Thermal Unfolding Process Research Articles

Collapse of the hydration shell of a protein prior to thermal unfolding

Based on high statistical quality wide-angle X-ray scattering data for the unfolding–refolding process of hen egg-white lysozyme (HEWL), we have analysed the change of the hydration shell as a function temperature using the program CRYSOL. The present results suggest that the decrease of the hydration-shell density starts from a lower temperature than the transition temperature of the collapse of the tertiary structure of HEWL. Although the use of CRYSOL for scattering data for proteins before the transition has an apparent limitation, the collapse of the hydration shell prior to the unfolding of HEWL agrees with a slight tendency of the radius of gyration to decrease during the thermal unfolding process.

Charge interaction and temperature effects on the solution structure of lysozyme as revealed by small-angle X-ray scattering

We have studied the structure of lysozyme as influenced by solution environment using small-angle X-ray scattering (SAXS). With an ellipsoid form factor and a structure factor derived using the mean spherical approximation to account for the electrostatic repulsion of lysozyme, we have extracted detailed structural information about the protein in aqueous solutions, including the size, shape, and net charge number. The SAXS data analysis shows that lysozyme in pure water, expressing an averaged net charge number of ~6, folds to an ellipsoid-like shape with a radius of gyration Rg = 16.6 A. Temperature-dependent SAXS for lysozyme in a buffer solution in which charge repulsion has been eliminated suggests that the protein may thermally unfold gradually along a preferred direction from the ellipsoidal shape with an aspect ratio of p ≃ 2 at 303 K to an elongated shape with p ≃ 3 at 343 K. The structural parameters of the unfolded lysozyme obtained using model fitting are compared with the envelope morphology simulated using a dummy-residues model. From the evolution of the volume of lysozyme during the thermal unfolding process, we deduce a free-energy profile for the protein thermally unfolded in water using a modified Ising model on the basis of a mean field approximation.

Thermal Unfolding Process of Dihydrolipoamide Dehydrogenase Studied by Fluorescence Spectroscopy

The thermal unfolding pathway for dihydrolipoamide dehydrogenase (LipDH) isolated from Bacillus stearothermophilus was investigated focusing on the transient intermediate state characterized through time-resolved fluorescence studies. The decrease in ellipticity in the far UV region in the CD spectrum, the fluorescence spectral change of Trp-91 and FAD, and the thermal enzymatic inactivation curve consistently demonstrated that LipDH unfolded irreversibly on heat treatment at higher than 65 degrees C. LipDH took a transient intermediate state during the thermal unfolding process which could refold back into the native state. In this state, the internal rotation of FAD was activated in the polypeptide cage and correspondingly LipDH showed a peculiar conformation. The transient intermediate state of LipDH characterized in time-resolved fluorescence depolarization studies showed very similar properties to the molten-globule state, which has been confirmed in many studies on protein folding.

Structural study of the catalytic domain of PKCζ using infrared spectroscopy and two‐dimensional infrared correlation spectroscopy

The secondary structure of the catalytic domain from protein kinase C zeta was studied using IR spectroscopy. In the presence of the substrate MgATP, there was a significant change in the secondary structure. After heating to 80 degrees C, a 14% decrease in the alpha-helix component was observed, accompanied by a 6% decrease in the beta-pleated sheet; no change was observed in the large loops or in 3(10)-helix plus associated loops. The maximum increase with heating was observed in the aggregated beta-sheet component, with an increase of 14%. In the presence of MgATP, and compared with the sample heated in its absence, there was a substantial decrease in the 3(10)-helix plus associated loops and an increase in alpha-helix. Synchronous 2D-IR correlation showed that the main changes occurred at 1617 cm(-1), which was assigned to changes in the intermolecular aggregated beta-sheet of the denaturated protein. This increase was mainly correlated with the change in alpha-helix. In the presence of MgATP, the main correlation was between aggregated beta-sheet and the large loops component. The asynchronous 2D-correlation spectrum indicated that a number of components are transformed in intermolecularly aggregated beta-sheet, especially the alpha-helix and beta-sheet components. It is interesting that changes in 3(10)-helix plus associated loops and in alpha-helix preceded changes in large loops, which suggests that the open loops structure exists as an intermediate state during denaturation. In summary, IR spectroscopy revealed an important effect of MgATP on the secondary structure and on the thermal unfolding process when this was induced, whereas 2D-IR correlation spectroscopy allowed us to show the establishment of the denaturation pathway of this protein.

Oligomerization and Aggregation of Bovine Pancreatic Ribonuclease A: Characteristic Events Observed by FTIR Spectroscopy

Nonnative protein aggregation, which is a common feature in biotechnology, is also a clinical feature in more than 20 serious degenerative diseases. We studied the specific events of bovine pancreatic ribonuclease A thermal aggregation by a combination of second derivative infrared analysis and two-dimensional infrared correlation spectroscopy. By comparing the events that occur in reversible and irreversible thermal unfolding processes, certain events that were related to protein aggregation were characterized. Particularly, a band that appeared at high temperatures was assigned to the cross β-structures in oligomers. The effect of pH, NaCl, and ethanol on ribonuclease A oligomerization as well as further aggregation induced by heat were studied and dissimilar effects of these additives were found. Basic pH and NaCl could accelerate the thermal aggregation but did not affect the formation of oligomers, whereas ethanol could increase both the aggregation rate and the population of oligomers. Our results suggested that the aggregation of RNase A might be initiated by hydrophobic interactions, controlled by oligomerization and mediated by electrostatic interactions. Moreover, the strategy of using second derivative and two-dimensional infrared analysis might provide a potential powerful tool to study the events that are directly related to the initiation of protein aggregation.

Effects of Ionic Strength on the Thermal Unfolding Process of Granulocyte-Colony Stimulating Factor

This paper reports the effect of ionic strength on the process of thermal unfolding of recombinant methionyl human granulocyte-colony stimulating factor (rmethuG-CSF) at acid pH. We previously reported that the protein aggregates were formed at the highest temperature at pD 2.1 in the pD range of 5.5-2.1 and that the aggregation proceeded a little at pD 2.1 because of the strong repulsive interaction between the unordered structures that play the role of a precursor for the aggregation. In the present study temperature-dependent IR spectra and far-UV CD spectra were measured for rmethuG-CSF in aqueous solutions containing various concentrations of NaCl at acid pH. Second derivative and curve-fitting analysis were performed to examine the obtained IR spectra. The results revealed that the structure of rmethuG-CSF becomes less stable with increasing ionic strength at all pDs investigated (pD 2.1, 2.5, and 4.0). We have also demonstrated that, at pD 2.1, the temperature at which the protein aggregation starts becomes lower and that the amount of the aggregates becomes larger with the addition of NaCl. This is probably because the addition of NaCl masks the repulsive electrostatic interaction between the unordered structures.

Thermal Unfolding Process of Proteins Depending on Structural Hierarchy Clarified by Wide-Angle X-Ray Scattering at a Third Generation Synchrotron Source

α-ラクトアルブミン（α-LA）と鶏卵白リゾチーム（HEWL）は互いに相同なタンパク質であり，三次構造は極めて類似していることが知られている．一方，それらのアンフォールディング•リフォールディング過程は異なった熱力学的特徴を示すことが明らかにされているが，その違いと構造転移との関係の詳細は明らかにされていない．そこで，この報告では，pH 7の溶媒中におけるα-LAとHEWLの熱構造転移の特徴をX線溶液散乱法によって比較検討する．放射光（SR）光源を用いる小角X線散乱法（SAXS）は溶液中でのタンパク質の構造解析，特にタンパク質のフォールディングの研究に利用されてきた．従来のSR-SAXS法では小角領域のデータから評価する回転半径などの低分解能の構造情報を解析するにとどまっていた．本研究では，第3世代SR光源を用いた広角X線散乱測定（WAXS）によって，2.5Åから200Åにわたる広い空間領域で観測し，全階層構造（四•三次構造，ドメイン構造，二次構造）に依存した構造転移過程の特徴の詳細を解析した．ここで示すSR-WAXSデータ解析法を用いると，タンパク質のアンフォールディング•リフォールディング過程における階層構造別の変化，階層構造間の転移の協同性，局所的な構造変化などを定量的に明らかにできる．我が国の高輝度光科学研究センター（SPring-8）のような第3世代SR光源の利用によって，実験データの時間•空間分解能が著しく改善された溶液散乱法の現状を示す．

Molecular Dynamics Simulations to Investigate the Thermal Unfolding Behaviors of the Tetramerization Domain of Shaker and Kv1.1 Potassium Channels

In our previous study, a set of homology models of the tetramerization (T1) domain of six eukaryotic potassium channels Kv1.1-Kv1.6 from Homo Sapiens was constructed based on the crystal structure of the Shaker T1 domain from Aplysia californica. The results reveal that the T1 domains of these Kv channels exhibit similar folds as those of Shaker K(superscript +) channel. In this study, several molecular dynamics (MD) simulations towards the Shaker and Kv1.1 T1 domains were conducted at various temperatures. Our results show that the Shaker T1 domain exhibit higher structural integrity than the Kvl.1 T1 domain at all temperatures examined. In addition, the thermal unfolding of the Shaker T1 domain begins at layer 3. In contrast, layers land 2 exhibit higher structural stability because layer 1 remains more hydrogen bonding interactions at elevated temperatures and layer 2 is located in the highly conserved hydrophobic core. Ile121 in the Shaker T1 domain plays an important role in disrupting the loop between helices 4 and 5. During the thermal unfolding process, the newly formed hydrophobic interactions between A1a120, Ile12l, Leu122, Leu13l, and Leu151 may distort the native contact between layers 2 and 3 of the T1 domain.

An Infrared Spectroscopy Study of Acid Stability and Thermal Unfolding Process of Granulocyte-Colony Stimulating Factor

Temperature-dependent (25-80 degrees C) infrared (IR) spectra were obtained for recombinant methionyl human granulocyte-colony stimulating factor (rmethuG-CSF) in aqueous solutions over the pD range of 5.5-2.1 to investigate its thermal stability at various pDs. Second derivative, Fourier self-deconvolution, and curve-fitting analyses were performed to analyze the obtained spectra. These spectral analyses demonstrated that in the thermal unfolding process the alpha-helix structure of rmethuG-CSF partially changes to an unordered structure and then the unordered structure forms aggregates. The temperature-dependent IR spectra revealed that the structure of rmethuG-CSF is the most stable at pD 2.5 in the pD range of 5.5-2.1. It has been suggested that the unordered structure formed before the marked structural change in the whole molecule is a perturbed form of the native structure of rmethuG-CSF and plays a role as a precursor for the aggregation. This alteration to the perturbed form is likely to be the first secondary structure change that occurs along the aggregation pathway. Of particular note is that the stability at pD 2.1 is slightly lower than that at pD 2.5, but that aggregates are formed at higher temperature at pD 2.1 than at pD 2.5, probably because the repulsive interaction between the unordered structure is stronger at pD 2.1.

Thermal unfolding of bovine β-lactoglobulin studied by UV spectroscopy and fluorescence quenching

In spite of numerous physical and biochemical studies on thermal denaturation of bovine β-lactoglobulin (β-LG), the definition of the N- and U-states, and the extent of reversibility assigned to the process are not clearly determined. The application of two-state model and dissociation coupled unfolding model to interpret the thermal unfolding profiles obtained by different authors using UV-difference absorbance at 293 nm, was analyzed and discussed. We conclude that both models fail to describe correctly the β-LG thermal unfolding process in the conditions assayed in these previous works (at protein concentrations below 4 mg mL −1, at low ionic strength and near neutral pH). The probable causes of this failure are: (1) below 55 °C, β-LG solutions are a mixture of monomers and dimers, (2) between 55 and 90 °C, β-LG heating induces irreversible changes in the monomeric protein structure and promotes the formation of aggregates of low molecular weight, and (3) above 90 °C, β-LG aggregates of high molecular weight are formed irreversibly. In this work, the presence of irreversible changes in β-LG heating was demonstrated by quenching the intrinsic fluorescence of the protein by added acrylamide.

Protein Thermal Aggregation Involves Distinct Regions: Sequential Events in the Heat-Induced Unfolding and Aggregation of Hemoglobin

Protein thermal aggregation plays a crucial role in protein science and engineering. Despite its biological importance, little is known about the mechanism and pathway(s) involved in the formation of aggregates. In this report, the sequential events occurring during thermal unfolding and aggregation process of hemoglobin were studied by two-dimensional infrared correlation spectroscopy. Analysis of the infrared spectra recorded at different temperatures suggested that hemoglobin denatured by a two-stage thermal transition. At the initial structural perturbation stage (30–44°C), the fast red shift of the band from α-helix indicated that the native helical structures became more and more solvent-exposed as temperature increased. At the thermal unfolding stage (44–54°C), the unfolding of solvent-exposed helical structures dominated the transition and was supposed to be responsible to the start of aggregation. At the thermal aggregation stage (54–70°C), the transition was dominated by the formation of aggregates and the further unfolding of the buried structures. A close inspection of the sequential events occurring at different stages suggested that protein thermal aggregation involves distinct regions.

2F1430 バクテリオロドプシンの熱変性過程で起こる構造変化と光誘起変性現象との関係

2F1430 バクテリオロドプシンの熱変性過程で起こる構造変化と光誘起変性現象との関係

Thermal unfolding of chitosanase from Streptomyces sp. N174: role of tryptophan residues in the protein structure stabilization

Tryptophan residues in chitosanase from Streptomyces sp. N174 (Trp 28, Trp 101, and Trp 227) were mutated to phenylalanine, and thermal unfolding experiments of the proteins were done in order to investigate the role of tryptophan residues in thermal stability. Four types of mutants (W28F, W101F, W227F and W28F/W101F) were produced in sufficient quantity in our expression system using Streptomyces lividans TK24. Each unfolding curve obtained by CD at 222 nm did not exhibit a two-state transition profile, but exhibited a biphasic profile: a first cooperative phase and a second phase that is less cooperative. The single tryptophan mutation decreased the midpoint temperature ( T m) of the first transition phase by about 7°C, and the double mutation by about 11°C. The second transition phase in each mutant chitosanase was more distinct and extended than that in the wild-type. On the other hand, each unfolding curve obtained by tryptophan fluorescence exhibited a typical two-state profile and agreed with the first phase of transition curves obtained by CD. Differential scanning calorimetry profiles of the proteins were consistent with the data obtained by CD. These data suggested that the mutation of individual tryptophan residues would partly collapse the side chain interactions, consequently decreasing T m and enhancing the formation of a molten globule-like intermediate in the thermal unfolding process. The tryptophan side chains are most likely to play important roles in cooperative stabilization of the protein.

The binding of 3,6-disubstituted bile salts to human serum albumin induces conformational change on the molecule of this protein

The binding of 3,6-hydroxy and keto disubstituted bile salts to human serum albumin was studied using differential scanning calorimetry, fluorescence spectroscopy and circular dichroism. The bile salts assayed did not produce any modification in the shape of the albumin thermogram, its thermal unfolding process in their presence being reversible; however, an increase in the enthalpy of unfolding and in the T m was observed in the presence of 3,6-diketo and 3-hydroxy-6-keto bile salts. These two derivatives induced a negative circular dichroism spectrum of the protein around 280–290 nm, quenched the native fluorescence of the buried tryptophan of albumin and induced energy transfer between 1 aniline-8-naphthalene sulfonate and the buried tryptophan 214 of albumin. The presence of a keto group at C 6 in the steroid ring of the bile salts plays an important role in producing slight movement of the albumin domains, increasing the distance between domains I and II.

Conformational study of a collagen peptide by 1H NMR spectroscopy: observation of the 14N- 1H spin-spin coupling of the Arg guanidinium moiety in the triple-helix structure

CB2, a CNBr peptide of 36 residues from type I collagen α1(I) chain has been studied by NMR spectroscopy as a function of temperature. At low temperature, the guanidinium protons of Arg 9 showed sharp 1:1:1 NMR triplets around 6.95 ppm, characteristic of 14N coupled protons ( 1 J NH=52 Hz) when the quadrupolar relaxation rate is drastically reduced. These spectral characteristics and the low temperature coefficient of the 1:1:1 triplets (Δδ/Δ T of −3.6 ppb/°C) suggest that the H atoms of the protonated guanidinium moiety of Arg 9 in the triple helix are slowly exchanging with bulk water, most likely involved in hydrogen bonds. On the basis of conformational energy computations on a model segment of type I collagen (Vitagliano, L., Némethy, G., Zagari, A. and Scheraga, H.A. (1993) Biochemistry 32, 7354–7359), similar to CB2, our data could indicate that the guanidinium group of Arg 9 form hydrogen bonds with a backbone carbonyl of an adjacent chain probably by using the N ϵ hydrogen, leaving the four N η hydrogens bound to water molecules that must be in slow exchange with bulk water and that could therefore be considered structural elements of the trimeric α1(I) CB2 triple helix. The behaviour of Arg 9 has been investigated also in terms of equilibrium between random monomer and helical trimer conformations controlled by temperature. The thermal unfolding process was found to be reversible and the melting point resulted to be 17°C.

Thermodynamic stability of two variants of xylanase (Xys1) from Streptomyces halstedii JM8.

In a continuation of our earlier study [Ruiz-Arribas, A., Santamaría, R.I., Zhadan, G. G., Villar, E. & Shnyrov, V. L. (1994) Differential scanning calorimetric study of the thermal stability of xylanase from Streptomyces halstedii JM8, Biochemistry 33, 13787-13791], we used high-sensitivity differential scanning microcalorimetry, intrinsic tryptophan fluorescence and far-ultraviolet circular dichroism to study the effect of regional sequence differences on the thermodynamic stability of xylanase (Xys1) from Streptomyces halstedii JM8 (1,4-beta-D-xylanohydrolase, EC 3.2.1.8). Thermal transitions were measured for original xylanase (Xys1S) and two variants. Thermal denaturation of all the xylanases studied revealed two structural domains, each of which, despite its partial irreversibility, follows a two-state thermal unfolding process under our experimental conditions. Both variants were found to exhibit slightly decreased stability, possessing the same activity as the original. The unfolding parameters for each domain of both variants, unlike the situation with wild-type xylanase (see our previous report), fit some correlations obtained for the most compact globular proteins. The values of enthalpy and entropy of unfolding/residue at 383 K were found to be inversely proportional to residual, well-regulated structures in unfolded states.

Membrane-bound Na+,K+-ATPase as the cardiac glycoside receptor

DSC studies are carried out to characterize Na+,K+-ATPase isolated from pig kidney in its natural membrane environment as well as in its purified state upon detergent treatment. The transition temperatures of the investigated thermal protein unfolding process, observed between 43 and 64.5° C, depend on the local membrane environment as well as onpH. In addition, the transition temperature is significantly increased upon binding of different cations and ligands which are known to interact with the enzyme. Evidence for a lipid phase transition around 23 °C in the original biological membrane is reported.

Acid and thermal unfolding of Escherichia coli dihydrofolate reductase.

The acid and thermal unfolding of Escherichia coli dihydrofolate reductase (DHFR) were studied by means of circular dichroism (CD) and fluorescence spectroscopy. There existed at least one intermediate around pH 4 in the acid unfolding process at 15 degrees C, in which the tertiary structure was disrupted before unfolding of the secondary structure. The fluorescence energy transfer from intrinsic tryptophan residues to 1-anilinonaphthalene-8-sulfonate suggested the disruption of the tertiary structure around some tryptophan residues of the intermediate. The thermal unfolding process at pH 7.0 also involved at least one intermediate having a disrupted tertiary structure and a folded secondary structure. The three-state thermodynamic analysis showed that the intermediate in thermal unfolding was less stable by 1.8 kcal/mol than the native state. The similarity of the far-ultraviolet CD spectra of acid and thermally unfolded forms suggests that both types of unfolding produce the same structure, which may be a molten globule intermediate such as that in the folding kinetics of DHFR. The acid and thermal unfolding were depressed in the presence of KCl due to stabilization of the native form.

A stable intermediate in the thermal unfolding process of a chimeric 3‐isopropylmalate dehydrogenase between a thermophilic and a mesophilic enzymes

The thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, enzymes was studied by CD spectrophotometry and differential scanning calorimetry (DSC). The enzyme is a homodimer with a subunit containing two structural domains. The DSC melting profile of the chimeric enzyme in 20 mM NaHCO3, pH 10.4, showed two endothermic peaks, whereas that of the T. thermophilus wild-type enzyme had one peak. The CD melting profiles of the chimeric enzyme under the same conditions as the DSC measurement, also indicated biphasic unfolding transition. Concentration dependence of the unfolding profile revealed that the first phase was protein concentration-independent, whereas the second transition was protein concentration-dependent. When cooled after the first transition, the intermediate was isolated, which showed only the second transition upon heating. These results indicated the existence of a stable dimeric intermediate followed by the further unfolding and dissociation in the thermal unfolding of the chimeric enzyme at pH 10-11. Because the portion derived from the mesophilic isopropylmalate dehydrogenase in the chimeric enzyme is located in the hinge region between two domains of the enzyme, it is probably responsible for weakening of the interdomain interaction and causing the decooperativity of two domains. The dimeric form of the intermediate suggested that the first unfolding transition corresponds to the unfolding of domain 1 containing the N- and C-termini of the enzyme, and the second to that of domain 2 containing the subunit interface.

Core domain of hirudin from the leech Hirudinaria manillensis: chemical synthesis, purification, and characterization of a Trp3 analog of fragment 1-47.

Hirudin is a small (approximately 7 kDa) disulfide-cross-linked polypeptide known as the most potent and specific thrombin inhibitor. We have previously shown that the N-terminal proteolytic fragment 1-47 of hirudin HM2 from Hirudinaria manillensis maintains inhibitory action toward thrombin [Vindigni, A., et al. (1994) Eur. J. Biochem. 226, 323-333]. Here we report the solid-phase chemical synthesis of an analog of fragment 1-47 bearing a Tyr3-->Trp exchange (Y3W analog). The crude, reduced peptide was purified by reverse-phase HPLC and subjected to oxidative folding to the disulfide-cross-linked species. The folding process of the Y3W analog was slower than that of the natural fragment 1-47, but nevertheless still occurred almost quantitatively as the natural species. The overall final yield of the synthetic product was approximately 35%, and its identity and homogeneity was established by a number of analytical techniques, including electrospray mass spectometry. The unique alignment of the three disulfide bridges of the Y3W analog was established by peptide mapping as Cys6-Cys14, Cys16-Cys28, and Cys22-Cys37 and shown to be identical to that of the natural fragment. The results of far- and near-ultraviolet circular dichroism and fluorescence emission measurements provided evidence that the Y3W analog retains the structural features of the natural species. The thermodynamic quantities (delta GD, delta Hm, delta Sm, and delta Cp) characterizing the reversible and cooperative thermal unfolding processes of the Y3W analog (Tm = 60.5 degrees C) and the natural fragment species (Tm = 62.5 degrees C) were evaluated. Despite the relatively high Tm values, the stability of both fragment species at 37 degrees C was only approximately 10 kJ mol-1, well below the average 50 kJ mol-1 typical of single-domain globular proteins. The synthetic Y3W species was found to be approximately 5-fold more active (KI = 30 +/- 5 nM) than the natural fragment 1-47 (KI = 150 +/- 20 nM) in inhibiting thrombin. Of interest was that the difference in the free energies of binding to thrombin at 37 degrees C, delta delta Gb, between the Y3W analog and natural species (4.2 kJ mol-1) was that expected for the difference in hydrophobicity between the two polypeptides resulting from the Tyr-->Trp exchange. The results of this study indicate that solid-phase chemical synthesis represents a convenient and high-yield procedure to prepare analogs of the biologically active, N-terminal core domain of hirudin with improved functional properties.