Temperature-dependent Folding Research Articles

Crystallization and melting of poly(butylene terephthalate) and poly(ethylene terephthalate) investigated by fast-scan chip calorimetry and small angle X-ray scattering

The crystallization and melting behaviors of poly(butylene terephthalate) (PBT) and poly(ethylene terephthalate) (PET) were examined by Hoffman-Weeks (H–W), Gibbs-Thomson (G-T), and thermal Gibbs-Thomson (t-G-T) plots constructed by using fast-scan calorimetry and small-angle X-ray scattering. With PBT and PET, neither an H–W nor a G-T plot could be utilized for the determination of the equilibrium melting point (TM0) of chain-extended infinite-size crystals. The thermal Gibbs-Thomson plot utilizes the change in the melting point and the heat of fusion of chain-folded crystals during the secondary stage of isothermal crystallization. TM0 was determined from a t-G-T plot, and the results were in good agreement with the literature values for PBT and PET. G-T and t-G-T plots suggested a temperature-dependent folding surface free energy (σe), as has been proposed by Hoffman et al. The σe values obtained with G-T and t-G-T plots support the consistency of the analysis.

Novel Heat-Promoted Folding Dynamics of the yybP-ykoY Manganese Riboswitch: Kinetic and Thermodynamic Studies at the Single-Molecule Level

Riboswitches are highly structured RNA elements that regulate gene expressions by undergoing conformational changes in response to their cognate ligands. Such regulatory strategies are particularly prevalent among bacteria, which need to be evolutionarily responsive to thermal fluctuations in the surrounding environment, for example, generating extremophiles evolved to survive anomalously high or low temperatures. As a consequence, the response of such riboswitches to thermal stress becomes of considerable interest. In this study, the temperature-dependent folding kinetics and thermodynamics of the manganese riboswitch ( yybP-ykoY) is studied by single-molecule FRET spectroscopy under external thermal control. Surprisingly, the folding of the manganese riboswitch is found to be strongly promoted by temperature. Detailed thermodynamic analysis of the equilibrium and forward/reverse kinetic rate constants reveal folding to be a strongly endothermic process (Δ H0) made feasible by an overall entropic lowering (- TΔ S0) in free energy. This is in contrast to a more typical picture of RNA folding achieving a more compact, highly ordered state (Δ S0) and clearly speaks to the significant role of solvent/cation reorganization in the folding thermodynamics. With the help of the transition-state theory, free energy landscapes for the manganese riboswitch are constructed from the temperature-dependent kinetic data, revealing two distinctive folding mechanisms promoted by Mg2+ and Mn2+, respectively. It is speculated that this unconventional temperature dependence for folding of the manganese riboswitch may reflect evolution of bacterial gene regulation strategies to survive environments with large-temperature variations.

Detailed Kinetics of RNA Folding Pathways and Thermodynamic Origins of Crowding by Single-Molecule FRET

Specific three dimensional folding is required for RNA function while RNA can be a highly dynamic system that actively samples different configurations under thermal fluctuations. For instance, in gene regulation of RNA riboswitch, the prompt structural transition in response to ligand is crucial for triggering transcriptional termination, making the time scale of folding and ligand response exceedingly relevant to RNA functions. In single-molecule FRET (smFRET) experiments, the conformational changes of RNA are visualized by the time-dependent energy transfer to determine folding and unfolding rates. Such kinetic information allows us to construct a model for manganese riboswitch folding and explore the detailed folding mechanisms, where Mg2+ binding is found to pre-fold the RNA, followed by ligand (Mn2+) association to further stabilize the folded structure. Moreover, the thermodynamic origins can be obtained from temperature dependent folding. The manganese riboswitch folding is found unexpectedly promoted by heat and the process is endothermic with net entropy gain, much contradictory to the conventional idea of folding into a more order state. In molecular crowding study where we try to understand the influence of fundamental excluded volume on RNA structures in highly concentrated intracellular environments, the chemically inert crowders are shown to entropically favor the more compact folded structure of RNA; a simple physical picture of osmotic pressure is employed to explain the crowder size dependence. Lastly, a hydraulic pressure generator is used to apply pressure (up to 5 kbar) to RNA sample through capillary. The free volume change (pΔV) during RNA folding may be related to structural response under crowding conditions.

Temperature-dependent folding allows stable dimerization of secretory and virus-associated E proteins of Dengue and Zika viruses in mammalian cells

Dengue and Zika are two of the most important human viral pathogens worldwide. In both cases, the envelope glycoprotein E is the main target of the antibody response. Recently, new complex quaternary epitopes were identified which are the consequence of the arrangement of the antiparallel E dimers on the viral surface. Such epitopes can be exploited to develop more efficient cross-neutralizing vaccines. Here we describe a successful covalent stabilization of E dimers from Dengue and Zika viruses in mammalian cells. Folding and dimerization of secretory E was found to be strongly dependent on temperature but independent of PrM co-expression. In addition, we found that, due to the close relationship between flaviviruses, Dengue and Zika viruses E proteins can form heterodimers and assemble into mosaic viral particles. Finally, we present new virus-free analytical platforms to study and screen antibody responses against Dengue and Zika, which allow for differentiation of epitopes restricted to specific domains, dimers and higher order arrangements of E.

Softenable composite boom for reconfigurable and self-deployable structures

ABSTRACTThis article investigates (experimentally and numerically) the time and temperature-dependent folding and unfolding behavior, including the structural nonlinearity, of thin-walled softenable composite structures. The composites are fabricated with woven fabric fibers and a heat softenable material of the shape memory polymer (SMP) resin. A numerical model is developed. The model is based on the elastic properties of the woven fabric fibers, their fabric geometry, and the thermo-viscoelastic properties of the SMP resin. A thin-walled cylindrical boom, which can be flexibly folded into an arbitrary configuration and be self-deployed, is fabricated using the heat softenable composite. The viscoelasticity and the shape memory behaviors are investigated during the folding and unfolding of the boom at elevated temperatures.

Aromatic oligoamides with increased backbone flexibility: improved synthetic efficiencies, solvent-dependent folding and cooperative conformational transitions

A 15-residue aromatic oligoamide with a backbone of increased flexibility exhibits solvent- and temperature-dependent folding and highly cooperative conformational transition.

Temperature-Dependent Hammond Behavior in a Protein-Folding Reaction: Analysis of Transition-State Movement and Ground-State Effects

Characterization of the transition-state ensemble and the nature of the free-energy barrier for protein folding are areas of intense activity and some controversy. A key issue that has emerged in recent years is the width of the free-energy barrier and the susceptibility of the transition state to movement. Here we report denaturant-induced and temperature-dependent folding studies of a small mixed α–β protein, the N-terminal domain of L9 (NTL9). The folding of NTL9 was determined using fluorescence-detected stopped-flow fluorescence measurements conducted at seven different temperatures between 11 and 40 °C. Plots of the log of the observed first-order rate constant versus denaturant concentration, “chevron plots,” displayed the characteristic V shape expected for two-state folding. There was no hint of deviation from linearity even at the lowest denaturant concentrations. The relative position of the transition state, as judged by the Tanford β parameter, β T, shifts towards the native state as the temperature is increased. Analysis of the temperature dependence of the kinetic and equilibrium m values indicates that the effect is due to significant movement of the transition state and also includes a contribution from temperature-dependent ground-state effects. Analysis of the Leffler plots, plots of Δ G ‡ versus Δ G°, and their cross-interaction parameters confirms the transition-state movement. Since the protein is destabilized at high temperature, the shift represents a temperature-dependent Hammond effect. This provides independent confirmation of a recent theoretical prediction. The magnitude of the temperature-denaturant cross-interaction parameter is larger for NTL9 than has been reported for the few other cases studied. The implications for temperature-dependent studies of protein folding are discussed.

Probing the Transition State Ensemble of a Protein Folding Reaction by Pressure-Dependent NMR Relaxation Dispersion

The F61A/A90G mutant of a redesigned form of apocytochrome b562 folds by an apparent two-state mechanism. We have used the pressure dependence of 15N NMR relaxation dispersion rate profiles to study the changes in volumetric parameters that accompany the folding reaction of this protein at 45 degrees C. The experiments were performed under conditions where the folding/unfolding equilibrium could be studied at each pressure without addition of denaturants. The exquisite sensitivity of the methodology to small changes in folding/unfolding rates facilitated the use of relatively low-pressure values (between 1 and 270 bar) so that pressure-induced changes to the unfolded state ensemble could be minimized. A volume change for unfolding of -81 mL/mol is measured (at 1 bar), a factor of 1.4 larger (in absolute value) than the volume difference between the transition state ensemble (TSE) and the unfolded state. Notably, the changes in the free energy difference between folded and unfolded states and in the activation free energy for folding were not linear with pressure. Thus, the difference in the isothermal compressibility upon unfolding (-0.11 mL mol(-1) bar(-1)) and, for the first time, the compressibility of the TSE relative to the unfolded state (0.15 mL mol(-1) bar(-1)) could be calculated. The results argue for a TSE that is collapsed but loosely packed relative to the folded state and significantly hydrated, suggesting that the release of water occurs after the rate-limiting step in protein folding. The notion of a collapsed and hydrated TSE is consistent with expectations based on earlier temperature-dependent folding studies, showing that the barrier to folding at 45 degrees C is entropic (Choy, W. Y.; Zhou, Z.; Bai, Y.; Kay, L. E. J. Am. Chem. Soc. 2005, 127, 5066-5072).

Persistent Glycoprotein Misfolding Activates the Glucosidase II/UGT1-Driven Calnexin Cycle to Delay Aggregation and Loss of Folding Competence

The UDP-glucose:glycoprotein glucosyltransferase (UGT) is a central player of glycoprotein quality control in the endoplasmic reticulum (ER). UGT reglucosylation of nonnative glycopolypeptides prevents their release from the calnexin cycle and secretion. Here, we compared the fate of a glycoprotein with a reversible, temperature-dependent folding defect in cells with and without UGT1. Upon persistent misfolding, tsO45 G was slowly released from calnexin and entered a second level of retention-based ER quality control by forming BiP/GRP78-associated disulfide-bonded aggregates. This correlated with loss in the ability to correct misfolding. Deletion of UGT1 did not affect the stringency of ER quality control. Rather, it accelerated release from calnexin and transfer to the second ER quality control level, but it did so after an unexpectedly long lag, showing that cycling in the calnexin chaperone system is not frenetic, as claimed by existing models, and is fully activated only upon persistent glycoprotein misfolding.

Investigation of Mg2+- and temperature-dependent folding of the hairpin ribozyme by photo-crosslinking: effects of photo-crosslinker tether length and chemistry

We have used photo-crosslinking to investigate the structure and dynamics of four-way junction hairpin ribozyme constructs. Four phenylazide photo-crosslinkers were coupled to 2′-NH2-modified U+2 in the substrate and irradiated at different Mg2+ concentrations and temperatures. Consistent with the role of divalent metal ions in hairpin ribozyme folding, we observed more interdomain crosslinks in the presence of Mg2+ than in its absence. In general, we observed intradomain crosslinks to nucleotides 2–11 and interdomain crosslinks to the U1A binding loop. Crosslinks to A26 and G36 in domain B were also observed when crosslinking was carried out at −78°C. In contrast to crosslinking results at higher temperatures (0, 25 and 37°C), similar crosslinks were obtained in the presence and absence of Mg2+ at −78°C, suggesting Mg2+ stabilizes a low-energy hairpin ribozyme conformation. We also evaluated the effects of photo-crosslinker structure and mechanism on crosslinks. First, most crosslinks were to unpaired nucleotides. Second, shorter and longer photo-crosslinkers formed crosslinks to intradomain locations nearer to and farther from photo-crosslinker modification, respectively. Finally, fluorine substitutions on the phenylazide ring did not change the locations of crosslinks, but rather decreased crosslinking efficiency. These findings have implications for the use of phenylazide photo-crosslinkers in structural studies of RNA.

Temperature-Dependent Conformational Transitions and Hydrogen-Bond Dynamics of the Elastin-Like Octapeptide GVG(VPGVG): A Molecular-Dynamics Study

A joint experimental/theoretical investigation of the elastin-like octapeptide GVG(VPGVG) was carried out. In this article a comprehensive molecular-dynamics study of the temperature-dependent folding and unfolding of the octapeptide is presented. The current study, as well as its experimental counterpart (see companion article in this issue) find that this peptide undergoes an inverse temperature transition (ITT), leading to a folding at ∼40–60°C. In addition, an unfolding transition is identified at unusually high temperatures approaching the normal boiling point of water. Due to the small size of the system, two broad temperature regimes are found: the ITT regime at ∼10–60°C and the unfolding regime at ∼ T > 60°C, where the peptide has a maximum probability of being folded at T ≈ 60°C. A detailed molecular picture involving a thermodynamic order parameter, or reaction coordinate, for this process is presented along with a time-correlation function analysis of the hydrogen-bond dynamics within the peptide as well as between the peptide and solvating water molecules. Correlation with experimental evidence and ramifications on the properties of elastin are discussed.

High-Throughput Studies of the Effects of Polymer Structure and Solution Components on the Phase Separation of Thermoresponsive Polymers

This paper describes a high-throughput method for characterization of the temperature-dependent folding of poly(N-isopropylacrylamide), PNIPAM, using dark field microscopy to measure this thermoresponsive polymer's lower critical solution temperature (LCST). The effect of ionic solution components (halide and alkali metal ions) on the polymer precipitation temperature follows the Hofmeister series whereas solution isotopic effects using D2O give rise to a roughly linear increase in the LCST with the mole fraction of D2O added. The polymer structure itself was also varied through the synthesis of N-alkylacrylamide copolymers. It was found that replacement of the isopropyl N-alkyl pendant groups of PNIPAM with varying amounts of N-n-propyl groups results in a monotonic decrease in the LCST. Repeated analyses of the same sample and of separately prepared samples show that this type of analysis is quite precise and that investigations of subtle effects of polymer microstructure and solvation are feasible even when the difference in LCST temperatures is <1 °C. Such effects may be difficult to study by other methods.

Lipid unsaturation determines the interaction of AFP type I with model membranes during thermotropic phase transitions

We have previously shown that antifreeze protein (AFP) type I from winter flounder interacts with the acyl chains of lipids in model membranes containing a mixture of dimyristoylphosphatidylcholine (DMPC) and the plant thylakoid lipid digalactosyldiacylglycerol (DGDG), most likely through hydrophobic interactions. By contrast, in studies with pure phospholipid membranes, no such interaction was seen. DGDG is a highly unsaturated lipid, which renders these studies quite different from the previous studies of AFP-membrane interaction where the lipids were saturated or trans-unsaturated. Therefore, it seemed possible that either the digalactose headgroups or the unsaturated DGDG acyl chains, or both, may be important for interactions of membranes with AFP type I. To distinguish between these possibilities, we catalytically hydrogenated the DGDG to obtain a galactolipid with completely saturated fatty acyl chains. The results with the hydrogenated DGDG were strikingly different from those obtained previously with the unsaturated DGDG; the clear binding of AFPs to the bilayer appeared to be lost. Nevertheless, the temperature-dependent folding of AFP type I was inhibited in the presence of liposomes containing either the unsaturated or the hydrogenated DGDG. The results indicate that the liposomes and protein still interact, even following hydrogenation of the acyl chains, perhaps at the membrane–solution interface.

RNA hairpin-folding kinetics.

Based on the complete ensemble of hairpin conformations, a statistical mechanical model that combines the eigenvalue solutions of the rate matrix and the free-energy landscapes has been able to predict the temperature-dependent folding rate, kinetic intermediates, and folding pathways for hairpin-forming RNA sequences. At temperatures higher than a "glass transition" temperature, T(g), the eigenvalues show a distinct time separation, and the rate-limiting step is a two-state single exponential process determined by the slowest eigenmode. At temperatures lower than T(g), no distinct time separation exists for the eigenvalues, hence multiple (slow) eigenmodes contribute to the rate-determining processes, and the folding involves the trapping and detrapping of kinetic intermediates. For a 21-nt sequence we studied, T(g) is lower than the transition temperature, T(m), for thermodynamic equilibrium folding. For T > T(m), starting from the native state, the chain undergoes a biphasic unfolding transition: a preequilibrated quasi-equilibrium macrostate is formed followed by a rate-limiting two-state transition from the macrostate to the unfolded state. For T(g) < T < T(m), the chain undergoes a two-state on-pathway folding transition, at which a nucleus is formed by the base stacks close to the loop region before a rapid assembly of the whole hairpin structure. For T < T(g), the multistate kinetics involve kinetic trapping, causing the roll-over behavior in the rate-temperature Arrhenius plot. The complex kinetic behaviors of RNA hairpins may be a paradigm for the folding kinetics of large RNAs.

Liposomes as formulation excipients for protein pharmaceuticals: a model protein study.

The advent of recombinant DNA technology has made possible the pharmaceutical use of a wide range of proteins and peptides. However, the complex structure of proteins renders them susceptible to physical instabilities such as denaturation, aggregation and precipitation. We tested the hypothesis that partial unfolding and exposure of hydrophobic domains leads to physical instability, and investigated approaches to stabilize protein formulations. KP6 beta, an 81 amino acid killer toxin from Ustilago maydis, was used as a model protean. Circular dichroism and fluorescence spectroscopy were used to study the temperature dependent folding/ unfolding characteristics of KP6 beta. ANS (1,8 anilinonaphthalene sulfonate), a fluorescent probe that partitions into hydrophobic domains, was used to detect exposure of hydrophobic domains. As the temperature was elevated, near-UV CD indicated progressive loss of KP6 beta tertiary structure, while far-UV CD indicated retention of secondary structure. Increasing exposure of hydrophobic domains was observed, as indicated by the penetration of ANS. At elevated temperatures (60 degrees C), KP6 beta3 conserved most secondary structural features. However, tertiary structure was disordered, suggesting the existence of a partially folded, structured intermediate state. Liposomes bound to partially unfolded structures and prevented the formation of aggregates. Partial unfolding resulted in increased exposure of hydrophobic domains and aggregation of KP6 beta, but with preservation of secondary structure. Liposomes interacted with the structured intermediate state, stabilizing the protein against aggregation. These results suggest a general formulation strategy for proteins, in which partially unfolded structures are stabilized by formulation excipients that act as molecular chaperones to avoid physical instability.

Localization and Suppression of a Kinetic Defect in Cystic Fibrosis Transmembrane Conductance Regulator Folding

A growing body of evidence indicates that the most common cystic fibrosis-causing mutation, DeltaF508, alters the ability of the cystic fibrosis transmembrane conductance regulator (CFTR) protein to fold and transit to the plasma membrane. Here we present evidence that the DeltaF508 mutation affects a step on the folding pathway prior to formation of the ATP binding site in the nucleotide binding domain (NBD). Notably, stabilization of the native state with 4 mM ATP does not alter the temperature-dependent folding yield of the mutant DeltaF508 NBD1 in vitro. In contrast, glycerol, which promotes DeltaF508-CFTR maturation in vivo, increases the folding yield of NBD1DeltaF and reduces the off pathway rate in vitro, although it does not significantly alter the free energy of stability. Likewise a second site mutation, R553M, which corrects the maturation defect in vivo, is a superfolder which counters the effects of DeltaF508 on the temperature-dependent folding yield in vitro, but does not significantly alter the free energy of stability. A disease-causing mutation, G551D, which does not alter the maturation of CFTR in vivo but rather its function as a chloride channel, and the S549R maturation mutation have no discernible effect on the folding of the domain. These results demonstrate that DeltaF508 is a kinetic folding mutation that affects a step early in the process, and that there is a significant energy barrier between the native state and the step affected by the mutation precluding the use of native state ligands to promote folding. The implications for protein folding in general are that the primary sequence may not necessarily simply define the most stable native structure, but rather a stable structure that is kinetically accessible.

Quality control in the secretory pathway: retention of a misfolded viral membrane glycoprotein involves cycling between the ER, intermediate compartment, and Golgi apparatus.

Proteins synthesized in the ER are generally transported to the Golgi complex and beyond only when they have reached a fully folded and assembled conformation. To analyze how the selective retention of misfolded proteins works, we monitored the long-term fate of a membrane glycoprotein with a temperature-dependent folding defect, the G protein of tsO45 vesicular stomatitis virus. We used indirect immunofluorescence, immunoelectron microscopy, and a novel Nycodenz gradient centrifugation procedure for separating the ER, the intermediate compartment, and the Golgi complex. We also employed the folding and recycling inhibitors dithiothreitol and AIF4-, and coimmunoprecipitation with calnexin antibodies. The results showed that the misfolded G protein is not retained in the ER alone; it can move to the intermediate compartment and to the cis-Golgi network but is then recycled back to the ER. In the ER it is associated with calnexin and BiP/GRP78. Of these two chaperones, only BiP/GRP78 seems to accompany it through the recycling circuit. Thus, the retention of this misfolded glycoprotein is the result of multiple mechanisms including calnexin binding in the ER and selective retrieval from the intermediate compartment and the cis-Golgi network.