both the U5-PBS and tRNA(Pro) primer that otherwise sequester residues necessary for annealing. This high-specificity and high-affinity binding by NC consequently liberates these sequestered residues--which are exactly complementary--for intermolecular interactions. Furthermore, NC utilizes a step-wise, entropy-driven mechanism to trigger both residue-specific destabilization and residue-specific release. Our structures of NC bound to U5-PBS and tRNA(Pro) reveal the structure-based mechanism for retroviral primer annealing and provide insights as to how ATP-independent chaperones can target specific RNAs amidst the cellular milieu of non-target RNAs.

Quantification of in situ granulation-induced changes in pre-compression, solubility, dose distribution and intrinsic in vitro release characteristics of ibuprofen-cationic dextran conjugate crystanules.

The direct effect of intermolecular association between ibuprofen and diethylaminoethyl dextran (Ddex) and the novel 'melt-in situ granulation-crystallization' technique on the solubility, dose distribution, in vitro dissolution kinetics and pre-compression characteristics of the ibuprofen-Ddex conjugate crystanules have been investigated using various mathematical equations and statistical moments. The research intention was to elucidate the mechanisms of ibuprofen solubilization, densification and release from the conjugate crystanules as well as its dose distribution in order to provide fundamental knowledge on important physicochemical, thermodynamic and system-specific parameters which are key indices for the optimization of drug-polymer conjugate design for the delivery of poorly soluble drugs. The process of melt-in situ-granulation-crystallization reduced the solubility slightly compared with pure ibuprofen, however, the ibuprofen-Ddex conjugate crystanules exhibited increased ibuprofen solubility to a maximum of 2.47×10(-1) mM (at 1.25×10(-4) mM Ddex) and 8.72×10(-1) mM (at 6.25×10(-4) mM Ddex) at 25 and 37 °C, respectively. Beyond these concentrations of Ddex ibuprofen solubility decreased steadily due to stronger bond strength of the conjugate crystanules. The enthalpy-entropy compensation plot suggests a dominant entropy-driven mechanism of solubilization. In the same vein, the addition of Ddex increased the rate and extent of in vitro ibuprofen release from the conjugate crystanule to 100% within 168 h at Ddex concentration of 1.56×10(-4) mM, followed by a decrease with Ddex concentration. The conjugate crystanules exhibited controlled and extended-complete release profile which appeared to be dictated by the concentration of the Ddex and its strong affinity for ibuprofen. A comparison of the real experimental with the predicted data using artificial neural network shows excellent correlation between solubility and dissolution profiles (average error=0.2348%). Heckel, Kawakita, Cooper-Eaton and Kuno equations were employed to determine the mechanism of densification during tapping process. Ddex in the crystanules consistently improved particle rearrangement in the order of 2.5-7 folds compared with pure ibuprofen and stabilized ibuprofen against fragmentation during tapping process. Primary and secondary particle rearrangements were the prominent mechanisms of densification while deformation and fragmentation did not occur. Lower concentrations of Ddex below its critical granular concentration (<6.25×10(-4) mM) hindered plastic deformation and fragmentation, however, the summation of primary and secondary rearrangement parameters was greater than unity suggesting that the overall rearrangement of the conjugate crystanules cannot be explained exclusively by these two steps. This study has demonstrated the formulation of a novel ibuprofen-polymer conjugate which exhibited improved dose distribution and pre-compression characteristics as well as controlled and extended-complete release profiles - a potential drug delivery strategy for poorly soluble drugs.

Ring-Opening Metathesis Polymerization of a Naturally Derived Macrocyclic Glycolipid

Lactonic sophorolipid (LSL) is a naturally occurring macrocyclic monomer that undergoes ring-opening metathesis polymerization (ROMP) via an entropy-driven mechanism (ED-ROMP). Typically, gel permeation chromatographic analysis of poly(LSL) showed products consist of about 70% polymer with Mn up to about 180K (Mw/Mn 1.6–1.8) coexisting with 10% of oligomer and 20% monomer. Detailed kinetic studies for LSL ROMP were performed using two classic metathesis catalysts (i.e., G2 and G3). G2 exhibited apparent first-order propagation, although its slow initiation caused subsequent events of secondary metathesis that decreased molecular weight. An induction period observed for G2 at 33 and 45 °C largely disappears at 60 °C with an increase in the apparent rate constant (kpapp) of 11 times. G3 gave fast initiation even at 33 °C while plots of ln{[M]0/[M]t} versus reaction time for G3 show that kp continuously decreased, implying a decline in G3 catalytic activity. Plots of ln{[M]0/[M]t} versus reaction time for G2 are linear, suggesting apparent first-order kinetic behavior. From analysis of an Arrhenius plot for G2-catalyzed LSL polymerization in THF, the activation energy (Ea) of propagation is 18 ± 3 kcal/mol. By keeping [LSL] constant at 0.54 M, G2-catalyzed LSL ED-ROMP (60 °C, THF) gave a plot of Mn versus [monomer]/[initiator] ratio close to that of the theoretical curve based on a living polymerization model. Hence, despite pronounced secondary metathesis in ED-ROMP, polymerization kinetics with G2 closely resembled living behavior. The length of the induction period for G2-catalyzed polymerizations is inversely proportional to the solvent dielectric constant (εDCM > εTHF > εCHCl3). Finally, this work provides an important example of how complex structures derived from nature can be transformed into unique macromolecules.

Computer simulation study of folding thermodynamics and kinetics of proteins in osmolytes and denaturants

In living cells, the presence of macromolecular crowders, such as osmolytes or denaturants, strongly affects the stability of folded proteins. The overall effects depend on the size, concentration, and chemical properties of the crowding agents. This work uses an all-atom Gō model of the Trp-cage in spherical solvents to probe the physical origin of protein stabilization/destabilization by osmolytes/denturants. The solvent quality is controlled by the solvent-protein contact εPS that can represent repulsive osmolytes (εPS > 0) or attractive denaturants (εPS < 0). The model is used to show that protein stabilization by osmolytes proceeds by an excluded volume, entropy-driven mechanism. Protein destabilization by denaturant is shown to be driven by changes in enthalpy. It is found that small osmolytes are the most effective stabilizer of proteins. Folding simulations of the Trp-cage in osmolytes observe a two-fold increase in folding rates, for small osmolytes. This is due to an osmolyte-induced shift to more compact unfolded protein conformations.

Entropy-Driven Mechanism of an E3 Ligase

Ubiquitin-like modifications are macromolecular chemistry for which our understanding of the enzymatic mechanisms is lacking. Most E3 ligases in ubiquitin-like modifications do not directly participate in chemistry but are thought to confer allosteric effects; however, the nature of the allosteric effects has been elusive. Recent molecular dynamics simulations suggested that an E3 binding enhances the population of the conformational states of the E2·SUMO thioester that favor reactions. In this study, we conducted the first temperature-dependent enzyme kinetic analysis to investigate the role of an E3 on activation entropy and enthalpy. The small ubiquitin-like modifier (SUMO) E3, RanBP2, confers unusually large, favorable activation entropy to lower the activation energy of the reaction. Mutants of RanBP2, designed to alter the flexibilities of the E2·SUMO thioester, showed a direct correlation of their favorable entropic effects with their ability to restrict the conformational flexibility of the E2·SUMO thioester. While the more favorable activation entropy is consistent with the previously suggested role of E3 in conformational selection, the large positive entropy suggests a significant role of solvent in catalysis. Indeed, molecular dynamics simulations in explicit water revealed that the more stable E2·SUMO thioester upon E3 binding results in stabilization of a large number of bound water molecules. Liberating such structured water at the transition state can result in large favorable activation entropy but unfavorable activation enthalpy. The entropy-driven mechanism of the E3 is consistent with the lack of structural conservation among E3s despite their similar functions. This study also illustrates how proteins that bind both SUMO and E2 can function as E3s and how intrinsically unstructured proteins can enhance macromolecular chemistry in addition to their known advantages in protein--protein interactions.

Structural disorder and dynamics of elastinThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular &amp; Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

Elastin is a self-assembling, extracellular-matrix protein that is the major provider of tissue elasticity. Here we review structural studies of elastin from over four decades, and draw together evidence for solution flexibility and conformational disorder that is inherent in all levels of structural organization. The characterization of disorder is consistent with an entropy-driven mechanism of elastic recoil. We conclude that conformational disorder is a constitutive feature of elastin structure and function.

Increase in Backbone Mobility of the VTS1p-SAM Domain on Binding to SRE–RNA

The sterile alpha motif (SAM) domain of VTS1p, a posttranscriptional gene regulator, belongs to a family of SAM domains conserved from yeast to humans. Even though SAM domains were originally classified as protein–protein interaction domains, recently, it was shown that the yeast VTS1p-SAM and the SAM domain of its Drosophila homolog Smaug can specifically recognize RNA hairpins termed Smaug recognition element (SRE). Structural studies of the SRE–RNA complex of VTS1p-SAM revealed that the SAM domain primarily recognizes the shape of the RNA fold induced by the Watson–Crick base-pairing in the RNA pentaloop. Only the central G nucleotide is specifically recognized. The VTS1p-SAM domain recognizes SRE–RNAs with a CNGGN pentaloop where N is any nucleotide. The C1–G4 base pair in the wild type can be replaced by any pair of nucleotides that can form base pairs even though the binding affinity is greatest with a pyrimidine in position 1 and a purine in position 4. The interaction thus combines elements of sequence-specific and non-sequence-specific recognitions. The lack of structural rearrangements in either partner following binding is rather intriguing, suggesting that molecular dynamics may play an important role in imparting relaxed specificity with respect to the exact combination of nucleotides in the loop, except for the central nucleotide. In this work, we extend our previous studies of SRE–RNA interaction with VTS1p, by comparing the dynamics of the VTS1p-SAM domain both in its free form and when bound to SRE–RNA. The 15N relaxation studies of backbone dynamics suggest the presence of a dynamic interaction interface, with residues associated with specific G3 recognition becoming more rigid on RNA binding while other regions attain increased flexibility. The results parallel the observations from our studies of dynamics changes in SRE–RNA upon binding to VTS1p-SAM and shows that molecular dynamics could play a crucial role in modulating binding affinity and possibly contribute to the free energy of the interaction through an entropy-driven mechanism.

Noise induced patterning in reaction-diffusion systems with non-Fickian diffusion

We have studied the entropy-driven mechanism leading to stationary patterns formation in stochastic systems with local dynamics and non-Fickian diffusion. It is shown that a multiplicative noise fulfilling a fluctuation-dissipation relation is able to induce and sustain stationary structures. It was found that at small and large noise intensities the system is characterized by unstable homogeneous states. At intermediate values of the noise intensity three types of patterns are possible: nucleation, spinodal decomposition and stripes with liner defects (dislocations). Our analytical investigations are verified by computer simulations.

Monitoring Aromatic Picosecond to Nanosecond Dynamics in Proteins via 13C Relaxation: Expanding Perturbation Mapping of the Rigidifying Core Mutation, V54A, in Eglin c

Long-range effects, such as allostery, have evolved in proteins as a means of regulating function via communication between distal sites. An NMR-based perturbation mapping approach was used to more completely probe the dynamic response of the core mutation V54A in the protein eglin c by monitoring changes in picosecond to nanosecond aromatic side-chain dynamics and H/D exchange stabilities. Previous side-chain dynamics studies on this mutant were limited to methyl-bearing residues, most of which were found to rigidify on the picosecond to nanosecond time scale in the form of a contiguous "network". Here, high precision (13)C relaxation data from 13 aromatic side chains were acquired by applying canonical relaxation experiments to a newly developed carbon labeling scheme [Teilum et al. (2006) J. Am. Chem. Soc. 128, 2506-2507]. The fitting of model-free parameters yielded S (2) variability which is intermediate with respect to backbone and methyl-bearing side-chain variability and tau e values that are approximately 1 ns. Inclusion of the aromatic dynamic response results in an expanded network of dynamically coupled residues, with some aromatics showing increases in flexibility, which partially offsets the rigidification in methyl side chains. Using amide hydrogen exchange, dynamic propagation on a slower time scale was probed in response to the V54A perturbation. Surprisingly, regional stabilization (slowed exchange) 10-12 A from the site of mutation was observed despite a global destabilization of 1.5 kcal x mol (-1). Furthermore, this unlikely pocket of stabilized residues colocalizes with increases in aromatic flexibility on the faster time scale. Because the converse is also true (destabilized residues colocalize with rigidification on the fast time scale), a plausible entropy-driven mechanism is discussed for relating colocalization of opposing dynamic trends on vastly different time scales.

Calcium-dependent Self-assembly of Human Centrin 2

Human centrin 2 (HsCen2) is a member of the EF-hand superfamily of calcium-binding proteins, often associated with the centrosomes and basal bodies. These organelles exhibit different morphological aspects, including a variety of centrin-containing fibers that connect the two centrioles or other structural elements of the pericentriolar space. The molecular basis of the Ca(2+)-sensitive fibers and their precise role in centrosome duplication are not known. To explore the possible structural role of HsCen2, we initiated a physicochemical study of the self-assembly properties of the purified protein in vitro. Using light scattering experiments, we investigated the temporal evolution of the assembly process and characterized the dependence on various chemical and physical factors, including temperature, di-cation concentration, ionic strength, protein concentration, and pH. The reversible self-assembly revealed many features of a large-size protein polymerization, with nucleation and elongation steps. Kinetic and equilibrium experiments show that a hydrophobic fluorescent probe (ANS) inhibits the polymerization by interfering with the nucleation step, probably through interactions with the apolar exposed sites on the protein surface. A truncated form of HsCen2, lacking the first 25 residues (Delta25HsCen2), shows no detectable self-assembly, pointing to the critical role played by the N-terminal fragment in the supermolecular organization of HsCen2. As revealed by isothermal titration experiments, the isolated N-terminal domains bind with a significant affinity (2 x 10(5) m(-1)) to preformed oligomers of Delta25HsCen2 through an entropy-driven mechanism.

Molecular dynamics simulation of the liquid–vapor interface: I. The orientational profile of 2-center Lennard–Jones and of Stockmayer fluid molecules

Molecular dynamics simulations were performed to study the structure and orientation of non-polar non-spherical 2-center Lennard–Jones and of dipolar spherical Lennard–Jones molecules in the vapor–liquid interface of a pure fluid. Our aim is a systematic and detailed investigation of the orientational behavior of dipolar spherical Stockmayer—and non-polar non-spherical 2-center Lennard–Jones molecules across the interface. The simulations were performed in a three-dimensional (3D) periodic system, the long range dipolar interactions were treated both by a modified Ewald summation and by a ‘cut-off’ procedure which takes into account only those direct interactions in the basic simulation cell. The results of both techniques agree very well in the case of the orientational behavior but they—naturally—deviate to some extent in the thermodynamic properties and interfacial tension. The orientational profiles were obtained for: (i) dipolar Lennard–Jones (Stockmayer) and (ii) for non-polar non-spherical 2-center Lennard–Jones molecules with varying dipolar strength and varying elongation over a range of temperatures. The resulting profiles exhibit the existence of two different types of orientational mechanism: an entropy-driven mechanism in the case of the non-spherical molecules, which results in S-shaped orientational profiles and an energy-driven mechanism that favors a parallel orientation of dipoles in the interface. The entropy-driven mechanism originates from the repulsive interactions of molecular cores whereas the energy-driven orientation is governed by the dipolar electrostatic field.

Massive magnetic-field-induced structural transformation in Gd5Ge4 and the nature of the giant magnetocaloric effect.

A massive magnetic-field-induced structural transformation in Gd5Ge4, which occurs below 30 K, was imaged at the atomic level by uniquely coupling high-resolution x-ray powder diffraction with magnetic fields up to 35 kOe. In addition to uncovering the nature of the magnetic field induced structural transition, our data demonstrate that the giant magnetocaloric effect, observed in low magnetic fields, arises from the amplification of a conventional magnetic entropy-driven mechanism by the difference in the entropies of two phases, borne by the concomitant structural transformation.

Tension and solute depletion in multilamellar vesicles.

We show that a metastable multilamellar vesicle ("onion"), in contact with excess solvent, can spontaneously deplete solute molecules from its interior through an unusual, entropy-driven mechanism. Fluctuation entropy is gained as the uneven partition of solute molecules helps the onion relieve tension in its lamellae. This mechanism accounts for recent experiments on the interaction between uncharged phospholipid onions and dissolved sugars.

Thermodynamically distinct high and low affinity states of the A(1) adenosine receptor induced by G protein coupling and guanine nucleotide ligation states of G proteins.

The influence of the receptor-G protein coupling state and the guanine nucleotide ligation state of the G protein on the binding mechanism of A(1) adenosine receptor ligands has been investigated in [(3)H]-1,3-dipropyl-8-cyclopentylxanthine ([(3)H]-DPCPX) binding studies in rat brain membranes. Thermodynamic parameters of binding of A(1) adenosine receptor ligands of different intrinsic activities were determined in the absence or presence of GDP and compared to the binding mechanism after receptor-G protein uncoupling. In agreement with previous studies, it was found that xanthine and non-xanthine antagonists showed an enthalpy- or enthalpy- and entropy-driven binding mechanism under all conditions. In contrast to antagonists, the binding mechanism of agonists was strongly affected by the G protein coupling state or the absence or presence of guanine nucleotides. Binding of full and partial agonists to the high-affinity state of the A(1) receptor was entropy-driven in the absence of GDP, and a good correlation between intrinsic activities and the contribution of entropy was observed. In the absence of GDP, binding of full and partial agonists and antagonists to the high affinity state of the receptor was thermodynamically discriminated. In contrast, no such discrimination was found in the presence of GDP. The binding mechanism of agonists to the low-affinity state of the receptor was identical to that of antagonists only after uncoupling of the receptor from G proteins by pretreatment with N-ethylmaleimide or guanosine-5'-(gamma-thio)-triphosphate (GTPgammaS). These results indicate the existence of two thermodynamically distinct high- and low-affinity states of the A(1) adenosine receptor.

Hypothetical entropy-driven mechanism for self-regulation of the size and division of primitive cells, suggesting the origin and nature of mesosomes

It is proposed that, when a critical cell radius was reached, spontaneous invaginations caused by adhesion of adjacent nodes in membranes rippled by Brownian bombardment, became progressive due to re-orientation of self-seeking molecules. Some circumstantial bacteriological evidence is adduced; the significance of mesosomes is discussed.