Kinetics of the helix/coil transition of the collagen-like peptide (Pro-Hyp-Gly)10

This article measures the rates of folding and unfolding of the collagen-like peptide (Pro-Hyp-Gly)(10) over overlapping concentration and temperature ranges. The data allow calculation of the orders of the folding and the unfolding reactions, the effective Arrhenius activation energies, and numerical solution of the differential equation controlling the helix/coil transition during temperature scanning. The resulting predictions of helicity closely followed DSC measurements of the peptide in both up- and down-scanning modes, confirming the validity of the theoretical equations governing the kinetics of the folding/unfolding process. In both up- and down-scanning, three regions were apparent: "quasistatic," "rate," and "mixed." At very low scanning rates, a quasistatic region revealed a broad, short endotherm that was independent of scanning rate, but dependent on concentration and equal to the equilibrium endotherm. At high up-scanning rates, the "rate region" endotherm was sharp and tall and T(max) increased with scanning rate. In down-scanning, the "rate peak" was very broad and very short and T(max) decreased with scanning rate. The "mixed region" showed nascent "rate" and nascent "quasistatic" peaks, which were evident in the same up-scan under certain conditions. Comparison of (Pro-Hyp-Gly)(10) and (Pro-Pro-Gly)(10) showed that the higher temperature stability of (Pro-Hyp-Gly)(10) is due mainly to its slower rate of unfolding and higher activation energy.     

Decrease in stability of human albumin with increase in protein concentration

The stability (reflected in denaturation temperature, Td) of defatted human albumin monomer, monitored by differential scanning calorimetry, decreases with increasing protein concentration. This is shown to be compatible with a simple model in which reversible polymerization of denatured monomer promotes unfolding. This model also predicts an increase in transition cooperativity with decreasing protein concentration whereas experimentally cooperativity decreases because the rate of thermally induced polymerization of unfolded monomer is slow relative to the scan rate of the calorimeter. The denaturation of undefatted human albumin monomer, subsaturated with high affinity endogenous long-chain fatty acid (LCFA), was previously observed by differential scanning calorimetry to be a biphasic process. Td for the first endotherm, associated with the denaturation of LCFA-poor species, decreases with increasing protein concentration similar to that for defatted monomer whereas Td for the second endotherm, associated with denaturation of LCFA-rich species, is independent of concentration. The magnitude of the concentration dependence of Td relates directly to the extent of polymerization of denatured monomer, which decreases with increasing level of bound ligand. The bimodal thermogram observed for undefatted monomer persists upon simultaneous extrapolation of Td values to low concentration and low scan rate thereby demonstrating that this biphasic denaturation arising from ligand redistribution during denaturation is a true thermodynamic phenomenon and not an artifact of specific experimental conditions or the method used to induce denaturation.     

Structural basis for differential insertion kinetics of dNMPs opposite a difluorotoluene nucleotide residue

We have recently challenged the widely held view that 2,4-difluorotoluene (dF) is a nonpolar isosteric analogue of the nucleotide dT, incapable of forming hydrogen bonds (HBs). To gain a further understanding for the kinetic preference that favors dAMP insertion opposite a templating dF, a result that mirrors the base selectivity that favors dAMP insertion opposite dT by RB69 DNA polymerase (RB69pol), we determined presteady-state kinetic parameters for incorporation of four dNMPs opposite dF by RB69pol and solved the structures of corresponding ternary complexes. We observed that both the F2 and F4 substituent of dF in these structures serve as HB acceptors forming HBs either directly with dTTP and dGTP or indirectly with dATP and dCTP via ordered water molecules. We have defined the shape and chemical features of each dF/dNTP pair in the RB69pol active site without the corresponding phosphodiester-linkage constraints of dF/dNs when they are embedded in isolated DNA duplexes. These features can explain the kinetic preferences exhibited by the templating dF when the nucleotide incorporation is catalyzed by wild type RB69pol or its mutants. We further show that the shapes of the dNTP/dF nascent base pair differ markedly from the corresponding dNTP/dT in the pol active site and that these differences have a profound effect on their incorporation efficiencies.     

Discrete reduction of type I collagen thermal stability upon oxidation

The oxidation of acid-soluble calf skin collagen type I caused by metal-dependent free radical generating systems, Fe(II)/H2O2 and Cu(II)/H2O2, was found to bring down in a specific, discrete way the collagen thermal stability, as determined by microcalorimetry and scanning densitometry. Initial oxidation results in splitting of the collagen denaturational transition into two components. Along with the endotherm at 41 degrees C typical for non-oxidized collagen, a second, similarly cooperative endotherm appears at 35 degrees C and increases in enthalpy with the oxidant concentration and exposure time, while the first peak correspondingly decreases. The two transitions at 35 and 41 degrees C were registered by densitometry as stepwise increases of the collagen-specific volume. Further oxidation results in massive collagen destruction manifested as abolishment of both denaturational transitions. The two oxidative systems used produce identical effects on the collagen stability but at higher concentrations of Cu(II) in comparison to Fe(II). The discrete reduction of the protein thermal stability is accompanied by a decrease of the free amino groups, suggestive of an oxidation attack of the side chains of lysine residues. Since the denaturation temperature of collagen shifts from above to below body temperature (41 degrees C-35 degrees C) upon oxidation, it appears important to account for this effect in a context of the possible physiological implications of collagen oxidation.     

Cooperative unfolding of a metastable serpin to a molten globule suggests a link between functional and folding energy landscapes

Alpha-1 antitrypsin (alpha(1)-AT) is a member of the serpin class of protease inhibitors, and folds to a metastable state rather than its thermodynamically most stable native state. Upon cleavage by a target protease, alpha(1)-AT undergoes a dramatic conformational change to a stable form, translocating the bound protease more than 70 A to form an inhibitory protease-serpin complex. Numerous mutagenesis studies on serpins have demonstrated the trade-off between the stability of the metastable state on the one hand and the inhibitory efficiency on the other. Studies of the equilibrium unfolding of serpins provide insight into this connection between structural plasticity and metastability. We studied equilibrium unfolding of wild-type alpha(1)-AT using hydrogen-deuterium/exchange mass spectrometry to characterize the structure and the stability of an equilibrium intermediate that was observed in low concentrations of denaturant in earlier studies. Our results show that the intermediate observed at low concentrations of denaturant has no protection from hydrogen-deuterium exchange, indicating a lack of stable structure. Further, differential scanning calorimetry of alpha(1)-AT at low concentrations of denaturant shows no heat capacity peak during thermal denaturation, indicating that the transition from the intermediate to the unfolded state is not a cooperative first-order-like phase transition.. Our results show that the unfolding of alpha(1)-AT involves a cooperative transition to a molten globule form, followed by a non-cooperative transition to a random-coil form as more guanidine is added. Thus, the entire alpha(1)-AT molecule consists of one cooperative structural unit rather than multiple structural domains with different stabilities. Furthermore, our results together with previous mutagenesis studies suggest a possible link between an equilibrium molten globule and a functional intermediate that may be populated during the protease inhibition.     

Differential scanning calorimetry of the irreversible denaturation of Rapana thomasiana (marine snail, Gastropod) hemocyanin

The thermal denaturation of the hemocyanin from gastropod Rapana thomasiana (RtH) at neutral pH was studied by means of differential scanning calorimetry (DSC). The denaturation was completely irreversible as judged by the absence of any endotherm on rescanning of previously scanned samples. Two transitions, with apparent transition temperatures (T(m)) at 83 and 90 degrees C, were detected by DSC using buffer 20 mM MOPS, containing 0.1 M NaCl, 5 mM CaCl(2) and 5 mM MgCl(2), pH 7.2. Both T(m) were dependent on the scanning rate, suggesting that the thermal denaturation of RtH is a kinetically controlled process. The activation energy (E(A)) of 597+/-20 kJ mol(-1) was determined for the main transition (at 83 degrees C). E(A) for the second transition was 615+/-25 kJ mol(-1). The T(m) and Delta H(cal) values for the thermal denaturation of RtH were found to be independent of the protein concentration, signifying that the dissociation of the protein into monomers does not take place before the rate-determining state of the process of thermal unfolding.     

Membrane binding induces lipid-specific changes in the denaturation profile of bovine prothrombin. A scanning calorimetry study.

Prothrombin denaturation was examined in the presence of Na2EDTA, 5mM CaCl2, and CaCl2 plus membranes containing 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC) in combination with either bovine brain phosphatidylserine (PS) or 1,2-dioleoyl-phosphatidylglycerol (DOPG). Heating denaturation of prothrombin produced thermograms showing two peaks, a minor one at approximately 59 degrees C previously reported to correspond to denaturation of the fragment 1 region (Ploplis, V. A., D. K. Strickland, and F. J. Castellino 1981. Biochemistry. 20:15-21), and a main one at approximately 57-58 degrees C, reportedly due to denaturation of the rest of the molecule (prethrombin 1). The main peak was insensitive to the presence of 5mM Ca2+ whereas the minor peak was shifted to higher temperature (Tm approximately 65 degrees C) by Ca2+. Sufficient concentrations of POPC/bovPS (75/25) large unilamellar vesicles to guarantee binding of 95% of prothrombin resulted in an enthalpy loss in the main endotherm and a comparable enthalpy gain in the minor endotherm accompanying an upward shift in peak temperature (Tm approximately 73 degrees C). Peak deconvolution analysis on the prothrombin denaturation profile and comparison with isolated prothrombin fragment 1 denaturation endotherms suggested that the change caused by POPC/PS vesicles reflected a shift of a portion of the enthalpy of the prethrombin 1 domain to higher temperature (Tm approximately 77 degrees C). The enthalpy associated with this high-temperature endotherm increased in proportion to the surface concentration of PS. By contrast, POPC/DOPG (50/50) membranes shifted the prethrombin 1 peak by 4 degrees C to a lower temperature and the fragment 1 peak by 5 degrees C to a higher temperature. The data lead to a hypothesis that the fragment 1 and prethrombin 1 domains of prothrombin do not denature quite independently and that binding of prothrombin to acidic-lipid membranes disrupts the interaction between these domains. It is further hypothesized that PS containing membranes exert the additional specific effect of decoupling the denaturation of two subdomains of the prethrombin 1 domain of prothrombin.     

Iron binding to conalbumin. Calorimetric evidence for two distinct species with one bound iron atom.

When thermal denaturation of conalbumin solutions partially saturated with Fe(III) is observed by differential scanning calorimetry, four endotherms are observed between 40 and 100 degrees. The relative size of these four endotherms is determined by the Fe(III) to conalbumin ration. At a heating rate of 10 degrees/min, in Tris buffer at pH 7.5, observed endotherm temperature maxima and enthalpies of denaturation are: conalbumin, 63 degrees, 320 kcal/mol; intermediate I, 68 degrees, intermediate I, 77 degrees; Fe2-conalbumin, 84 degrees, 630 kcal/mol. These four endotherms are observed over a range of protein concentration from 7 to 100 mg/ml and are unchanged when excess bicarbonate is present. Stoichiometric calculations of both total protein and total iron indicate that each intermediate endotherm results from denaturation of conalbumin molecules containing only one ferric ion. These experimental results are thus consistent with the presence of two different monomeric one-iron conalbumin intermediates. They strongly suggest that the two iron binding sites of conalbumin are not equivalent.     

Identification of an intermediate state in the helix-coil degradation of collagen by ultraviolet light

Differential scanning calorimetry has revealed the presence of a new denaturation endotherm at 32 degrees C following UV irradiation of collagen, compared with 39 degrees C for the native triple helix. Kinetic analyses showed that the new peak was a previously unknown intermediate state in the collagen helix-coil transition induced by UV light, and at least 80% of the total collagen was transformed to random chains via this state. Its rate of formation was increased by hydrogen peroxide and inhibited by free radical scavengers. SDS-polyacrylamide gels showed evidence of competing reactions of cross-linking and random primary chain scission. The cross-linking was evident from initial gelling of the collagen solution, but there was no evidence for a dityrosine cross-link. Primary chain scission was confirmed by end group analysis using fluorescamine. Electron microscopy showed that the segment long spacing crystallites formed from the intermediate state were identical to the native molecules. Clearly, collagen can undergo quite extensive damage by cleavage of peptide bonds without disorganizing the triple helical structure. This leads to the formation of a damaged intermediate state prior to degradation of the molecules to short random chains.     

The role of copper in the stability of ascorbate oxidase towards denaturing agents

The susceptibility of native, type-2 Cu-depleted and fully Cu-depleted ascorbate oxidase to thermal and chemical denaturation has been probed by differential scanning calorimetry, fluorimetry and circular dichroism. The data indicate that copper affects the stability, but not the protein conformation. The unfolding of ascorbate oxidase is characterized by a single endotherm. Calorimetric domains revealed by deconvolution are consistent with the domains identified by X-ray crystallography.     

Differential scanning calorimetry of the unfolding of myosin subfragment 1, subfragment 2, and heavy meromyosin

The thermal unfolding of rabbit skeletal heavy meromyosin (HMM), myosin subfragment 1, and subfragment 2 has been studied by differential scanning calorimetry (DSC). Two distinct endotherms are observed in the DSC scan of heavy meromyosin. The first endotherm, with a Tm of 41 degrees C at pH 7.9 in 0.1 M KCl, is assigned to the unfolding of the subfragment 2 domain of HMM based on scans of isolated subfragment 2. The unfolding of the subfragment 2 domain is reversible both in the isolated form and in HMM. The unfolding of subfragment 2 in HMM can be fit as a single two-state transition with a delta Hvh and delta Hcal of 161 kcal/mol, indicating that subfragment 2 exists as a single domain in HMM. The unfolding of subfragment 2 is characterized by an extraordinarily large delta Cp of approximately 30,000 cal/(deg.mol). In the presence of nucleotides, the high-temperature HMM endotherm with a Tm of 48 degrees C shifts to higher temperature, indicating that this peak corresponds to the unfolding of the subfragment 1 domain. This assignment has been confirmed by comparison with isolated subfragment 1. The stabilizing effect of AMPPNP was significantly greater than that of ADP. The vanadate-trapped ADP species was slightly more stable than M.AMPPNP with a Tm at 58 degrees C. The unfolding of subfragment 1, both in the isolated form and in HMM, was irreversible. Only a single endotherm was noted in the DSC scans of the subfragment 1 domain of HMM and in freshly prepared subfragment 1 complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biphasic denaturation of human albumin due to ligand redistribution during unfolding

Denaturation of defatted human albumin monomer, monitored by differential scanning calorimetry, is monophasic as reflected by the single, resulting endotherm. With low levels of various ligands, biphasic or monophasic unfolding processes are manifested as bimodal or unimodal thermograms, respectively. The greater the affinity of native protein for ligand, the greater is the tendency for biphasic denaturation. We propose that such a biphasic unfolding process arises from a substantial increase in stability (transition temperature) of remaining native protein during denaturation. This increase in stability derives from the free energy of ligand binding becoming more negative due to the release of high affinity ligand by unfolding protein. The tendency for biphasic denaturation is greatest at low (subsaturating) levels of ligand where greatest increases in stability occur. Biphasic unfolding arising from such ligand redistribution results from denaturation of different kinds of protein molecules, ligand-poor and ligand-rich species, and not from sequential unfolding of domains within the same molecule. Differentiating between these two mechanisms is necessary for the correct interpretation of biphasic denaturation data. Furthermore, biphasic unfolding due to ligand redistribution occurs independently of the means used to effect denaturation. The maximum increase in stability due to ligand binding relative to the stability of defatted albumin monomer alone occurs with the intermediate affinity ligand octanoate (22 degrees C) and not with the high affinity ligand hexadecanoate (15 degrees C). This indicates a much greater affinity of denatured albumin for hexadecanoate since increase in stability derives from the difference between free energy of ligand binding to folded and unfolded protein forms.     

Gas exchange parameters, muscle blood flow and electromechanical properties of the plantar flexors

Sixteen men were tested to determine VO2max (ml X kg-1 X min-1), anaerobic threshold VO2 (ATVO2) and oxygen kinetics (time constant, T.C.) during running on a treadmill. For measuring maximal calf blood flow (maxBF, ml X 100 ml-1 X min-1), venous occlusion plethysmography was employed immediately following a combination of arterial occlusion and toe raising exercise to exhaustion. In addition, supramaximal electrical stimulations were given to determine maximal calf twitch force (Fmax, N), maximal rate of twitch force development (dF/dt) and relaxation (R X dF/dt, N X ms-1) and electro-mechanical delay time (EMD, ms). Results demonstrated that VO2max, ATVO2 and maxBF were all inversely related to T.C. (p less than 0.05). MaxBF and ATVO2 showed the highest correlation (r = 0.89, p less than 0.01). Stepwise multiple linear regression analyses revealed that variance in VO2max (60%) and ATVO2 (84%) could be accounted for by the combined effects of the following peripheral factors: VO2max = 51,25-3.24(dF/dt) + 0.14(maxBF), and ATVO2 = 11.68 + 0.42(maxBF) - 0.2(Fmax). These findings, together with the results of cluster analysis, suggest a tight link between ATVO2 and peripheral blood flow capacity. On the other hand, a moderate correlation (r = 0.64, p less than 0.01) between VO2max and maxBF might be due in part to individual differences in oxygen extraction-utilization capacity during heavy exercise above anaerobic threshold.     

Proteolytic Scanning Calorimetry: A Novel Methodology that Probes the Fundamental Features of Protein Kinetic Stability

We introduce proteolytic scanning calorimetry, a modification of the differential scanning calorimetry approach to the determination of protein stability in which a proteolytic enzyme (thermolysin) is used to mimic a harsh environment. This methodology allows the straightforward calculation of the rate of irreversible denaturation as a function of temperature and concentration of proteolytic enzyme and, as a result, has the potential to probe efficiently the fundamental biophysical features of protein kinetic stability. In the particular case of Escherichia coli thioredoxin (used as an illustrative example in this article), we find that the rate of irreversible denaturation is determined by 1), the global unfolding mechanism at low thermolysin concentrations, indicating that thermodynamic stability may contribute directly to the kinetic stability of thioredoxin under moderately harsh conditions and 2), the rate of unfolding at high thermolysin concentrations, indicating that the free-energy barrier for unfolding may act as a safety mechanism that ensures significant kinetic stability, even in very harsh environments. This thioredoxin picture, however, is by no means expected to be general and different proteins may show different patterns of kinetic stabilization. Proteolytic scanning calorimetry is particularly well-suited to probe this diversity at a fundamental biophysical level.     

High-resolution differential scanning calorimetric study of myosin, functional domains, and supramolecular structures

High-resolution differential scanning calorimetry (DSC) has been employed to study the thermal stability of myosin, its major constitutive fragments (S-1, light chains, and rod), and also reconstituted thick filaments. The thermal denaturation of soluble myosin was complex and was characterized by a multistep endothermic process for the temperature range from 41 to 60 degrees C. The shape of the endotherm was highly dependent on the pH and the ionic strength of the solution, although the delta Hcal (calorimetric enthalpy) of denaturation (1715 +/- 75 kcal/mol) was insensitive to these changes for the solvent conditions used in this study. This value also agrees, within experimental error, with the sum of the denaturation enthalpies obtained for isolated fragments (1724 +/- 79 kcal/mol). In identical conditions of ionic strength, pH, and heating rate, the computer-calculated differential endotherms of domains belonging to S-1 and light chains were superimposable with those of the isolated fragments. Their responses to changes in the solvent condition were also similar. We suggest that the observed functional independence of the major domains in myosin reflects also the independence of their structural stability. The thermal unfolding of the isolated rod was multiphasic and readily reversible (95%). It occurred between 41 and 60 degrees C, with an delta Hcal of 1058 +/- 59 kcal/mol. The melting of S-1 showed a single peak at 46.3 +/- 0.1 degrees C with an delta Hcal of 255 +/- 12 kcal/mol. Light chains melted at 51.0 +/- 0.2 degrees C with an delta Hcal of 85 +/- 15 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic Hysteresis in Collagen Folding

The triple helix of collagen shows a steep unfolding transition upon heating, whereas less steep and more gradual refolding is observed upon cooling. The shape of the hysteresis loop depends on the rate of temperature change as well as the peptide concentration. Experimental heating and cooling rates are usually much faster than rates of unfolding and refolding. In this work, collagen model peptides were used to study hysteresis quantitatively. Their unfolding and refolding profiles were recorded at different heating and cooling rates, and at different peptide concentrations. Data were fitted assuming kinetic mechanisms in which three chains combine to a helix with or without an intermediate that acts as a nucleus. A quantitative fit was achieved with the same kinetic model for the forward and backward reactions. Transitions of exogenously trimerized collagen models were also analyzed with a simplified kinetic mechanism. It follows that true equilibrium transitions can only be measured at high concentrations of polypeptide chains with slow scanning rates, for example, 0.1°C/h at 0.25 mM peptide concentration of (Gly-Pro-Pro)₁₀. (Gly-Pro-4(R)Hyp)₁₀ folds ∼2000 times faster than (Gly-Pro-Pro)₁₀. This was explained by a more stable nucleus, whereas the rate of propagation was almost equal. The analysis presented here can be used to derive kinetic and thermodynamic data for collagenous and other systems with kinetically controlled hysteresis.     

Kinetically robust monomeric protein from a hyperthermophile

Equilibrium and kinetic studies were carried out under denaturation conditions to clarify the energetic features of the high stability of a monomeric protein, ribonuclease HII, from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII). Guanidine hydrochloride (GdnHCl)-induced unfolding and refolding were measured with circular dichroism at 220 nm, and heat-induced denaturation was studied with differential scanning calorimetry. Both GdnHCl- and heat-induced denaturation are very reversible. It was difficult to obtain the equilibrated unfolding curve of Tk-RNase HII below 40 degrees C, because of the remarkably slow unfolding. The two-state unfolding and refolding reactions attained equilibrium at 50 degrees C after 2 weeks. The Gibbs energy change of GdnHCl-induced unfolding (DeltaG(H(2)O)) at 50 degrees C was 43.6 kJ mol(-1). The denaturation temperature in the DSC measurement shifted as a function of the scan rate; the denaturation temperature at a scan rate of 90 degrees C h(-1) was higher than at a scan rate of 5 degrees C h(-1). The unfolding and refolding kinetics of Tk-RNase HII were approximated as a first-order reaction. The ln k(u) and ln k(r) values depended linearly on the denaturant concentration between 10 and 50 degrees C. The DeltaG(H(2)O) value obtained from the rate constant in water using the two-state model at 50 degrees C, 44.5 kJ mol(-1), was coincident with that from the equilibrium study, 43.6 kJ mol(-1), suggesting the two-state folding of Tk-RNase HII. The values for the rate constant in water of the unfolding for Tk-RNase HII were much smaller than those of E. coli RNase HI and Thermus thermophilus RNase HI, which has a denaturation temperature similar to that of Tk-RNase HII. In contrast, little difference was observed in the refolding rates among these proteins. These results indicate that the stabilization mechanism of monomeric protein from a hyperthermophile, Tk-RNase HII, with reversible two-state folding is characterized by remarkably slow unfolding.     

A calorimetric study of the influence of calcium on the stability of bovine alpha-lactalbumin

Bovine alpha-lactalbumin has been studied by differential scanning calorimetry with various concentrations of calcium to elucidate the effect of this ligand on its thermal properties. In the presence of excess calcium, alpha-lactalbumin unfolds upon heating with a single heat-absorption peak and a significant increase of heat capacity. Analysis of the observed heat effect shows that this temperature-induced process closely approximates a two-state transition. The transition temperature increases in proportion with the logarithm of the calcium concentration, which results in an increase in the transition enthalpy as expected from the observed heat capacity increment of denaturation. As the total concentration of free calcium in solution is decreased below that of the proteins, there are two temperature-induced heat absorption peaks whose relative area depends on the calcium concentration, such that further decrease of calcium concentration results in a increase of the low-temperature peak and a decrease of the high-temperature one. The high-temperature peak occurs at the same temperature as the unfolding of the holo-protein, while the low-temperature peak is within the temperature range associated with the unfolding of the apo-protein. Statistical thermodynamic modeling of this process shows that the bimodal character of the thermal denaturation of bovine alpha-lactalbumin at non-saturated calcium concentrations is due to a high affinity of Ca2+ for alpha-lactalbumin and a low rate of calcium exchange between the holo- and apo-forms of this protein. Using calorimetric data, the calcium-binding constant for alpha-lactalbumin has been determined to be 2.9 x 10(8) M-1.     

Differential scanning calorimetry of bovine rhodopsin in rod-outer-segment disk membranes.

Rhodopsin-containing retinal rod disk membranes from cattle have been examined by differential scanning calorimetry. Under conditions of 67 mM phosphate pH 7.0, unbleached rod outer segment disk membranes gave a single major endotherm with a temperature of denaturation (Tm) of 71.9 +/- 0.4 degrees C and a thermal unfolding calorimetric enthalpy change (delta Hcal) of 700 +/- 17 kJ/mol rhodopsin. Bleached rod outer segment disk membranes (membranes that had lost their absorbance at 498 nm after exposure to orange light) gave a single major endotherm with a Tm of 55.9 +/- 0.3 degrees C and a delta Hcal of 520 +/- 17 kJ/mol opsin. Neither bleached nor unbleached rod outer segment disk membranes gave endotherms upon thermal rescans. When thermal stability is examined over the pH range of 4-9, the major endotherms of both bleached and unbleached rod outer segment disk membranes were found to show maximum stability at pH 6.1. The observed delta Hcal values for bleached and unbleached rod outer segment disk membranes exhibit membrane concentration dependences which plateau at protein concentrations beyond 1.5 mg/mL. For partially bleached samples of rod outer segment disk membranes, the calorimetric enthalpy change for opsin appears to be somewhat dependent on the degree of bleaching, indicating intramembrane nearest neighbor interactions which affect the unfolding of opsin. Delta Hcal and Tm are particularly useful for assessing stability and testing for completeness of regeneration of rhodopsin from opsin. Other factors such as sample preparation and the presence of low concentrations of ethanol also affect the delta Hcal values while the Tm values remain fairly constant. This shows that the delta Hcal is a sensitive parameter for monitoring environmental changes of rhodopsin and opsin.     

Equilibrium heat-induced denaturation of chitinase 40 from Streptomyces thermoviolaceus

High-precision differential scanning calorimetry (DSC) and circular dichroism (CD) have been employed to study the thermal unfolding of chitinase 40 (Chi40) from Streptomyces thermoviolaceus. Chi40 belongs to family 18 of glycosyl hydrolase superfamily bearing a catalytic domain with a "TIM barrel"-like fold, which exhibits deviations from the (beta/alpha)8 fold. The thermal unfolding is reversible at pH = 8.0 and 9.0. The denatured state is characterized by extensive structural changes with respect to the native. The process is characterized by slow relaxation kinetics. Even slower refolding rates are recorded upon cooling. It is shown that the denaturation calorimetric data obtained at slow heating rate (0.17 K/min) are in excellent agreement with equilibrium data obtained by extrapolation of the experimental results to zero scanning rate. Analysis of the DSC results reveals that the experimental data can be successfully fitted using either a non-two-state sequential model involving one equilibrium intermediate, or an independent transitions model involving the unfolding of two Chi40 energetic domains to intermediate states. The stability of the native state with respect to the final denatured state is estimated, deltaG = 24.0 kcal/mol at 25 degrees C. The thermal results are in agreement with previous findings from chemical denaturation studies of a wide variety of (beta/alpha)8 barrel proteins, that their unfolding is a non-two-state process, always involving at least one unfolding intermediate.