Temperature-induced Conformational Changes Research Articles

Differential Scanning Calorimetry: A Powerful and Versatile Tool for Analyzing Proteins and Peptides

Differential Scanning Calorimetry (DSC) is an efficient and versatile analytical technique widely used to study the thermal properties and stability of proteins. This review article provides a comprehensive overview of the principles and applications of DSC in protein research. DSC measures the heat flow associated with temperature-induced conformational changes in proteins, allowing for the direct determination of key thermodynamic parameters such as melting temperature (Tm), enthalpy change (ΔH), and heat capacity change (ΔCp). These parameters provide valuable insights into protein and peptide stability, folding mechanisms, and interactions with ligands. The review discusses the methodological aspects of DSC, including sample preparation, experimental setup, and data analysis techniques. It also highlights the application of DSC in various aspects, such as drug discovery, biopharmaceutical development, and the study of protein-ligand interactions. By elucidating the thermal behavior of proteins, DSC contributes significantly to our understanding of protein and peptide structure, function, and stability, making it an indispensable tool in biochemical and biophysical research.

EIF4G as a switch for heat shock mRNA translation

The heat shock response is crucial for cell survival. In this issue of Molecular Cell, Desroches Altamirano etal.1 demonstrate that a temperature-induced conformational change in the translation initiation factor eIF4G is a key mechanism regulating translation during the heat shock response.

Crystallization of Dimensional Isomers in Covalent Organic Frameworks.

Dimensional isomers, defined in reticular chemistry as frameworks consisting of identical molecular building blocks but extended in two or three dimensions (2D or 3D), are an important type of framework isomers that have never been isolated. Herein, we report the crystallization of dimensional isomers in covalent organic frameworks (COFs) for the first time. By polymerization of the same molecular building blocks at different temperatures, both 2D and 3D COFs were successfully constructed due to the temperature-induced conformational changes of precursors from planar to tetrahedral. In addition, the non-fluorescent 2D COF can be gradually converted into the fluorescent 3D COF by increasing the temperature under solvothermal conditions. Therefore, it is reasonable to crystallize the dimensional isomers of reticular materials by controlling the conformation of molecular building blocks, and more examples can be expected. Since the obtained dimensional isomers show different properties and functions, this work will definitely motivate us to design reticular materials for target applications in the future.

A mid-infrared lab-on-a-chip for dynamic reaction monitoring

Mid-infrared spectroscopy is a sensitive and selective technique for probing molecules in the gas or liquid phase. Investigating chemical reactions in bio-medical applications such as drug production is recently gaining particular interest. However, monitoring dynamic processes in liquids is commonly limited to bulky systems and thus requires time-consuming offline analytics. In this work, we show a next-generation, fully-integrated and robust chip-scale sensor for online measurements of molecule dynamics in a liquid solution. Our fingertip-sized device utilizes quantum cascade technology, combining the emitter, sensing section and detector on a single chip. This enables real-time measurements probing only microliter amounts of analyte in an in situ configuration. We demonstrate time-resolved device operation by analyzing temperature-induced conformational changes of the model protein bovine serum albumin in heavy water. Quantitative measurements reveal excellent performance characteristics in terms of sensor linearity, wide coverage of concentrations, extending from 0.075 mg ml−1 to 92 mg ml−1 and a 55-times higher absorbance than state-of-the-art bulky and offline reference systems.

Role of an intramolecular H-bond in lidocaine conformer distribution and polymorph stability

• Solubility of lidocaine in scCO 2 in wide range of phase diagram was measured. • Analysis of the conformations of lidocaine in scCO 2 was performed. • Stability of intramolecular hydrogen bond in lidocaine molecule was found. In this work, we study conformational equilibria of lidocaine molecules in a saturated lidocaine solution in scCO 2 under heating conditions in the temperature range of 35–80 °C along three isochores corresponding to the scCO 2 density of 1.1, 1.3, and 1.5 of its critical value. It is established that among of 68 conformers obtained by quantum chemical calculations, it is only conformers with an intramolecular hydrogen bond that are realized within the studied range of parameters of state. The IR spectroscopy data show that the lidocaine solubility in scCO 2 increases significantly with the fluid phase temperature and density growth, reaching the concentration about 10 −1 mol·L −1 . However, this does not lead to the formation of intermolecular hydrogen bonds between the lidocaine molecules in the solution. Moreover, the heating changes the conformational equilibrium of lidocaine molecules in the scCO 2 phase, but this change is not related to the destruction of the intramolecular hydrogen bond. Higher scCO 2 phase density is found to reduce the temperature effect on the conformational equilibrium. Finally, we establish that the temperature-induced conformational changes in the lidocaine molecules in the scCO 2 phase are completely reversible. It allows us to assume that, regardless of the SCF solution parameters of state in the considered phase diagram region, lidocaine crystallization from this solution will lead to the formation of the same stable polymorph.

Design of a Toolbox of RNA Thermometers.

RNA thermometers are RNA regulatory elements that convert temperature into a functional biological response through a temperature-induced conformational change. These regulatory elements have been investigated in numerous natural contexts and have been designed for synthetic biology as well. A basic challenge has been the design of an RNA thermometer whose final activity in response to temperature matches a prespecified response, in terms of its sensitivity, threshold, and leakiness. This chapter provides a methodology for the design of a toolbox of RNA thermometers. We describe considerations for the conceptual design, a computational assessment, and strategies for experimental synthesis and measurement.

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.

Drying and temperature induced conformational changes of nucleic acids and stallion sperm chromatin in trehalose preservation formulations

Even though dried sperm is not viable, it can be used for fertilization as long as its chromatin remains intact. In this study, we investigated drying- and temperature-induced conformational changes of nucleic acids and stallion sperm chromatin. Sperm was diluted in preservation formulations with and without sugar/albumin and subjected to convective drying at elevated temperatures on glass substrates. Accumulation of reactive oxygen species was studied during storage at different temperatures, and the sperm chromatin structure assay was used to assess DNA damage. Fourier transform infrared spectroscopy was used to identify dehydration and storage induced conformational changes in isolated DNA and sperm chromatin. Furthermore, hydrogen bonding in the preservation solutions associated with storage stability were investigated. Reactive oxygen species and DNA damage in dried sperm samples were found to accumulate with increasing storage temperature and storage duration. Non-reducing disaccharides (i.e., trehalose, sucrose) and albumin counteracted oxidative stress and preserved sperm chromatin during dried storage, whereas glucose increased DNA damage during storage. When sperm was dried in the presence of trehalose and albumin, no spectral changes were detected during storage at refrigeration temperatures, whereas under accelerated aging conditions, i.e., storage at 37 °C, spectral changes were detected indicating alterations in sperm chromatin structure.

Thermodynamic stability of hnRNP A1 low complexity domain revealed by high-pressure NMR.

We have investigated the pressure- and temperature-induced conformational changes associated with the low complexity domain of hnRNP A1, an RNA-binding protein able to phase separate in response to cellular stress. Solution NMR spectra of the hnRNP A1 low-complexity domain fused with protein-G B1 domain were collected from 1 to 2500 bar and from 268 to 290 K. While the GB1 domain shows the typical pressure-induced and cold temperature-induced unfolding expected for small globular domains, the low-complexity domain of hnRNP A1 exhibits unusual pressure and temperature dependences. We observed that the low-complexity domain is pressure sensitive, undergoing a major conformational transition within the prescribed pressure range. Remarkably, this transition has the inverse temperature dependence of a typical folding-unfolding transition. Our results suggest the presence of a low-lying extended and fully solvated state(s) of the low-complexity domain that may play a role in phase separation. This study highlights the exquisite sensitivity of solution NMR spectroscopy to observe subtle conformational changes and illustrates how pressure perturbation can be used to determine the properties of metastable conformational ensembles.

Understanding The Role of Reline, a Natural DES, on Temperature-Induced Conformational Changes of C-Kit G-Quadruplex DNA: A Molecular Dynamics Study.

The noncanonical guanine-rich DNAs have drawn particular attention to the scientific world due to their controllable diverse and polymorphic structures. Apart from biological and medical significance, G-quadruplex DNAs are widely used in various fields such as nanotechnology, nanomachine, biosensors, and biocatalyst. So far, the applications of the G-quadruplex DNA are mainly limited in the water medium. Recently, a new generation of solvent named deep eutectic solvent (DES) has become very popular and has been widely used as a reaction medium of biocatalytic reactions and long-term storage medium for nucleic acids, even at high temperature. Hence, it is essential to understand the role of DES on temperature-induced conformational changes of a G-quadruplex DNA. In this research work, we have explored the temperature-mediated conformational dynamics of c-kit oncogene promoter G-quadruplex DNA in reline medium in the temperature range of 300-500 K, using a total of 10 μs unbiased all-atom molecular dynamics simulation. Here, from RMSD, RMSF, Rg and principal component analyses, we notice that the c-kit G-quadruplex DNA is stable up to 450 K in reline medium. However, it unfolds in water medium at 450 K. It is found that the hydrogen bonding interactions between c-kit G-quadruplex DNA and reline play a key role in the stabilization of the G-quadruplex DNA even at high temperature. Furthermore, in this work we have observed a very interesting and distinctive phenomenon of the central cation of the G-quadruplex DNA. Its position was seen to fluctuate between the two tetrad cores, that is, the region between tetrad-1 and tetrad-2 and that between tetrad-2 and tetrad-3 and vice versa at 450 and 500 K in reline medium which is absent in water medium at 450 K. Moreover, the rate of its oscillation is increased when temperature is increased.

Temperature-Resolved Cryo-EM Uncovers Structural Bases of Temperature-Dependent Enzyme Functions.

Protein functions are temperature-dependent, but protein structures are usually solved at a single (often low) temperature because of limitations on the conditions of crystal growth or protein vitrification. Here we demonstrate the feasibility of solving cryo-EM structures of proteins vitrified at high temperatures, solve 12 structures of an archaeal ketol-acid reductoisomerase (KARI) vitrified at 4-70 °C, and show that structures of both the Mg2+ form (KARI:2Mg2+) and its ternary complex (KARI:2Mg2+:NADH:inhibitor) are temperature-dependent in correlation with the temperature dependence of enzyme activity. Furthermore, structural analyses led to dissection of the induced-fit mechanism into ligand-induced and temperature-induced effects and to capture of temperature-resolved intermediates of the temperature-induced conformational change. The results also suggest that it is preferable to solve cryo-EM structures of protein complexes at functional temperatures. These studies should greatly expand the landscapes of protein structure-function relationships and enhance the mechanistic analysis of enzymatic functions.

Spectroscopic Studies on the Conformational Stability of Hemocyanin of Pila virens (Lamarck, 1822) in the Presence of Temperature and Detergents

Gastropods are endowed with a soluble copper containing, blue coloured respiratory protein known as hemocyanin. In the present study, for the first time, hemocyanin from Pila virens, a fresh water gastropod endemic to Indian sub-continent was studied for its conformational stability in the presence of temperature and various detergents by different spectroscopic techniques viz. UV–Vis, fluorescence and circular dichroism spectroscopy. Results from UV–Vis spectroscopy revealed that, absorbance corresponding to active site moieties were decreased with increase in temperature from 30 to 70 °C. Whereas ‘thermal reversibility’ studies showed the increase in absorbance of active site with decrease in temperature from 70 to 20 °C (is an indicative of the characteristic feature of hemocyanins—that oxygen is reversibly bound to the copper in the active site). Results from CD spectroscopy revealed that the temperature-induced conformational changes on the secondary structure of hemocyanin was reversible up to 70 °C. Beyond 80 °C, the protein was completely denatured and assumed an increased random coil conformation. Hemocyanin in the presence of high concentration of detergents lost its structure as evident from UV–Vis fluorescence (evident from the red shifts in the emission maxima accompanied by quenching in emission intensities) and CD spectroscopy. Stability of active site moieties in the presence of different detergents was also studied by UV–Vis spectroscopy. The biophysical techniques gave insights into the varied effects of each of the different denaturants on the conformational stability of hemocyanin from Pila virens.

Study on the Amyloid Aβ42 with Accelerated Molecular Dynamics Simulations**Supported by the National Natural Science Foundation of China under Grant Nos. 11574184, 11774207; the Natural Science Foundation of Shandong Province under Grant No. ZR2016JL003; and Primary Research & Development Plan of Shandong Province under Grant No. 2017GSF18108

One major cause of Alzheimer’s disease (AD) is evidently due to the aggregation and deposition of amyloid β peptides (Aβ) in the brain tissue of the patient. Preventing misfolding and self-aggregation of Aβ protein can reduce the formation of highly toxic polymer, which is important for the treatment of AD. Among them, the α-helix consisting of 42 residues (Aβ42) is the main component of senile plaques in AD. In this paper, 500 ns accelerated molecular dynamics are performed at different temperatures (300 K, 350 K, 400 K, 450 K) to study of the effect of temperature-induced conformation changes of Aβ42 protein during the unfolding process respectively.

On the mechanism of payload release from liposomes bound to temperature-sensitive microgel particles

Thermosensitive N-isopropylacrylamide based cationic microgels (μG) were synthesized and complexed with anionic liposomes containing encapsulated antitumor antibiotic Doxorubicin (Dox). Compositions of the resulting complexes, in terms of a liposome-to-μG number ratio N, varied from N = 0.5 to a saturated complex with N = 30 where the surface of μG particles is fully covered with liposomes. The microgels collapse when heated from 25 up to 50 °C and the surface area of a microgel particle decreases. The thermo-induced contraction is accompanied with release of Dox from the μG-bound liposomes while a significant fraction of DOX is released for both the unsaturated and saturated complexes. Therefore, two mechanisms are hypothesized for the thermo-induced Dox release. The first one is due to temperature-induced conformational changes of polymer chains, subsequent rearrangements at liposome-microgel interface via liposome-microgel interaction. This mechanism can work for the complexes of any composition. The second process, namely a squeezing of liposomes via liposome-liposome interaction followed by Dox release can become significant for the saturated complex. The results we have obtained are of great interest for constructing multi-liposomal drug carriers, diagnostic systems and catalysts.

Temperature-induced large amplitude conformational change in the complex of oxatub[4]arene revealed via rotaxane synthesis

Oxatub[4]arene was revealed via rotaxane synthesis to show a large amplitude conformational response to temperature change.

Temperature Dependence of Chlorophyll Triplet Quenching in Two Photosynthetic Light-Harvesting Complexes from Higher Plants and Dinoflagellates.

Chlorophyll (Chl) triplet states generated in photosynthetic light-harvesting complexes (LHCs) can be quenched by carotenoids to prevent the formation of reactive singlet oxygen. Although this quenching occurs with an efficiency close to 100% at physiological temperatures, the Chl triplets are often observed at low temperatures. This might be due to the intrinsic temperature dependence of the Dexter mechanism of excitation energy transfer, which governs triplet quenching, or by temperature-induced conformational changes. Here, we report about the temperature dependence of Chl triplet quenching in two LHCs. We show that both the effects contribute significantly. In LHC II of higher plants, the core Chls are quenched with a high efficiency independent of temperature. A different subpopulation of Chls, which increases with lowering temperature, is not quenched at all. This is probably caused by the conformational changes which detach these Chls from the energy-transfer chain. In a membrane-intrinsic LHC of dinoflagellates, similarly two subpopulations of Chls were observed. In addition, another part of Chl triplets is quenched by carotenoids with a rate which decreases with temperature. This allowed us to study the temperature dependence of Dexter energy transfer. Finally, a part of Chls was quenched by triplet-triplet annihilation, a phenomenon which was not observed for LHCs before.

Protective Effect of Biological Osmolytes against Heat- and Chaotropic Agent-Induced Denaturation of Bacillus licheniformis γ-Glutamyl Transpeptidase.

In the present study, the stabilizing effect of four different biological osmolytes on Bacillus licheniformis γ-glutamyl transpeptidase (BlGGT) was investigated. BlGGT appeared to be stable under temperatures below 40°C, but the enzyme retained less than 10% of its activity at 60°C. The tested osmolytes exhibited different degrees of effectiveness against temperature inactivation of BlGGT, and sucrose was found to be the most effective among these. The use of circular dichroism spectroscopy for studying the secondary structure of BlGGT revealed that the temperature-induced conformational change of the protein molecule could be prevented by the osmolytes. Consistently, the molecular structure of the enzyme was essentially conserved by the osmolytes at elevated temperatures as monitored by fluorescence spectroscopy. Sucrose was further observed to counteract guanidine hydrochloride (GdnHCl)- and urea-induced denaturation of BlGGT. Taken together, we observed evidently that some well-known biological osmolytes, especially sucrose, make a dominant contribution to the structural stabilization of BlGTT.

Investigation on temperature-induce conformational change of immobilized β2 adrenergic receptor

The β2 adrenergic receptor (β2-AR) is a prototypical family A G protein-coupled receptor (GPCR) and an excellent model system for studying the mechanism of GPCR activation. Purified β2-AR was immobilized on macroporous silica gel to obtain liquid chromatographic stationary phase. The resulting phase was packed into a stainless steel column (4.6 × 50 mm, 7 μm) and used for on-line chromatographic system. When column oven temperature increased from 20.0 °C to 40.0 °C, uncomplete separate chromatographic peaks of ephedrine and pseudoephedrine as receptor conformational probe were gradually merged into one peak, meanwhile retention time and resolution of the probes were reduced correspondingly, which suggested that temperature could regulate protein conformation. Temperature-induced conformational change of immobilized β2-AR, especially changes at higher temperatures, indicated that constructed receptor chromatography could simulate fever disease state of human body and clarify receptor conformation change at pathological condition. At the same time this study could also provide new ideas for screening active components in pathological conditions.

Harnessing Cooperative Interactions between Thermoresponsive Aptamers and Gels To Trap and Release Nanoparticles.

We use computational modeling to design a device that can controllably trap and release particles in solution in response to variations in temperature. The system exploits the thermoresponsive properties of end-grafted fibers and the underlying gel substrate. The fibers mimic the temperature-dependent behavior of biological aptamers, which form a hairpin structure at low temperatures (T) and unfold at higher T, consequently losing their binding affinity. The gel substrate exhibits a lower critical solution temperature and thus, expands at low temperatures and contracts at higher T. By developing a new dissipative particle dynamics simulation, we examine the behavior of this hybrid system in a flowing fluid that contains buoyant nanoparticles. At low T, the expansion of the gel causes the hairpin-shaped fibers to extend into the path of the fluid-driven particle. Exhibiting a high binding affinity for these particles at low temperature, the fibers effectively trap and extract the particles from the surrounding solution. When the temperature is increased, the unfolding of the fiber and collapse of the supporting gel layer cause the particles to be released and transported away from the layer by the applied shear flow. Since the temperature-induced conformational changes of the fiber and polymer gel are reversible, the system can be used repeatedly to "catch and release" particles in solution. Our findings provide guidelines for creating fluidic devices that are effective at purifying contaminated solutions or trapping cells for biological assays.

Triggering activity of catalytic rod-like supramolecular polymers.

Supramolecular polymers based on benzene-1,3,5-tricarboxamides (BTAs) functionalized with an L- or D-proline moiety display high catalytic activity towards aldol reactions in water. High turnover frequencies (TOF) of up to 27×10(-4) s(-1) and excellent stereoselectivities (up to 96% de, up to 99% ee) were observed. In addition, the catalyst could be reused and remained active at catalyst loadings and substrate concentrations as low as 0.1 mol % and 50 mM, respectively. A temperature-induced conformational change in the supramolecular polymer triggers the high activity of the catalyst. The supramolecular polymer's helical sense in combination with the configuration of the proline (L- or D-) is responsible for the observed selectivity.