Heat-induced Conformational Changes Research Articles

Temperature Affects the Supramolecular Structures Resulting from α-Lactalbumin−Lysozyme Interaction

The interaction between alpha-lactalbumin and lysozyme, two globular proteins with highly homologous tertiary structures but opposite electric charges, was investigated. As assessed by isothermal titration calorimetry, lysozyme did not bind to the native form of alpha-lactalbumin but did interact with calcium-depleted alpha-lactalbumin (apo alpha-LA). This interaction leads to the formation of different supramolecular structures depending on the temperature. Heterogeneous, amorphous aggregates are formed at 5 degrees C, while droplets, coacervate-like structures, exist at 45 degrees C. The coacervates are formed by equimolar quantities of the two proteins, but their size and number depend on the initial protein molar ratio. These supramolecular structures are found to be stable when the temperature is decreased to 5 degrees C, while prolonged heating at 45 degrees C induces the formation of larger coacervates through a coalescence phenomenon. Surprisingly, interplay occurs between aggregates and coacervates when the temperature is increased from 5 to 45 degrees C. We discuss the results in terms of subtle heat-induced conformational changes in apo alpha-LA. In conclusion, our results show an association between globular proteins that leads to the formation of a variety of supramolecular structures in a temperature-dependent manner and confirm the primordial role of certain alpha-lactalbumin unfolding intermediates in protein-driven assembly.

Heat-induced conformational changes of egg yolk proteins

Heat-induced conformational changes of egg yolk proteins

Ion transport across model lipid membranes containing light-harvesting complex II: an effect of light

The effect of light on proton transport across lipid membranes of small unilamellar liposomes containing incorporated major light-harvesting pigment–protein complex of Photosystem II (LHCII) has been studied with the application of pH-sensitive dyes entrapped inside vesicles. Proton permeability coefficient for LHCII-modified membranes was found to be about twice as high as in the case of the control pure lipid vesicles. Illumination of the samples with light absorbed by the LHCII-bound photosynthetic pigments considerably affects the kinetics of proton transport: it increases the rate and decreases the steady-state level of proton gradient across the membranes. The effect was interpreted in terms of heat-induced conformational changes of LHCII molecular structures that affect proton buffering capacity of this protein. Both the control and the LHCII-modified lipid membranes have been found to be practically impermeable to Ca ++ ions, as demonstrated by fluorescence of liposome-entrapped calcium-sensitive probe calcium crimson. The slight differences in the proton transport across the LHCII-containing membranes under the presence of Ca ++ suggest calcium binding to this antenna protein.

Mapping Temperature-induced Conformational Changes in the Escherichia coli Heat Shock Transcription Factor σ32 by Amide Hydrogen Exchange

Stress conditions such as heat shock alter the transcriptional profile in all organisms. In Escherichia coli the heat shock transcription factor, sigma 32, out-competes upon temperature up-shift the housekeeping sigma-factor, sigma 70, for binding to core RNA polymerase and initiates heat shock gene transcription. To investigate possible heat-induced conformational changes in sigma 32 we performed amide hydrogen (H/D) exchange experiments under optimal growth and heat shock conditions combined with mass spectrometry. We found a rapid exchange of around 220 of the 294 amide hydrogens at 37 degrees C, indicating that sigma 32 adopts a highly flexible structure. At 42 degrees C we observed a slow correlated exchange of 30 additional amide hydrogens and localized it to a helix-loop-helix motif within domain sigma 2 that is responsible for the recognition of the -10 region in heat shock promoters. The correlated exchange is shown to constitute a reversible unfolding with a half-life of about 30 min due to a temperature-dependent decrease in stabilization energy. We propose that this gradual decrease in stabilization energy of domain sigma 2 with increasing temperatures facilitates the unfolding of sigma 32 by the AAA+ protease FtsH thereby decreasing its half-life. Taken together our data show that the sigma 2 domain of sigma 32 can act as a thermosensor, which might be important for the heat shock regulation.

Study of conformational changes in poly(3-dodecylthiophene) dependent on backbone stereoregularity using a spin-probe technique

Poly(3-dodecylthiophene) (P3DDT), with different amounts of head-to-head configuration defects, was characterized by ultraviolet–visible, 1H-NMR, photoluminescence, and Raman spectra. The heat-induced conformational sample changes were studied by electron paramagnetic resonance (EPR). For the study of these changes, a spin-probe technique was used, in which 5-doxyl-stearic acid methyl ester was applied as a spin probe. From the EPR spin-probe spectra, rotational correlation times and order parameters were calculated. The heat-induced conformational changes of P3DDT were accompanied by a monotonic decrease in the rotational correlation time up to approximately −33°C (240 K) and then by an increase in the range of the glass-transition temperatures, with the maximum being near room temperature and depending on the effective conjugation length. Afterward, the rotational correlation times had a decreasing tendency up to 90°C (363 K). © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 88: 2215–2223, 2003

Native and hydrophobized human IGG: Enthalpies of heat-induced structural changes and adsorption onto silica

Abstract Differential scanning calorimetry (DSC) and isothermal calorimetric batch technique were used to monitor the heat-induced structural changes and adsorption properties of human immunoglobulin G (IgG), in native and hydrophobized states. The transition temperature (T max) and enthalpy of heat-induced conformational changes (cal H) of IgG in solution as well as the enthalpy change accompanying the adsorption of IgG onto hydrophilic silica (ads H), were shown to depend on the degree of the protein hydrophobicity (number of covalently attached alkyl chains). The adsorption enthalpy for all forms of IgG at all surface concentrations was found to be endothermic, that is the process is entropy driven. Factors affecting the IgG adsorption onto silica are discussed.

Protein micelles from lipoxygenase 3.

Heat-induced conformational changes in lipoxygenase 3 were characterized by differential scanning calorimetry. The positions of the observed transitions were sensitive to the composition of the buffer. In particular, lipoxygenase 3 heated in carbonate buffer at pH 8.0 formed large soluble aggregates. Variable-temperature circular dichroism revealed that the formation of the aggregates was not accompanied by the unfolding of the C-terminal domain, which is composed primarily of alpha-helix. The aggregates were investigated using size exclusion chromatography, native polyacrylamide gel electrophoresis, dynamic light scattering, and electron microscopy. The data were consistent with the formation of roughly spherical particles with an average hydrodynamic radius of 26 nm and an approximate composite molecular weight of 10,000,000 Da. To account for the formation of soluble aggregates from lipoxygenase 3, we propose that hydrophobic amino acid residues are exposed by unfolding of the N-terminal beta-barrel domain of the protein resulting in the formation of protein micelles with a hydrophilic surface composed of the C-terminal domains.

Electric Conductivity of Poly(3-dodecylthiophene) and Its Dependence on the Conformational Changes of Polymer Chains

The influence of the heat-induced conformational changes in side alkyls and in the backbone of poly(3-dodecylthiophene) (PDDT) with a higher content of configurational head-to-head defects (39 studied. It has been found that conformational changes affect the electric conductivity values of PDDT doped with FeCl3 which attain at 0.021 mol of Fe/mol dodecylthiophene more than 108 S m-1 within the whole temperature range (from 78 up to 320 K). For the analysis of conformational changes the entropy temperature dependence was calculated from the experimental data on the specific heat capacity. In this paper dependences between the resistance and the conformational changes of PDDT were discussed.

Heat-induced Conformational Changes of Ara h 1, a Major Peanut Allergen, Do Not Affect Its Allergenic Properties

Ara h 1, a major peanut allergen was isolated, and its structure on secondary, tertiary, and quaternary level at ambient temperature was investigated using spectroscopic and biochemical techniques. Ara h 1 appeared to be a highly structured protein on a secondary level, possesses a clear tertiary fold, and is present as a trimeric complex. Heat treatment of purified Ara h 1 results in an endothermic, irreversible transition between 80 and 90 degreesC, leading to an increase in beta-structures and a concomitant aggregation of the protein. Ara h 1 from peanuts that were heat-treated prior to the purification procedure exhibited a similar denatured state with an increased secondary folding and a decreased solubility. The effect of heat treatment on the in vitro allergenic properties of Ara h 1 was investigated by means of a fluid-phase IgE binding assay using serum from patients with a clinically proven peanut allergy. Ara h 1 purified from peanuts heated at different temperatures exhibited IgE binding properties similar to those found for native Ara h 1, indicating that the allergenicity of Ara h 1 is heat-stable. We conclude that the allergenicity of Ara h 1 is unaffected by heating, although native Ara h 1 undergoes a significant heat-induced denaturation on a molecular level, indicating that the recognition of conformational epitopes of Ara h 1 by IgE either is not a dominant mechanism or is restricted to parts of the protein that are not sensitive to heat denaturation.

Heat-Induced Gelation of Globular Protein Mixtures

Heat-induced conformational changes and heat-induced gels of whey and egg white albumen, and their major components were studied under physicochemical conditions that favour protein-protein interactions. We used differential scanning calorimetry (DSC) to compare their conformational heat stability, through the characteristic temperature (Θmax) corresponding to the maximal heat flow and the overall calorimetric heat of reaction (ΔrHcal). Times needed to observe sol-gel state transitions at various temperatures were determined by a tilting test and the corresponding time-temperature experimental points were best fitted to two successive Arrhenius plots intersecting at Θ∼Θmax corresponding to the major protein component for whey proteins and to a minor protein component for egg white albumen. Observations of gel-networks by scanning electron microscopy indicated a wide range of stuctural patterns, depending on the composition of protein solutions. The results are discussed in terms of the temperature of maximal rate of heat-induced conformational changes and of sol-gel state transitions of protein molecules.

Heat-induced conformational changes of patatin, the major potato tuber protein.

This paper presents a first structural characterization of isolated patatin, the major potato tuber protein, at ambient and elevated temperatures. Isolated patatin at room temperature is a highly structured molecule at both secondary and tertiary levels. It is estimated from far-ultraviolet circular dichroism data that about 33% of the residues adopts an alpha-helical and 46% a beta-stranded structure. Patatin is thermally destabilized at temperatures exceeding 28 degrees C, as was indicated by near-ultraviolet circular dichroism. It was shown that parts of the alpha-helical contributions unfold in the 45-55 degrees C region, whereas the beta-stranded parts unfold more gradually at temperatures of 50-90 degrees C. This was confirmed with Fourier-transform infrared spectroscopy. Differential scanning calorimetry indicated a cooperative transition between 50-60 degrees C, most likely reflecting the unfolding of alpha-helical parts of the molecule. Furthermore, fluorescence spectroscopy confirmed a global unfolding of the protein between 45-55 degrees C. The observed unfolding of the protein coincides with the inactivation of the patatin enzyme activity and with the precipitation as occurs in the potato fruit juice upon heating. At high temperatures, patatin still contains some helical and stranded structures. Upon cooling the protein partly refolds, it was observed that mainly alpha-helical structures were formed.

Reversibility of heat-induced conformational changes and surface exposed hydrophobic clusters of β-lactoglobulin: their role in heat-induced sol–gel state transition

The effects of heat-treatment on the conformational changes of β-lactoglobulin were monitored by differential scanning calorimetry (DSC), binding properties to 1-anilino-8-naphtalenesulphonic acid (ANS) and to 5,5′-dithio-bis (2-nitrobenzoic acid) (DTNB). The thermal transition of β-lactoglobulin was 100% reversible on re-heating and its binding properties to the ANS fluorescent-dye and to the DTNB probe did not significantly change when the first heating was made to a temperature T< T max, that of the DSC maximal peak deviation of unheated solutions. When the solutions were heated to higher temperatures, the degree of reversibility of the thermal transition decreased, while the β-lactoglobulin surface hydrophobicity increased. Furthermore, the time ( tg) needed for the sol–gel state transitions was highly temperature-dependent for the solutions showing no significant reactivity with the DTNB probe, a high percentage of residual tertiary structure but a low surface hydrophobicity. For β-lactoglobulin showing <50% residual tertiary structure but high surface hydrophobicity, tg values were hardly temperature-dependent. The results are discussed in terms of the role of hydrophobic interactions in the aggregation process of denatured β-lg molecules.

Evaluation of heat-induced conformational changes in proteins by nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry

The characterization of heat-induced conformational changes of proteins by nanoelectrospray mass spectrometry is shown. The used device is the first to make a simple and effective investigation of the denaturation of proteins possible using minimum sample consumption, as enabled by nanoelectrospray mass spectrometry. As indicated by average charge states at several solution temperatures, a thermal induced denaturation of ubiquitin, in the temperature range between 25 and 90°C, was investigated. A single structure transition of ubiquitin was observed. Heating and unassisted cooling by air convection of the sample solution occurs directly inside the nanospray capillary during the electrospray measurement. Therefore, this new device is also suitable for probing for the reversibility of conformational changes in proteins.

Effect of Heat-Induced Structural Perturbation of Secondary and Tertiary Structures on the Chaperone Activity of α-Crystallin

α-Crystallin, a major protein of the lens, is known to have chaperone activity to protect other proteins against thermal aggregation. Heat-induced structural change of α-crystallin was previously shown to increase its chaperone activity. In this report, we studied the thermal reversibility of α-crystallin and the effect of change in secondary structure on its chaperone functionin vitro.The heat-induced conformational changes in the aromatic region of near-UV CD spectra showed only a small degree of reversibility. The structural transitions from 50 to 70°C were largely reversible if the incubation time was short. However, the protective ability to inhibit thermal aggregation of alcohol dehydrogenase by α-crystallin was essentially similar at 48 and 70°C. Under long-term heating at high temperatures, there was a time-dependent irreversibility of structural change in α-crystallin as revealed by CD spectroscopy. Such denatured α-crystallin by long-term heating can still preserve its ability to prevent UV-induced aggregation of γ-crystallin at room temperature, indicating relatively little effect of heat-induced changes in secondary structure on the chaperone activity of α-crystallin.

Study on the heat-induced conformational changes of β-conglycinin by FTIR and CD analysis

The influence of temperature on conformational changes was investigated for β-conglycinin, which is one of the main storage proteins of soybean seeds, using Fourier transform infrared (FTIR) and circular dichroism (CD) methods. FTIR spectra have been measured at the temperature range 25–80° C for 10% β-conglycinin in 35 mmolldm 3 phosphate buffer at pD 7.6. The band at 1639 cm −1 indicated the degree of denaturation and the band at 1618 cm −1 was considered to reflect the formation of intermolecular β-sheet structures. The decrease in the band at 1639 cm −1 indicated that heat-denaturation of β-conglycinin occurred at ~65°C and proceeded with increasing temperature in the range 65–80°C. On the other hand, with increasing temperature, the band at 1618 cm −1 began to increase at ~65°C, and seemed to saturate when the temperature was >75°C. The results of CD spectroscopies are in good agreement with results from FTIR. These results show that the conformational states of heat-denatured β-conglycinin are different depending on temperature. However, once heat-denaturated β-conglycinin was cooled, partial refolding of the unfolded structure was induced, and it was difficult to elucidate conformational differences between β-conglycinin which was treated with different temperatures by CD analysis.

The Plasmodium falciparum protein RESA interacts with the erythrocyte cytoskeleton and modifies erythrocyte thermal stability

The ring-infected erythrocyte surface antigen (RESA) associates with spectrin in the erythrocyte membrane (Foley, M., Tilley, L., Sawyer, W.H. and Anders, R.F. (1991) Mol. Biochem. Parasitol., 46, 137–148). A fragment of the RESA protein, which was expressed in Escherichia coli, was found to bind to inside-out vesicles of erythrocyte membranes in an apparently saturable manner. Upon extraction of inside-out vesicles with Triton X-100, the RESA fragment remained associated with the erythrocyte cytoskeleton. Using the technique of steady-state fluorescence polarisation, we have studied the thermal denaturation of fluorescein-labelled spectrin in the presence of recombinant RESA. We found that the RESA fragment partially protected spectrin against heat-induced conformational changes. Furthermore, erythrocytes infected with a RESA (−) laboratory strain (FCR3) were shown to be more susceptible to heat-induced fragmentation than erythrocytes infected with a RESA (+) strain of the parasite. RESA does not, however, appear to play an essential role in the invasion process per se as erythrocytes resealed to contain anti-RESA antibodies were efficiently invaded.

Heat-Induced Conformational Changes in Whey Protein Isolate and Its Relation to Foaming Properties

Heat-induced changes in the physicochemical properties of whey protein isolate (WPI) have been studied. WPI (5%) heated at 70 o C underwent rapid conformational changes within 1 min. The aperiodic structure content increased primarily at the cost of β-sheet structure. The hydrophobic character, as measured by changes in the pH-solubility profile and the solubility profile at pH 4.6 in NaCl solutions, of the protein surface increased. However, the surface hydrophobicity, as measured by the cis-parinaric acid binding method, decreased. In contrast, WPI (9%) heated at 90 o C did not exhibit significant changes in the secondary structure content

Heat-induced conformational changes in proteins studied by electrospray ionization mass spectrometry.

A simple and effective device for investigating heat-induced denaturation of proteins by electrospray ionization mass spectrometry is described. Results are presented for the denaturation as a function of temperature and solution pH of bovine ubiquitin and bovine cytochrome c. These results are in concert with and extend the earlier results of LeBlanc et al. (Org. Mass Spectrom. 1991, 26, 831). The cooperative effects of pH and temperature on the denaturation of ubiquitin and cytochrome c were investigated. Electrospray ionization mass spectrometry is also shown to be a useful probe of the reversibility of heat-induced denaturation of proteins. Finally, it is demonstrated that heat-induced denaturation can be used to improve the mass spectrometric response of proteins that do not normally yield useful spectra when the solubilized protein is electrosprayed at ambient temperatures.

Heat-induced conformational changes in phaseolin and its relation to proteolysis

Heat-induced conformational changes and their relationship to trypsinolysis of phaseolin, the major storage globulin of Phaseolus vulgaris, were studied using far- and near-UV circular dichroism (CD), second-derivative UV absorption spectroscopy and intrinsic fluorescence quenching of Trp fluorescence by an ionic (iodide) and neutral (acrylamide) quencher. Thermal denaturation did not cause any significant changes in the secondary structure of phaseolin. However, near-UV CD data indicated a 0.8–3-fold decrease in the intensity of Phe and Tyr peaks. At least 22 out of 31 Tyr residues in the native phaseolin, while 29 in the 30-min heated phaseolin were exposed to aqueous solvent. The fluorescent Trp residues in both the native and heated phaseolins were little accessible to iodide, while upon heating, 70–90% of the fluorescence was quenched by acrylamide. The surface hydrophobicity of the protein increased 7–9-fold upon thermal denaturation. The results presented here suggest that a disruption upon heating of the tertiary and quaternary structures of phaseolin, and not changes in the secondary structure, is crucial for its enhanced susceptibility to trypsin.

Stabilization of poliovirus against heat inactivation

Fatty acids and related compounds, as well as many salts, stabilize poliovirus against heat inactivation. Addition of myristate to poliovirus prevents heat-induced conformational changes which are detected by trypsin degradation of the virion. Using equilibrium dialysis, we found that several molecules of myristate bind per virion. The relative stabilizing potencies of the salts can be explained by the Hofmeister effect.