Temperature-dependent Conformational Change Research Articles

Temperature-dependent structural changes in xylanase II from Trichoderma longibrachiatum

Endo-β-1,4-xylanases degrade heteroxylans that constitute the lignocellulosic plant cell wall. This enzyme is widely used in the food, paper, textile, and biorefinery industries. Temperature affects the optimum activity of xylanase and is an important factor in its application. Various structural analyses of xylanase have been performed, but its structural influence by temperature is not fully elucidated. To better understand the structural influence of xylanase due to temperature, the crystal structure of xylanase II from Trichoderma longibrachiatum (TloXynII) at room and cryogenic temperatures was determined at 2.1 and 1.9 Å resolution, respectively. The room-temperature structure of TloXynII (TloXynIIRT) showed a B-factor value 2.09 times higher than that of the cryogenic-temperature structure of TloXynII (TloXynIICryo). Subtle movement of the catalytic and substrate binding residues was observed between TloXynIIRT and TloXynIICryo. In TloXynIIRT, the thumb domain exhibited high flexibility, whereas in TloXynIICryo, the finger domain exhibited high flexibility. The substrate binding cleft of TloXynIIRT was narrower than that of TloXynIICryo, indicating a distinct finger domain conformation. Numerous water molecule networks were observed in the substrate binding cleft of TloXynIICryo, whereas only a few water molecules were observed in TloXynIIRT. These structural analyses expand our understanding of the temperature-dependent conformational changes in xylanase.

Exploring temperature-dependent conformational changes in P38 MAP kinases involved in inflammation and acute lung injury

Exploring temperature-dependent conformational changes in P38 MAP kinases involved in inflammation and acute lung injury

Variation of CI-2 Conformers upon Addition of Methanol to Water: An IMS-MS-Based Thermodynamic Analysis.

Chymotrypsin inhibitor 2 (CI-2) is a well-studied, textbook example of a cooperative, two-state, native ↔ denatured folding transition. A recent hybrid ion mobility spectrometry (IMS)/mass spectrometry (MS) thermal denaturation study of CI-2 (the well-studied truncated 64-residue model) in water reported evidence that this two-state transition involves numerous (∼41) unique native and non-native (denatured) solution conformations. The characterization of so many, often low-abundance, states is possible because of the very high dynamic range of IMS-MS measurements of ionic species that are produced upon electrospraying CI-2 solutions from a variable temperature electrospray ionization source. A thermodynamic analysis of these states revealed large changes in enthalpy (ΔH) and entropy (ΔS) at different temperatures, and it was suggested that such variation might arise because of temperature-dependent conformational changes of the protein in response to changes in the conformational entropy and the dielectric permeability of water, which drops from a value of ε ∼ 79 at 24 °C to ∼ 60 at 82 °C. Herein, we examine how adding methanol to water influences the distributions of CI-2 conformers and their ensuing stabilities. The dielectric constant of a 60:40 water:methanol (MeOH) drops from ε ∼ 60 at 24 °C to ∼ 51 at 64 °C. Although the same set of conformers observed in water appears to be present in 60:40 water:MeOH, the abundance of each is substantially altered by the presence of methanol. Relative free energy values (ΔG) and thermodynamic values [ΔH and ΔS and heat capacities (ΔCp)] are derived from a Gibbs-Helmholtz analysis. A comparison of these data from water and water:MeOH systems allows rare insight into how variations in solvation and temperature affect many-state protein equilibria. While these studies confirm that variations in solvent dielectric constant with temperature affect the distributions of conformers that are observed, our findings suggest that other solvent differences may also affect abundances.

Characterization of a FourU RNA Thermometer in the 5' Untranslated Region of Autolysin Gene blyA in the Bacillus subtilis 168 Prophage SPβ.

RNA thermometers are noncoding RNA structures located in the 5' untranslated regions (UTRs) of genes that regulate gene expression through temperature-dependent conformational changes. The fourU class of RNA thermometers contains a specific motif in which four consecutive uracil nucleotides are predicted to base pair with the Shine-Dalgarno (SD) sequence in a stem. We employed a bioinformatic search to discover a fourU RNA thermometer in the 5'-UTR of the blyA gene of the Bacillus subtilis phage SPβc2, a bacteriophage that infects B. subtilis 168. blyA encodes an autolysin enzyme, N-acetylmuramoyl-l-alanine amidase, which is involved in the lytic life cycle of the SPβ prophage. We have biochemically validated the predicted RNA thermometer in the 5'-UTR of the blyA gene. Our study suggests that RNA thermometers may play an underappreciated yet critical role in the lytic life cycle of bacteriophages.

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.

ВЛИЯНИЕ ЗАМОРАЖИВАНИЯ-ОТТАИВАНИЯ НА ТЕМПЕРАТУРНОЕ ПОВЕДЕНИЕ ГЕМОГЛОБИНА

The use of blood preservation at low temperatures methods in medical practice makes it necessary to study the temperature effect on hemoglobin. The effect of freezing-thawing to a liquid nitrogen temperature of -196C and subsequent thawing on hemoglobin A was studied, using the methods of temperature-perturbation differential spectrophotometry and analysis of absorption spectra first derivatives. After the hemoglobin A solution freezing-thawing, a smoothing of the breaks in the S-shaped temperature intensity dependence at the maximum of the temperature-perturbation differential spectra at 286 nm is noted. This probably means that temperature-dependent conformational changes in the hemoglobin A molecule are smoothed out and become less noticeable. It should be noted that after freezing-thawing, this dependence S-shaped form is preserved, i.e. conformational changes take place, but they are less pronounced than before freezing-thawing. Thus, on the one hand, freezing the hemoglobin A solution leads to conformational changes in the hemoglobin A molecule affecting the polar regions, namely, to an increase in the polar regions availability to the solvent, which is consistent with the data on the protein globule partial unfolding, accompanied by the globin aromatic amino acids exposure to the solvent, and with the data that the freezing-thawing process leads in most cases to conformational changes in the protein, which consist in the molecule weakening and increasing the protein active sites accessibility. On the other hand, freezing of hemoglobin A solutions leads to compaction of the hemoglobin A molecule subunits. This can probably be explained by the fact that, presumably, an increase in the availability of the hemoglobin A molecule absorbing chromophores located in the polar regions to the solvent leads to the compaction of the hemoglobin molecule A other parts, not involved in increasing the amino acid residues availability to the solvent. That is, some parts of the hemoglobin A molecule become more accessible to the solvent, while others become compact.

Synthesis and characterization of novel thermo- and salt-sensitive amphoteric terpolymers based on acrylamide derivatives

A novel linear amphoteric terpolymers based on neutral monomer — N-isopropylacrylamide (NIPAM), ani- onic monomer — 2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt (AMPS), and cationic mono- mer — (3-acrylamidopropyl) trimethylammonium chloride (APTAC) were synthesized by free-radical polymerization in aqueous solution and characterized by methods of 1H NMR and FTIR spectroscopy, TGA, GPC, Dynamic light scattering (DLS) and zeta-potential. The thermal and salt sensitivity of amphoteric ternary polymers of various compositions, particularly, [NIPAM]:[AMPS]:[APTAC] = 90:2.5:7.5; 90:5:5; 90:7.5:2.5 mol.% were studied in aqueous and aqueous-salt solutions in the temperature range from 25 to 60 C and at the NaCl ionic strength  interval from 10–3 to 1M. It was found that due to hydrophobic/hydrophilic balance, the temperature dependent conformational and phase change of macromolecular chains becomes sensitive to salt addition and allows the fine-tuning of the phase transition. In aqueous and aqueous-salt solutions, the average hydrodynamic size of amphoteric terpolymers is varied from 8 to 300 nm exhibiting bimodal distribution at room temperature. The number average (Mn) and weight average (Mw) molecular weights, polydispersity index (PDI), and zeta-potentials of amphoteric terpolymers in aqueous solu- tions were determined

Mapping temperature-dependent conformational change in the voltage-sensing domain of an engineered heat-activated K+ channel

Temperature-dependent regulation of ion channel activity is critical for a variety of physiological processes ranging from immune response to perception of noxious stimuli. Our understanding of the structural mechanisms that underlie temperature sensing remains limited, in part due to the difficulty of combining high-resolution structural analysis with temperature stimulus. Here, we use NMR to compare the temperature-dependent behavior of Shaker potassium channel voltage sensor domain (WT-VSD) to its engineered temperature sensitive (TS-VSD) variant. Further insight into the molecular basis for temperature-dependent behavior is obtained by analyzing the experimental results together with molecular dynamics simulations. Our studies reveal that the overall secondary structure of the engineered TS-VSD is identical to the wild-type channels except for local changes in backbone torsion angles near the site of substitution (V369S and F370S). Remarkably however, these structural differences result in increased hydration of the voltage-sensing arginines and the S4-S5 linker helix in the TS-VSD at higher temperatures, in contrast to the WT-VSD. These findings highlight how subtle differences in the primary structure can result in large-scale changes in solvation and thereby confer increased temperature-dependent activity beyond that predicted by linear summation of solvation energies of individual substituents.

Evidence that the TRPV1 S1-S4 membrane domain contributes to thermosensing

Sensing and responding to temperature is crucial in biology. The TRPV1 ion channel is a well-studied heat-sensing receptor that is also activated by vanilloid compounds, including capsaicin. Despite significant interest, the molecular underpinnings of thermosensing have remained elusive. The TRPV1 S1-S4 membrane domain couples chemical ligand binding to the pore domain during channel gating. Here we show that the S1-S4 domain also significantly contributes to thermosensing and couples to heat-activated gating. Evaluation of the isolated human TRPV1 S1-S4 domain by solution NMR, far-UV CD, and intrinsic fluorescence shows that this domain undergoes a non-denaturing temperature-dependent transition with a high thermosensitivity. Further NMR characterization of the temperature-dependent conformational changes suggests the contribution of the S1-S4 domain to thermosensing shares features with known coupling mechanisms between this domain with ligand and pH activation. Taken together, this study shows that the TRPV1 S1-S4 domain contributes to TRPV1 temperature-dependent activation.

Compensatory Mechanisms in Temperature Dependence of DNA Double Helical Structure: Bending and Elongation.

Changes in the structure of double-stranded (ds) DNA with temperature affect processes in thermophilic organisms and are important for nanotechnological applications. Here we investigate temperature-dependent conformational changes of dsDNA at the scale of several helical turns and at the base pair step level, inferred from extensive all-atom molecular dynamics simulations of DNA at temperatures from 7 to 47 °C. Our results suggest that, contrary to twist, the overall bending of dsDNA without A-tracts depends only very weakly on temperature, due to the mutual compensation of directional local bends. Investigating DNA length as a function of temperature, we find that the sum of distances between base pair centers (the wire length) exhibits a large expansion coefficient of ∼2 × 10-4 °C-1, similar to values reported for thermoplastic materials. However, the wire length increase with temperature is absorbed by expanding helix radius, so the length measured along the helical axis (the spring length) seems to suggest a very small negative thermal expansion coefficient. These compensatory mechanisms contribute to thermal stability of DNA structure on the biologically relevant scale of tens of base pairs and longer.

Structural and conformational behavior of MurE ligase from Salmonella enterica serovar Typhi at different temperature and pH conditions

MurE ligase is known to play a significant role in peptidoglycan biosynthesis. It catalyzes the addition of meso-diaminopimelic acid to nucleotide precursor. The protein can adopt different conformations for its proper functioning. Different environmental conditions can alter the stability and function of enzyme due to their ability to disrupt interactions between different domains. We have explored the pH and temperature dependent conformational changes in MurE ligase from Salmonella Typhi and estimated the protein stability. The study enabled us to decipher the effect of different milieu condition in the enzyme activity. At acidic pH 3.0, StMurE ligase forms molten globule (MG) state and at alkaline pH it is in unfolded state. The different states of StMurE ligase were characterized using various spectroscopic techniques. These techniques including near-UV CD, far-UV CD, ANS fluorescence, differential scanning calorimetry and fluorescence spectroscopy helped to determine the secondary structural changes and detect local conformational modifications. The structural analysis using StMurE ligase homology model revealed the variations in ionization states of catalytic amino acid residues involved in substrate binding. This study provides an insight into the dynamics states of StMurE ligase at different environmental conditions during bacterial pathogenesis.

Supramolecular Polymer Network-Mediated Structural Phase Transitions within Polymeric Micelles in Aliphatic Alcohols.

Self-complementary supramolecular polymers (SCSPs), an efficient combination of sextuple hydrogen-bonded dimer moieties and a temperature-responsive polymer, can promote the construction of stable supramolecular polymer networks (SPNs) that enable the formation of well-defined nanospherical micelles in aliphatic alcohols. These micelles undergo tailorable, thermoresponsive phase transitions at the upper critical solution temperature (UCST) and have a desirable spherical morphology and size ranges, thus, are potential candidates for applications in interfacial engineering and biomedical fields. Moreover, concentration-dependent UCST measurements and variable-temperature experiments indicated that the hydrogen-bonded complexes are strong enough to form stable intermolecularly entangled SPNs within the micelles, even above the UCST or at low concentrations in solution, which enables the micelles to undergo reversible temperature-dependent conformational changes between insoluble and soluble globules without significant changes in particle size or size distribution. Thus, this newly discovered system offers a new approach toward the development of next-generation temperature-responsive SCSPs with the desired structural stability that undergoes UCST transitions.

Influence of urea on methyl upbeta-D-glucopyranoside in alkali at different temperatures

The dissolution efficiency plays an important role on the properties of regenerated cellulose-based products. Urea is known to be one of the additives aiding to improve cellulose dissolution in the NaOH(aq) system. The acting mechanism caused by urea has been debated and one of the hypothesis is that urea could induce a conformational change on cellulose, which promotes dissolution. Here we used NMR spectroscopy on a model system for cellulose, namely, methyl upbeta-D-glucopyranoside (upbeta-MeO-Glcp) and compared chemical shifts and J couplings, which both are indicators for conformational changes, as a function of temperature and upon the addition of urea. We found that in NaOH(aq), the hydroxymethyl group changes its conformation in favour of the population of the gt rotamer, while the presence of urea induced temperature dependent conformational changes. Heteronuclear Overhauser effect experiments showed that urea associates with cellulose but in a non-specific manner. This suggests that urea rather than binding to the carbohydrate, changes the chemical environment inducing a change in conformation of upbeta-MeO-Glcp and likely also for cellulose when dissolved in NaOH(aq) with urea.

A temperature-dependent conformational shift in p38α MAPK substrate–binding region associated with changes in substrate phosphorylation profile

Febrile-range hyperthermia worsens and hypothermia mitigates lung injury, and temperature dependence of lung injury is blunted by inhibitors of p38 mitogen-activated protein kinase (MAPK). Of the two predominant p38 isoforms, p38α is proinflammatory and p38β is cytoprotective. Here, we analyzed the temperature dependence of p38 MAPK activation, substrate interaction, and tertiary structure. Incubating HeLa cells at 39.5 °C stimulated modest p38 activation, but did not alter tumor necrosis factor-α (TNFα)-induced p38 activation. In in vitro kinase assays containing activated p38α and MAPK-activated kinase-2 (MK2), MK2 phosphorylation was 14.5-fold greater at 39.5 °C than at 33 °C. By comparison, we observed only 3.1- and 1.9-fold differences for activating transcription factor-2 (ATF2) and signal transducer and activator of transcription-1α (STAT1α) and a 7.7-fold difference for p38β phosphorylation of MK2. The temperature dependence of p38α:substrate binding affinity, as measured by surface plasmon resonance, paralleled substrate phosphorylation. Hydrogen-deuterium exchange MS (HDX-MS) of p38α performed at 33, 37, and 39.5 °C indicated temperature-dependent conformational changes in an α helix near the common docking and glutamate:aspartate substrate-binding domains at the known binding site for MK2. In contrast, HDX-MS analysis of p38β did not detect significant temperature-dependent conformational changes in this region. We observed no conformational changes in the catalytic domain of either isoform and no corresponding temperature dependence in the C-terminal p38α-interacting region of MK2. Because MK2 participates in the pathogenesis of lung injury, the observed changes in the structure and function of proinflammatory p38α may contribute to the temperature dependence of acute lung injury.

Plasma protein adsorption on TiO2 nanoparticles: Impact of surface adsorption on temperature-dependent structural changes

Protein adsorption on metal oxide nanoparticle surfaces is crucial to the behavior of oxide nanoparticles in biological systems. In this study, attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used for the first time to probe differences in the temperature-dependent conformational changes of two plasma proteins, bovine serum albumin (BSA) and fibrinogen (Fib), in solution and adsorbed on titanium dioxide nanoparticle (ca. 20 nm in diameter) surfaces. ATR-FTIR spectra and autocorrelation moving window two-dimensional correlation (MW2D) spectroscopy were used to identify the denaturation temperature range of these two proteins in aqueous solution and adsorbed. Generalized two-dimensional correlation spectroscopy (2DCOS) was done to investigate the protein unfolding pathway. The results show that the thermostability of BSA changed when adsorbed on the surface whereas there was no difference for solution phase Fib and adsorbed on TiO2. The most notable fact was that solution BSA underwent significant structural changes upon adsorption whereas adsorbed Fib did not. Upon heating adsorbed BSA, no further changes occurred while BSA in solution thermally denatured. In contrast, solution and adsorbed Fib had identical denaturation temperatures and similar sequential secondary structural changes with increasing temperature. These studies show quantitatively, for the first time, that there are differences in the temperature-dependent structural changes of proteins adsorbed on nanoparticles surfaces and these changes depend on the initial interaction with the nanoparticle surface. Overall, the results of this study provide new insights into understanding the effects of temperature on the structure of adsorbed protein on nanoparticle surfaces and the range of different behaviors that can occur.

Maslinic acid conjugate with 7-amino-4-methylcoumarin as probe to monitor the temperature dependent conformational changes of human serum albumin by FRET.

Synthesis, characterization and spectroscopic investigation of maslinic acid labeled with fluorescent 7-amino-4-methylcoumarin is reported. It was found that the coumarin-maslinic derivative (MaCo) forms an excellent fluorescence resonance energy transfer (FRET) pair with the tryptophan (Trp) residue of human serum albumin (HSA). This feature allowed for monitoring HSA conformational alterations by measuring the distance between donor (Trp) and acceptor (MaCo) through Förster energy transfer mechanism. Displacement experiments confirmed that MaCo binds to subdomain IIA of HSA with independence of temperature. It was observed that, in the temperature range 35-45 °C, the fluorescence emission maximum of HSA-MaCo complex decreased, whereas in the range 45 °C-65 °C, an increment was detected. The concomitant change in the polarity of environment surrounding Trp was confirmed by red edge excitation shift experiments. Thermal denaturation of HSA was followed by time-resolved fluorescence spectroscopy. Average lifetime of Trp residue decreased with temperature due to the increment of solvent collisions and changes in the solvent exposure of Trp. To discriminate the importance of each effect, lifetime of N-Acetyl-L-tryptophanamide (NATA) at different temperatures was measured. Circular dichroism (CD) studies confirmed the loss of secondary structure of HSA with increasing temperature and showed a different trend in the conformational transformation below and above 45 °C, in agreement with steady-state and time-resolved fluorescence experiments.

Single-Molecule Insights into the Temperature Dependent Conformational Changes of a RNA Thermometer in the Presence of Crowders and Osmolytes

Single-Molecule Insights into the Temperature Dependent Conformational Changes of a RNA Thermometer in the Presence of Crowders and Osmolytes

Structural study reveals the temperature-dependent conformational flexibility of Tk-PTP, a protein tyrosine phosphatase from Thermococcus kodakaraensis KOD1.

Protein tyrosine phosphatases (PTPs) originating from eukaryotes or bacteria have been under intensive structural and biochemical investigation, whereas archaeal PTP proteins have not been investigated extensively; therefore, they are poorly understood. Here, we present the crystal structures of Tk-PTP derived from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, in both the active and inactive forms. Tk-PTP adopts a common dual-specificity phosphatase (DUSP) fold, but it undergoes an atypical temperature-dependent conformational change in its P-loop and α4−α5 loop regions, switching between the inactive and active forms. Through comprehensive analyses of Tk-PTP, including additional structural determination of the G95A mutant form, enzymatic activity assays, and structural comparison with the other archaeal PTP, it was revealed that the presence of the GG motif in the P-loop is necessary but not sufficient for the structural flexibility of Tk-PTP. It was also proven that Tk-PTP contains dual general acid/base residues unlike most of the other DUSP proteins, and that both the residues are critical in its phosphatase activity. This work provides the basis for expanding our understanding of the previously uncharacterized PTP proteins from archaea, the third domain of living organisms.

Functional Modification of Silica through Enhanced Adsorption of Elastin-Like Polypeptide Block Copolymers.

A powerful tool for controlling interfacial properties and molecular architecture relies on the tailored adsorption of stimuli-responsive block copolymers onto surfaces. Here, we use computational and experimental approaches to investigate the adsorption behavior of thermally responsive polypeptide block copolymers (elastin-like polypeptides, ELPs) onto silica surfaces, and to explore the effects of surface affinity and micellization on the adsorption kinetics and the resultant polypeptide layers. We demonstrate that genetic incorporation of a silica-binding peptide (silaffin R5) results in enhanced adsorption of these block copolymers onto silica surfaces as measured by quartz crystal microbalance and ellipsometry. We find that the silaffin peptide can also direct micelle adsorption, leading to close-packed micellar arrangements that are distinct from the sparse, patchy arrangements observed for ELP micelles lacking a silaffin tag, as evidenced by atomic force microscopy measurements. These experimental findings are consistent with results of dissipative particle dynamics simulations. Wettability measurements suggest that surface immobilization hampers the temperature-dependent conformational change of ELP micelles, while adsorbed ELP unimers (i.e., unmicellized block copolymers) retain their thermally responsive property at interfaces. These observations provide guidance on the use of ELP block copolymers as building blocks for fabricating smart surfaces and interfaces with programmable architecture and functionality.

SHAPE analysis of the htrA RNA thermometer from Salmonella enterica.

RNA thermometers regulate expression of some genes involved in virulence of pathogenic bacteria such as Yersinia, Neisseria, and Salmonella. They often function through temperature-dependent conformational changes that alter accessibility of the ribosome-binding site. The 5′-untranslated region (UTR) of the htrA mRNA from Salmonella enterica contains a very short RNA thermometer. We have systematically characterized the structure and dynamics of this thermometer at single-nucleotide resolution using SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) assays. Our results confirm that the htrA thermometer adopts the predicted hairpin conformation at low temperatures, with conformational change occurring over a physiological temperature regime. Detailed SHAPE melting curves for individual nucleotides suggest that the thermometer unfolds in a cooperative fashion, with nucleotides from both upper and lower portions of the stem gaining flexibility at a common transition temperature. Intriguingly, analysis of an extended htrA 5′ UTR sequence revealed not only the presence of the RNA thermometer, but also an additional, stable upstream structure. We generated and analyzed point mutants of the htrA thermometer, revealing elements that modulate its stability, allowing the hairpin to melt under the slightly elevated temperatures experienced during the infection of a warm-blooded host. This work sheds light on structure–function relationships in htrA and related thermometers, and it also illustrates the utility of SHAPE assays for detailed study of RNA thermometer systems.