Temperature-induced Conformational Transition Research Articles

A suicidal mechanism for the exquisite temperature sensitivity of TRPV1

The vanilloid receptor TRPV1 is an exquisite nociceptive sensor of noxious heat, but its temperature-sensing mechanism is yet to define. Thermodynamics dictate that this channel must undergo an unusually energetic allosteric transition. Thus, it is of fundamental importance to measure directly the energetics of this transition in order to properly decipher its temperature-sensing mechanism. Previously, using submillisecond temperature jumps and patch-clamp recording, we estimated that the heat activation for TRPV1 opening incurs an enthalpy change on the order of 100 kcal/mol. Although this energy is on a scale unparalleled by other known biological receptors, the generally imperfect allosteric coupling in proteins implies that the actual amount of heat uptake driving the TRPV1 transition could be much larger. In this paper, we apply differential scanning calorimetry to directly monitor the heat flow in TRPV1 that accompanies its temperature-induced conformational transition. Our measurements show that heat invokes robust, complex thermal transitions in TRPV1 that include both channel opening and a partial protein unfolding transition and that these two processes are inherently coupled. Our findings support that irreversible protein unfolding, which is generally thought to be destructive to physiological function, is essential to TRPV1 thermal transduction and, possibly, to other strongly temperature-dependent processes in biology.

Linking B-factor and temperature-induced conformational transition

The crystallographic B-factor, also called temperature factor or Debye-Waller factor, has long been used as a surrogate for local protein flexibility. However, the use of the absolute B-factor as a probe for protein motion requires reproducible validation against conformational changes against chemical and physical variables. Here we report the investigation of the thermal dependence of the crystallographic B-factor and its correlation with conformational changes of the protein. We obtained the crystal protein structure coordinates and B-factors at high resolution (1.5 Å) over a broad temperature range (100 K to 325 K). The exponential thermal dependence of B-factor as a function of temperature was equal for both the diffraction intensity data (Wilson B-factor) and for all modeled atoms of the system (protein and non-protein atoms), with a thermal diffusion constant of about 0.0045 K−1, similar for all atoms. The extrapolated B-factor at zero Kelvin (or zero-point fluctuation) varies among the atoms, although with no apparent correlation with temperature-dependent protein conformational changes. These data suggest that the thermal vibration of the atom does not necessarily correlate with the conformational dynamics of the protein.

Model for the Temperature-Induced Conformational Change in Xanthan Polysaccharide.

Xanthan is an extracellular bacterial polysaccharide. It is manufactured commercially by fermentation of Xanthomonas campestris and used extensively in food and other industries to control the viscosity and texture of various products. Its useful properties stem from its occurrence both as a relatively rigid double-helical polymer and as a branched polymer network presumably crosslinked by the same noncovalent interactions that stabilize the double-helical form. Interconversion of these two forms can be achieved through heating and cooling processes. This paper describes a model for this thermally induced transformation under conditions of very dilute aqueous polymer concentration, where the characteristics of double-helical and crosslinked aggregates can be studied experimentally using light scattering. Because xanthan is a regularly repeating copolymer, there is no requirement for specific registration of the two strands of the duplex structure as is required in naturally occurring nucleic acid double helices. Here, we demonstrate the important role of the resulting xanthan structural degeneracy in dictating the characteristics of the temperature-induced conformational transition.

Dissecting the conformational determinants of chitosan and chitlac oligomers.

Chitosan and its highly hydrophilic 1-deoxy-lactit-1-yl derivative (Chitlac) are polysaccharides with increasing biomedical applications. Aimed to unravel their conformational properties we have performed a series of molecular dynamics simulations of Chitosan/Chitlac decamers, exploring different degrees of substitution (DS) of lactitol side chains. At low DS, two conformational regions with different populations are visited, while for DS ≥ 20% the oligomers remain mostly linear and only one main region of the glycosidic angles is sampled. These conformers are (locally) characterized by extended helical "propensities". Helical conformations 32 and 21, by far the most abundant, only develop in the main region. The accessible conformational space is clearly enlarged at high ionic strength, evidencing also a new region accessible to the glycosidic angles, with short and frequent interchange between regions. Simulations of neutral decamers share these features, pointing to a central role of electrostatic repulsion between charged moieties. These interactions seem to determine the conformational behavior of the chitosan backbone, with no evident influence of H-bond interactions. Finally, it is also shown that increasing temperature only slightly enlarges the available conformational space, but certainly without signs of a temperature-induced conformational transition.

Temperature‐induced conformational transition of bovine serum albumin in neutral aqueous solution by reversed‐phase liquid chromatography

The temperature-induced conformational transition of bovine serum albumin (BSA) in neutral aqueous solution was studied using intrinsic fluorescence emission spectrum, reversed-phase liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the conformation transition thermodynamic parameters were determined in the temperature range 12-50 °C. The results showed that, in the temperature range 12-20 °C, BSA only existed in a single conformation state A, while in the temperature range 22-50 °C, it existed in two different conformation states: A and B. The percentage of conformation state A decreased while that of conformation state B increased with the increase in temperatures, and when temperature approached 50 °C conformation state B accounted for approximately 25% of all conformation states of BSA. In the conformational transition of BSA from conformation state A to conformation state B, the positive enthalpy change, entropy change and free energy changes demonstrated that the conformational transition was endothermic, nonspontaneous and mainly entropy-driven.

Temperature-Induced Conformational Transition of a Model Elastin-like Peptide GVG(VPGVG)3 in Water

The conformation of a single elastin-like peptide GVG(VPGVG)3 in liquid water is studied by computer simulations in the temperature interval between 280 and 440 K. Two main conformational states of the peptide can be distinguished: a rigid conformational state, dominating at low temperatures, and a flexible conformational state, dominating at high temperatures. A temperature-induced transition between these states occurs at about 310 K, rather close to a transition temperature seen in experiments. This transition is accompanied by the thermal breaking of the hydrogen-bonded spanning network of the hydration water via a percolation transition upon heating. This finding indicates that the H-bond clustering structure of the hydration water plays an important role in the conformational stability of biomolecules. A second important observation is the Gaussian distribution of the end-to-end distance in the high-temperature state, which supports the idea of a rubber-like elasticity of the studied elastin-like peptide. Finally our results challenge the idea of the folding of elastin-like peptides upon heating.

Fluorescence Spectroscopic Studies of Solvent- and Temperature-Induced Conformational Transition in Segmented Poly[2-methoxy-5-(2‘-ethylhexyl)oxy-1,4-phenylenevinylene] (MEHPPV)

Segmented poly[2-methoxy-5-(2'-ethylhexyl)oxy-1,4-phenylenevinylene] (MEHPPV-x, where x is the mole percent of conjugated segments) represent unique polymeric systems in which a large number of chromophores of different molecular conjugation lengths (different excitation/emission energies) are strung together in a single polymer chain, which thereby forces them to occupy a relatively small volume that is determined by the hydrodynamic size of the macromolecule. Changing the hydrodynamic volume by varying either solvent composition or temperature, therefore, provides a straightforward approach to modulate the interaction between the chromophores. Fluorescence spectroscopic studies of very dilute solutions (in dichloromethane/1,2-dichloroethane) of segmented MEHPPVs with increasing amounts of a nonsolvent (methanol/cyclohexane/ethanol) reveal an interesting inverted S-shaped variation in the emission yield with increasing nonsolvent composition, which is accompanied by a red shift of the emission maxima. Both these changes are indications of a conformational collapse of individual polymer chains, resulting in enhanced energy transfer from highly emissive short conjugation length segments to weakly emissive longer ones and/or to the formation of weakly emissive interchromophore excitons. The solvent composition at the onset of the steep decline in the S-shaped curve, termed the chain-collapse point, was found to vary with the extent of conjugation in a manner which suggests that the polymers bearing longer chromophores undergo collapse earlier than those bearing shorter ones. In samples with very low levels of conjugation, such as in MEHPPV-10, a rather unexpected observation of an initial increase in the emission yield prior to the sudden decline was seen. This is consistent with the unusual conjugation length dependence of the fluorescence quantum yield of oligoPPVs (OPV) reported by earlier workers. Fluorescence spectral variation as a function of temperature also reveals several interesting features of these systems, the most interesting of which is the observation that either an increase or a decrease of emission yields with increase in temperature could be observed, depending on the initial conformation of the polymer chain. When the chain is in a highly collapsed (or a highly solvated) state, an increase in temperature causes the expected decrease in emission yield, while in an intermediate partially collapsed conformation, a significant increase in the emission yield with temperature is observed. The latter observation is ascribed to polymer coil expansion that results in depletion of aggregated species and/or a reduction in energy transfer to weakly emitting chromophoric segments.

The wild type bacterial Co 2+/Co 2+-phosphotriesterase shows a middle-range thermostability

The phosphotriesterase (PTE) from Pseudomonas diminuta, a metalloenzyme that catalyses the hydrolysis of organophosphorus pesticides and nerve agents, has been described as a remarkably heat-stable protein [Grimsley et al., Biochemistry 36 (1997), 14366–14374]. Because substitution of the naturally occurring zinc ions by cobalt ions was found to enhance the enzyme catalytic activity, we investigated the thermal stability of the Co 2+/Co 2+-PTE. This study, carried out using capillary electrophoresis under optimised conditions in the pH range 9–10 compatible with optimal enzyme activity, provided evidence for irreversible denaturation according to the Lumry–Eyring model. A temperature-induced conformational transition ( T m≈58°C) and an early growing of aggregates were observed. Comparison of UV spectra with heat-induced inactivation data clearly demonstrated that the PTE state populated above T m was neither native nor active. Differential scanning calorimetry showed only an exothermic trace due to aggregation of the denatured protein at T=76°C. Accordingly, the temperature-induced denaturation process of the PTE could be described by a consecutive reaction model, including formation of an intermediate with enhanced activity at T≈45°C and an inactive unfolded state populated at T≈58°C, which leads to denatured aggregates. Thus, the wild type Co 2+/Co 2+-PTE displays a middle-range thermostability. Hence, for decontamination purposes under extreme Earth temperatures, wild type and engineered mutants of PTE substituted with other metal cations should be evaluated.

Fourier-transform infrared spectroscopic investigation of the thermal denaturation of hen egg-white lysozyme dissolved in aqueous buffer and glycerol

The thermal denaturation of lysozyme dissolved in aqueous phosphate buffer (pH 5.1) and glycerol was studied by Fourier-transform infrared (FTIR) spectroscopy. In both solvents, a single temperature-induced conformational transition was observed but at the distinctly different temperatures of 73 °C in aqueous buffer and 94 ± 2 °C in glycerol. No changes in the secondary structure were observed in glycerol up to 90 °C. Thus, FTIR data were consistent with the formation of a highly ordered molten globule state at temperatures below 90 °C followed by lysozyme unfolding at higher temperatures in glycerol.

Temperature-induced conformational transition of intestinal fatty acid binding protein enhancing ligand binding: a functional, spectroscopic, and molecular modeling study.

Intestinal fatty acid binding protein (IFABP) undergoes a reversible thermal transition between 35 and 50 degreesC, as revealed by circular dichroism spectroscopy in the near-UV region. For the apoprotein, the molar ellipticity measured at 254 nm (possibly implicating the environment around F17 and/or F55) decreases significantly in this temperature range, while in the holoprotein (bound to oleic acid), this phenomenon is not observed. Concomitantly, an increase in the activity of binding to [14C]oleic acid occurs. Nevertheless, other spectroscopic evidence indicates that the beta-barrel structure, the major motif of this protein, is highly stable up to 70 degreesC. No changes associated with conformation were detected for both structures by fourth-derivative analysis of the UV absorption spectra, circular dichroism in the far-UV region, and intrinsic fluorescence measurements. Further structural information arises from experiments in which binding to the anionic fluorescent probes 1-anilinonaphthalene-8-sulfonic acid (ANS) and its dimer bisANS was examined. The fluorescence intensity of bound ANS diminishes monotonically, whereas that of bisANS increases slightly in the temperature range of 35-50 degreesC. Given the different size of these probes, model building suggests that ANS would be able to sense regions located deeply inside the cavity, while bisANS could also reach the vicinity of the small helical domain of this protein. In light of these results, we believe that this subtle conformational transition of IFABP, which positively influences the binding activity, would involve fluctuations at the peripheral "entry portal" region for the ligand. This interpretation is compatible with the discrete disorder observed in this place in apo-IFABP, as evidenced by NMR spectroscopy [Hodsdon, M. E., and Cistola, D. P. (1997) Biochemistry 36, 1450-1460].

Effect of temperature on the intrinsic viscosity and conformation of chitosans in dilute HCl solution

The effects of temperature on the intrinsic viscosity and on the conformation of chitosans in dilute HCl solution were studied. Ten chitosans with the same degree of deacetylation but different molecular weights were produced by alkali deacetylation of chitin which was prepared from red shrimp wastes. The degree of deacetylation at 83% and weight average molecular weight of the chitosans ranging 78–914 kDa were determined by infrared spectroscopy and static light scattering, respectively. The intrinsic viscosities ([ η]) of these 10 chitosans in 0.01 M hydrochloric acid were measured at 10, 20, 30, 40, and 50°C. Then, d ln [ η]/d(1/ T) and the Mark-Houwink exponents were calculated as the indices for chain flexibility and molecule conformation, respectively. These results showed: the intrinsic viscosities decreased linearly with increasing temperature, therefore, a temperature-induced conformational transition did not occur for all 10 different molecular weight chitosans in the temperature range studied. Values of d ln [ η]/d(1/ T) were between 633 and 1334 and increased with decreasing molecular weight, indicating that higher molecular weight chitosans are more flexible. Between 10° and 50°C, the Mark-Houwink exponents ranged 0.64–0.76 and increased with increasing temperature, indicating that the conformation of these chitosans were all in random coil, and a temperature-induced conformational transition did not occur. The a* and a** Mark-Houwink exponents represent those chitosans whose molecular weights are larger and smaller than 223 kDa, respectively, and were obtained by using 223 kDa as the break point in the double logarithmic plots of the intrinsic viscosities and weight average molecular weight. Values of a** were between 0.41 and 0.54, while the a* values were from 0.96 to 1.07. These values for a** and a* indicate that larger and smaller molecular weight chitosans were in random coil and rod shape, respectively.

PH and temperature-induced molten globule-like denatured states of equinatoxin II: a study by UV-melting, DSC, far- and near-UV CD spectroscopy, and ANS fluorescence.

Thermal denaturation of equinatoxin II (EqTxII) in glycine buffer solutions (pH 1.1, 2.0, 3.0, and 3.5) and in triple distilled water (pH 5.5-6.0) was examined by differential scanning calorimetry, UV and CD spectroscopy and fluorescence emission spectroscopy of the added hydrophobic fluorescent probe ANS. At pH 5.5-6.0 and at temperatures below 60 degrees C, the protein exists in a native state characterized by a pronounced tertiary structure, a beta-rich secondary structure and a low degree of ANS-binding. At higher temperatures, it undergoes a two-state conformational transition, (delta H degree)VH = (delta H degree)DSC, into an unfolded state, which is characterized by a complete collapse of its tertiary structure and an incomplete denaturation of its secondary structure. At acidic pH, the EqTxII temperature-induced conformational transition appears at lower temperatures as non-two-state transition accompanied by the formation of an intermediate state which shows characteristics of molten globules, i.e., absence of defined tertiary structure, increase in alpha-rich secondary structure, and high affinity for ANS. At pH 2.0, the low-temperature initial state of EqTxII is already partially denatured; the tertiary structure is partially disrupted, and a pronounced inequality (delta H degree)VH > (delta H degree)DSC is observed. At pH value of 1.1 and below 60 degrees C, EqTxII exists in a stable acid-denatured compact state which shows all the characteristics of a molten globule, which even at 95 degrees C is not completely denatured. According to numerous studies on the pore forming toxins, such acid-denatured compact states may contribute to the protein's ability to penetrate into biological membranes.

Time-resolved fluorescence anisotropy studies of the temperature-induced intramolecular conformational transition of poly( N-isopropylacrylamide) in dilute aqueous solution

Time-resolved fluorescence anisotropy measurements have been performed upon an acenaphthylene-labelled (0.5 mol%) sample of poly( N-isopropylacrylamide), PNIPAM, in dilute solutions in both methanol and water as solvents. In methanol, segmental relaxation of PNIPAM follows an Arrhenius dependence upon temperature over the range investigated (279–323K) characterized by an ‘activation energy’ of 13.4(±0.5) kJ mol −1. This is only slightly greater (by ca 2.4 kJ mol −1) than that of solvent flow and it is likely that specific dipolar interactions between the PNIPAM and the methanol determine the macromolecular dynamics in this solvent. In aqueous solution, the segmental mobility of PNIPAM exhibits a dramatic thermoreversible discontinuity at ca 32°C. This change in conformational behaviour occurs at the polymer's lower critical solution temperature in aqueous media. This observation, supports the proposition (Winnik, F. M. Polymer 1990, 31, 2125) that the thermally-induced separation in this system occurs by a ‘dual mode’ mechanism wherein intermolecular aggregation is preceded by intramolecular conformational shrinkage of the polymer coils.

N.m.r. study of conformational changes in lysozyme around the thermal transition point.

Natural abundance carbon-13 n.m.r. at 50.3 MHz has been used to further document the thermal transition that hen egg-white lysozyme undergoes in solution between 20 degrees and 30 degrees. The study focuses on the temperature sensitivity of more than 50 carboxylic, aromatic and aliphatic single carbon resonances for which unambiguous assignments to specific residues are known. The analysis of selective perturbations in chemical shifts indicates that residues located on both edges of the active site cleft and in the hydrophobic box are primarily involved in the temperature-induced conformational transition. N.m.r. results are compared with crystallographic data on low temperature (form A) and high temperature (form B) interconverting lysozyme crystals, taking advantage of the recent availability of quality high resolution maps for B form orthorhombic crystals. In most cases, a good correlation is found at the atomic level between residues involved in the thermal transition in solution and in the crystalline state. Discrete discrepancies are noted for some residues such as Trp-62 and His-15.

Side-Chain and Main-Chain Conformational Transitions of Poly(L-1- and 2-naphthylalanine)

Solvent and temperature-induced conformational transitions of poly(L-1- and 2-naphthylalanine) were investigated by circular dichroism in a 1,2-dichloroethane (DCE)/trifluoroacetic acid (TFA) mixed solvent. The two polypeptides were in helical conformation in DCE in which the side chain chromophores are also arranged helically. Poly(L-1-naphthylalanine) showed a two-stage conformational transition upon the addition of TFA to the polymer solution in DCE. An intermediate conformation, which appeared at a TFA content between 0.5 and 3.0 (v/v) %, was tentatively assigned to another helical conformation in which the orientation of the side-chain chromophores is different from that of the helical conformation in DCE. The final state, which appeared at higher TFA content than 3%, was assigned to an extended β-structure. Poly(L-2-naphthylalanine) showed a single conformational transition at a TFA content of 2—3%. The final state was assigned to a randomly-coiled conformation. A temperature-induced conformational transition was observed for poly(L-2-naphthylalanine) in DCE/TFA mixed solvent with a TFA content of 1—5%. In the mixed solvent, the polymer was in a randomly-coiled state at low temperatures and in a helical conformation at high temperatures.

Temperature-induced conformational transition in rabbit muscle aldolase studied by temperature dependence of sulfhydryl reactivity.

A temperature-induced non-denaturing conformational transition in rabbit muscle aldolase has been as subject of discussion and controversy for some period of time. In this study the temperature dependence of the reactivity of aldolase SH groups is investigated in order to detect subtle changes in the enzyme conformation. For model thiol-containing systems such as cysteine, glutathione and bovine serum albumin, linear Arrhenius plots have been obtained for the reaction with 5,5'-dithiobis(2-nitrobenzoic acid). On the other hand, for rabbit muscle pyruvate kinase, a protein which undergoes temperature-induced conformational transition, the plot obtained is nonlinear with a break at the temperature (18 degrees C) close to that reported earlier. In the case of aldolase the Arrhenius plots for three slowly reacting SH groups (Cys-72, 289, 338) and a fast reacting group (Cys-239) are nonlinear with a break at about 26-27 degrees C. The fluorescence measurements show that a plot of the fluorescence intensity of tryptophan residues versus temperature exhibits a break at the same temperature. It is shown that the observed conformational change is fully reversible. In the presence of the competitive inhibitor hexitol 1,6-bisphosphate, which is known to protect Cys-72 and Cys-338 from chemical modification, the Arrhenius plot exhibits a break for the fast reacting Cys-239 residue and is linear for the slowly reacting Cys-289. It is found that 0.6 M urea increases the transition temperature for all exposed SH groups of aldolase. The above results show that at several points in the aldolase molecule, including the active-site region, an abrupt change of microenvironments takes place with temperature. The competitive inhibitor protects a portion of aldolase molecule against the thermal transition.