The influence of formamide on thermal denaturation profiles of DNA and metaphase chromosomes in suspension

Systematic photometric studies are presented to analyze the thermal denaturation behaviour with and without formamide of metaphase chromosome suspensions in comparison to DNA solutions. Temperature dependent hyperchromicity measurements at 256 nm and 313 nm were performed using an appropriately designed computer-controlled photometer device. Due to an upright optical axis, this allowed absorbance measurements with negligible sedimentation effects not only for solutions of pure DNA, but also for particle suspensions of isolated metaphase chromosomes. This device has a temperature resolution of +/- 0.5 degrees C and an optical sensitivity of 10(-3) to 10(-4) optical density. For calf thymus DNA the reduction of the melting point with the increase of formamide in the solution was measured at pH 7.0 and pH 3.2. The good correlation of the theoretical approximation to experimental data indicated the suitability of the apparatus to quantitatively describe DNA conformation changes induced by thermal denaturation. For metaphase chromosome preparations of Chinese hamster culture cells, absorbance changes were measured between 20 degrees C and 95 degrees C with a temperature gradient of 1 degrees C/min. These measurements were performed at pH 7.0 and at pH 3.2. The denaturation profiles (= first derivative of the absorbance curve) resulted in a highly variable peak pattern at 256 nm and 313 nm indicating complex conformation changes. A statistical evaluation of the temperature values of the peak maxima resulted in temperature ranges typical for chromosomal conformation changes during thermal treatment. Especially the range of highest temperature values was independent from pH modifications. For pH 3.2 the influence of formamide on the denaturation behaviour of metaphase chromosome preparations was analyzed. In contrast to pure DNA solutions, a reduction of the "melting point" (i.e. the maximum temperature at which a conformation change takes place) was not found. However, the denaturation behaviour depended on the duration of formamide treatment before the measurement.     

Thermal denaturation of nucleosomal core particles.

Thermal denaturation of very homogeneous preparations of core particles from chicken erythrocyte chromatin is studied by several techniques. The change in absorbance, which is very closely paralleled by changes in heat capacity, which is very closely paralleled by changes in heat capacity, is a biphasic process with inflexions at 60 degrees C and 74 degrees C. In contrast, isolated DNA of the same length denatures in a single transition around 44 degrees C. Monitoring the circular dichroism of the cores during thermal denaturation reveals biphasic changes in the secondary structure of the DNA, preceding the base unstacking by 10 degrees C in the first and 3 degrees C in the second phase. However, measurable alterations in the secondary structure of the histones are confined to the second phase with a melting temperature at 71 degrees C. Increase in the ionic strength of the buffer from 1 mM to 10 mM leads to almost monophasic melting curves as measured by absorbance and CD, while not causing any measurable conformational changes at room temperature. The melting of core particles is interpreted as a denaturation of about 40 base pairs in the first phase, followed by a massive breakdown of the native structure of a tight histone-DNA complex, which frees the remaining 100 base pairs for unstacking.     

Studies on interaction between poly(L-lysine58, L-phenylalanine42) and deoxyribonucleic acids.

A random copolymer of 58% L-lysine and 42% L-phenylalanine, poly(Lys58Phe42), was used as a model protein for studying the role of phenylalanine residues in protein-DNA interaction. Complexes between this copolypeptide and DNA, made by direct mixing, were studied by absorbance, circular dichroism (CD), fluorescence, and thermal denaturation. Complex formation results in an increase in absorbance, and an enhancement, red-shift, and broadening of phenylalanine fluorescence. The fluorescence enhancement is opposite to the quenching observed when a tyrosine copolypeptide is bound to DNA (R. M. Santella and H.J. Li (1974), Biopolymers 13, 1909). The positive CD band of DNA near 275 nm is reduced and red-shifted by the binding of the phenylalanine copolypeptide to a greater extent than by the tyrosine copolypeptide. Thermal denaturation of the complexes in 2.5 times 10(-4) M EDTA (pH 8.0) shows three characteristic melting bands. For complexes with calf thymus DNA, free base pairs melt at Tm,I (47-49 degrees) and copolypeptide-bound base pairs show two melting bands (Tm,II at 73-75 degrees, and Tm,III at 88 -90 degrees). Similar thermal denaturation results have been observed for complexes with Micrococcus luteus DNA. The fluorecence intensity of the complexes is greatly increased when the temperature is raised to the Tm,II region. In addition to fluorescence measurements, the effects of increasing temperature on absorption and CD spectra of the complexes were also studied. Stacking interaction between the phenylalanine chromophore and DNA bases, either partial or full intercalation, is implicated by the experimental results. Several mechanisms are proposed to describe the reaction between the copolypeptide and DNA, and thermal denaturation of the complex.     

Thermal denaturation of engineered tet repressor proteins and their complexes with tet operator and tetracycline studied by temperature gradient gel electrophoresis

The effects of Trp to Phe exchanges in the Tet repressor on the thermal stability of the proteins and their complexes with operator DNA and inducer have been studied by temperature gradient polyacrylamide gel electrophoresis. The denaturation temperatures obtained by this method are compared with the results from temperature-dependent fluorescence and binding activities of the proteins. It is established that exchanging the interior Trp75 to Phe reduces the thermal stability of the Tet repressor by 8 degrees C while exchanging the exterior Trp43 to Phe has no effect on the stability of the protein. Binding of the inducer tetracycline increases the thermal stability of wild-type and Trp43 to Phe mutant Tet repressors by 5 degrees C, while the ones with the Trp75 to Phe mutation are stabilized by 10 degrees C. The stabilizing effect of operator binding is 20 degrees C in the Trp75 to Phe mutant and only 9 degrees C in the ones with the Trp43 to Phe exchange. In addition to the denaturation temperatures, the gel mobility shifts observed in temperature gradient gel electrophoresis reveal also information about the intermediates of the denaturation reaction. The free proteins and their complexes with the inducer tetracycline exhibit monophasic transitions upon denaturation. The operator complexes of wild-type and Trp75 to Phe mutant repressors denature in more complex reactions. At low temperature they exhibit a stoichiometry of two repressor dimers per tandem tet operator DNA. Upon elevating the temperature they form complexes with only one repressor dimer per DNA fragment. When the temperature is further increased the double-stranded DNA begins to melt from one end resulting in a complex with partially single-stranded DNA which exists only in a narrow temperature range. Finally, the denatured protein and single-stranded DNA are formed at high temperature. The associated mobility shifts are analyzed by changing the ionic strength and characterizing multiphasic melting of a pure DNA fragment by temperature gradient gel electrophoresis.     

Unfolding and inactivation during thermal denaturation of an enzyme that exhibits phytase and acid phosphatase activities

The thermostability of an enzyme that exhibits phytase and acid phosphatase activities was studied. Kinetics of inactivation and unfolding during thermal denaturation of the enzyme were compared. The loss of phytase activity on thermal denaturation is most suggestive of a reversible process. As for acid phosphatase activities, an interesting phenomenon was observed; there are two phases in thermal inactivation: when the temperature was between 45 and 50 degrees C, the thermal inactivation could be characterized as an irreversible inactivation which had some residual activity and when the temperature was above 55 degrees C, the thermal inactivation could be characterized as an irreversible process which had no residual activity. The microscopic rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method [Adv. Enzymol. Relat. Areas Mol. Biol. 61 (1988) 381]. Fluorescence analyses indicate that when the enzyme was treated at temperatures below 60 degrees C for 60 min, the conformation of the enzyme had no detectable change; when the temperatures were above 60 degrees C, some fluorescence red-shift could be observed with a decrease in emission intensity. The inactivation rates (k(+0)) of free enzymes were faster than those of conformational changes during thermal denaturation at the same temperature. The rapid inactivation and slow conformational changes of phytase during thermal denaturation suggest that inactivation occurs before significant conformational changes of the enzyme, and the active site of this enzyme is situated in a relatively fragile region which makes the active site more flexible than the molecule as a whole.     

Rapid cycle DNA amplification: time and temperature optimization

Rapid temperature cycling with hot air allows rigorous optimization of the times and temperatures required for each stage of the polymerase chain reaction. A thermal cycler based on recirculating hot air was used for rapid temperature control of 10-microliters samples in thin glass capillary tubes with the sample temperature monitored by a miniature thermocouple probe. The temperatures and times of denaturation, annealing and elongation were individually optimized for the amplification of a 536-base pair beta-globin fragment from human genomic DNA. Optimal denaturation at 92 degrees-94 degrees C occurred in less than one second; yield decreased with denaturation times greater than 30 seconds. Annealing for one second or less at 54 degrees-56 degrees C gave the best product specificity and yield. Non-specific amplification was minimized with a rapid denaturation to annealing temperature transition (9 seconds) as compared to a longer transition (25 seconds). An elongation temperature of 75 degrees-79 degrees C gave the greatest yield and increased yields were obtained with longer elongation times. Product specificity was improved with rapid air cycling when compared to slower conventional heat block cycling. Rapid thermal control of the temperature-dependent reactions in DNA amplification can improve product specificity significantly while decreasing the required amplification time by an order of magnitude.     

Thermal analysis of CHL V79 cells using differential scanning calorimetry: implications for hyperthermic cell killing and the heat shock response

Differential scanning calorimetry (DSC) was used to assay thermal transitions that might be responsible for cell death and other responses to hyperthermia or heat shock, such as induction of heat shock proteins (HSP), in whole Chinese hamster lung V79 cells. Seven distinct peaks, six of which are irreversible, with transition temperatures from 49.5 degrees C to 98.9 degrees C are detectable. These primarily represent protein denaturation with minor contributions from DNA and RNA melting. The onset temperature of denaturation, 38.7 degrees C, is shifted to higher temperatures by prior heat shock at 43 degrees and 45 degrees C, indicative of irreversible denaturation occurring at these temperatures. Thus, using DSC it is possible to demonstrate significant denaturation in a mammalian cell line at temperatures and times of exposure sufficient to induce hyperthermic damage and HSP synthesis. A model was developed based on the assumption that the rate limiting step of hyperthermic cell killing is the denaturation of a critical target. A transition temperature of 46.3 degrees C is predicted for the critical target in V79 cells. No distinct transition is detectable by DSC at this temperature, implying that the critical target comprises a small fraction of total denaturable material. The short chain alcohols methanol, ethanol, isopropanol, and t-butanol are known hyperthermic sensitizers and ethanol is an inducer of HSP synthesis. These compounds non-specifically lower the denaturation temperature of cellular protein. Glycerol, a hyperthermic protector, non-specifically raises the denaturation temperature for proteins denaturing below 60 degrees C. Thus, there is a correlation between the effect of these compounds on protein denaturation in vivo and their effect on cellular sensitivity to hyperthermia.     

Cold shock and its effect on ribosomes and thermal tolerance in Listeria monocytogenes

Differential scanning calorimetry (DSC) and fatty acid analysis were used to determine how cold shocking reduces the thermal stability of Listeria monocytogenes. Additionally, antibiotics that can elicit production of cold or heat shock proteins were used to determine the effect of translation blockage on ribosome thermal stability. Fatty acid profiles showed no significant variations as a result of cold shock, indicating that changes in membrane fatty acids were not responsible for the cold shock-induced reduction in thermal tolerance. Following a 3-h cold shock from 37 to 0 degrees C, the maximum denaturation temperature of the 50S ribosomal subunit and 70S ribosomal particle peak was reduced from 73.4 +/- 0.1 degrees C (mean +/- standard deviation) to 72.1 +/- 0.5 degrees C (P < or = 0.05), indicating that cold shock induced instability in the associated ribosome structure. The maximum denaturation temperature of the 30S ribosomal subunit peak did not show a significant shift in temperature (from 67.5 +/- 0.4 degrees C to 66.8 +/- 0.5 degrees C) as a result of cold shock, suggesting that either 50S subunit or 70S particle sensitivity was responsible for the intact ribosome fragility. Antibiotics that elicited changes in maximum denaturation temperature in ribosomal components also elicited reductions in thermotolerance. Together, these data suggest that ribosomal changes resulting from cold shock may be responsible for the decrease in D value observed when L. monocytogenes is cold shocked.     

Thermal stability and DNA binding activity of a variant form of the Sso7d protein from the archeon Sulfolobus solfataricus truncated at leucine 54

Sso7d is a 62-residue, basic protein from the hyperthermophilic archaeon Sulfolobus solfataricus. Around neutral pH, it exhibits a denaturation temperature close to 100 degrees C and a non-sequence-specific DNA binding activity. Here, we report the characterization by circular dichroism and fluorescence measurements of a variant form of Sso7d truncated at leucine 54 (L54Delta). It is shown that L54Delta has a folded conformation at neutral pH and that its thermal unfolding is a reversible process, represented well by the two-state N <=> D transition model, with a denaturation temperature of 53 degrees C. Fluorescence titration experiments indicate that L54Delta binds tightly to calf thymus DNA, even though the binding parameters are smaller than those of the wild-type protein. Therefore, the truncation of eight residues at the C-terminus of Sso7d markedly affects the thermal stability of the protein, which nevertheless retains a folded structure and DNA binding activity.     

Thermal instability in the endoplasmic reticulum of the rat hepatocyte

The rates of heat denaturation of a protein component of endoplasmic reticulum, NADPH cytochrome c reductase, measured in the temperature range of 37-47 degrees C, show that this protein is highly unstable in the range of temperatures used in clinical hyperthermia. The rate of enzyme inactivation increases some 20-fold as the temperature increases from 37 degrees C to 45 degrees C. The enzyme has an in vitro half-life of only 7 h due to thermal inactivation at 37 degrees C. This suggests a general scheme for the physiological degradation pathway of intracellular proteins in which the first step is rapid thermal denaturation of the molecule followed by a second and slower step of proteolytic degradation. The rapid physiological turnover of intracellular proteins may be an unavoidable cost of maintaining metabolic precision in the presence of thermal noise at normal body temperature.     

Oligonucleotide--minor groove binder 1:2 conjugates: side by side parallel minor groove binder motif in stabilization of DNA duplex

Synthetic polycarboxamides consisting of N-methylpyrrole (Py), N-methylimidazole (Im), N-methyl-3-hydroxypyrrole (Hp) and beta-alanine (beta) show strong and sequence-specific interaction with the DNA minor groove when they form hairpin structures with side-by-side antiparallel motifs. In the present paper, new conjugates containing two ligands linked to the same terminal phosphate of DNA strand were constructed. The paper describes optimized synthesis and properties of oligonucleotide-linked polyamide strands that insert into the minor groove of a duplex in a parallel or antiparallel orientation. Strong stabilization of DNA duplexes by two attached minor groove ligands is demonstrated by the thermal denaturation method. The unmodified duplex 5'-CGTTTATTp-3'/5'-AATAAACG-3' melts at 20 degrees C. When one tetra(Py) residue was attached to the first strand of this duplex, denaturation temperature was increased to 46 degrees C; attachment of the second tetra(Py) in a parallel orientation resulted in denaturation temperature of 60 degrees C. It is even higher than in case of "classic" octapyrrole hairpin ligand (Tm = 58 degrees C). Sequence-specific character of stabilization by two conjugated ligands was demonstrated for G:C-containing oligonucleotides attached to tetracarboxamide and octacarboxamide ligands constructed from Py, Im and beta units according to established recognition rules (deltaTm = 20 degrees C). The two-strand parallel minor groove binder constructions attached to addressing oligonucleotides could be considered as site-specific ligands recognizing single- and double-stranded DNA similarly to already described hairpin MGB structures with antiparallel orientation of carboxamide units.     

Freezing denaturation of ovalbumin at acid pH

The effects of rapid freezing and thawing at acid pH on the physiochemical properties of ovalbumin were examined. At low pH (around 2), UV difference spectra showed microenvironmental changes around the aromatic amino acid residues; elution curves by gel permeation chromatography showed decreasing numbers of monomers after neutralization. These changes depended on the incubation temperature (between -196 and -10 degrees C) and the protein concentration (0.5-10 mg/ml), and a low concentration of ovalbumin incubated at around -40 degrees C suffered the most damage to its conformation. With freezing and then incubation at -40 degrees C, three of the four sulfhydryl groups in the ovalbumin molecule reacted with 2,2'-dithiodipyridine. The CD spectra showed these changes in the secondary structure, but they were smaller than those when guanidine hydrochloride was used for denaturation. Supercooling at -15 degrees C or freezing at -196 degrees C had little or no effect on the conformation of the ovalbumin molecule. Thus, irreversible conformational changes of ovalbumin were caused under the critical freezing condition at an acid pH. These changes arose from partial denaturation and resembled those with thermal denaturation of ovalbumin at neutral pH.     

Glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus. Thermal denaturation and activation.

Pyrococcus furiosus is a marine hyperthermophile that grows optimally at 100 degrees C. Glutamate dehydrogenase (GDH) from P. furiosus is a hexamer of identical subunits and has an M(r) = 270,000 +/- 5500 at 25 degrees C. Electron micrographs showed that the subunit arrangement is similar to that of GDH from bovine liver (i.e. 3/2 symmetry in the form of a triangular antiprism). However, GDH from P. furiosus is inactive at temperatures below 40 degrees C and undergoes heat activation above 40 degrees C. Both NAD+ and NADP+ are utilized as cofactors. Apparently the inactive enzyme also binds cofactors, since the enzyme maintains the ability to bind to an affinity column (Cibacron blue F3GA) and is specifically eluted with NADP+. Conformational changes that accompany activation and thermal denaturation were detected by precision differential scanning microcalorimetry. Thermal denaturation starts at 110 degrees C and is completed at 118 degrees C. delta(cal) = 414 Kcal [mol GDH]-1. Tm = 113 degrees C. This increase in heat capacity indicates an extensive irreversible unfolding of the secondary structure as evidenced also by a sharp increase in absorbance at 280 nm and inactivation of the enzyme. The process of heat activation of GDH from 40 to 80 degrees C is accompanied by a much smaller increase in absorbance at 280 nm and a reversible increase in heat capacity with delta(cal) = 187 Kcal [mol GDH]-1 and Tm = 57 degrees C. This absorbance change as well as the moderate increase in heat capacity suggest that thermal activation leads to some exposure of hydrophobic groups to solvent water as the GDH structure is opened slightly. The increase in absorbance at 280 nm during activation is only 12% of that for denaturation. Overall, GDH appears to be well adapted to correspond with the growth response of P. furiosus to temperature.     

Thermal denaturation of Micrococcus lysodeikticus adenosine triphosphatase. Influence of temperature on the circular dichroism, fluroescence and enzymic activity of the protein.

The soluble ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus underwent a major unfolding transition when solutions of the enzyme at pH 7.5 were heated. The midpoint occurred at 46 degrees C when monitored by changes in enzymic activity and intrinsic fluorescence, and at 49 degrees C when monitored by circular dichroism. The products of thermal denaturation retained much secondary structure, and no evidence of subunit dissociation was detected after cooling at 20 degrees C. The thermal transition was irreversible, and thiol groups were not involved in the irreversibility. The presence of ATP, adenylyl imidodiphosphate, CaCl2 or higher concentrations of ATPase conferred stability against thermal denaturation, but did not prevent the irreversibility one denaturation had taken place. In the presence of guanidinium chloride, thermal denaturation occurred at lower temperatures. The midpoints of the transition were 45 degrees C in 0.25 M-, 38 degrees C in 0.5 M-and 30 degrees C in 0.75 M-denaturant. In the highest concentration of guanidinium chloride a similar unfolding transition induced by cooling was observed. Its midpoint was 9 degrees C, and the temperature of maximum stability of the protein was 20 degrees C. The discontinuities occurring the the Arrhenius plots of the activity of this enzyme had no counterpart in variations in the far-u.v. circular dichroism or intrinsic fluorescence of the protein at the same temperature.     

Thermodynamics of thermal and athermal denaturation of gamma-crystallins: changes in conformational stability upon glutathione reaction

The conformational stabilities of bovine lens gamma-crystallin fractions II, IIIA, IIIB, and IVA and those modified with glutathione were compared by studying the thermal and guanidine hydrochloride (Gdn-HCl) denaturation behavior. The conformational state was monitored by both far-UV CD and fluorescence measurements. All the gamma-crystallins studied showed a sigmoidal order-disorder transition with varied melting temperatures. The thermal denaturation of these proteins is reversible up to a temperature 3 or 4 degrees C above T 1/2; above this temperature, irreversible aggregation occurs. The validity of a two-state approximation of both thermal and Gdn-HCl denaturation was tested for all four crystallins, and the presence of one or more intermediates was evident in the unfolding of IVA. delta GDH2O values of these crystallins range from 4 to 9 kcal/mol. Upon glutathione treatment IVA showed the maximum decrease in T 1/2 by approximately 9 degrees C and in delta GDH2O value by 29%; the smallest decrease in T 1/2 was for IIIA by 2 degrees C and in delta GDH2O by 15%. We have demonstrated that the glutathione reaction can dramatically reduce the conformational stability of gamma-crystallins and, thus, that the thermodynamic quantities of the unreacted crystallins can be used to evaluate the stability of these proteins when modified during cataract formation.     

The catabolite activator protein stabilizes its binding site in the E. coli lactose promoter.

The effect of catabolite activator protein, CAP, on the thermal stability of DNA was examined. Site specific binding was studied with a 62 bp DNA restriction fragment containing the primary CAP site of the E. coli lactose (lac) promoter. A 144 bp DNA containing the lac promoter region and a 234 bp DNA from the pBR322 plasmid provided other DNA sites. Thermal denaturation of protein-DNA complexes was carried out in a low ionic strength solvent with 40% dimethyl sulfoxide, DMSO. In this solvent free DNA denatured below the denaturation temperature of CAP. The temperature stability of CAP for site specific binding was monitored using an acrylamide gel electrophoresis assay. Results show that both specific and non-specific CAP binding stabilize duplex DNA. Site specific binding to the 62 bp DNA produced a 13.3 degrees C increase in the transition under conditions where non-specific binding stabilized this DNA by 2-3 degrees C.     

Irreversible Denaturation of Maltodextrin Glucosidase Studied by Differential Scanning Calorimetry,Circular Dichroism,and Turbidity Measurements

Thermal denaturation of Escherichia coli maltodextrin glucosidase was studied by differential scanning calorimetry, circular dichroism (230 nm), and UV-absorption measurements (340 nm), which were respectively used to monitor heat absorption, conformational unfolding, and the production of solution turbidity. The denaturation was irreversible, and the thermal transition recorded at scan rates of 0.5–1.5 K/min was significantly scan-rate dependent, indicating that the thermal denaturation was kinetically controlled. The absence of a protein-concentration effect on the thermal transition indicated that the denaturation was rate-limited by a mono-molecular process. From the analysis of the calorimetric thermograms, a one-step irreversible model well represented the thermal denaturation of the protein. The calorimetrically observed thermal transitions showed excellent coincidence with the turbidity transitions monitored by UV-absorption as well as with the unfolding transitions monitored by circular dichroism. The thermal denaturation of the protein was thus rate-limited by conformational unfolding, which was followed by a rapid irreversible formation of aggregates that produced the solution turbidity. It is thus important to note that the absence of the protein-concentration effect on the irreversible thermal denaturation does not necessarily means the absence of protein aggregation itself. The turbidity measurements together with differential scanning calorimetry in the irreversible thermal denaturation of the protein provided a very effective approach for understanding the mechanisms of the irreversible denaturation. The Arrhenius-equation parameters obtained from analysis of the thermal denaturation were compared with those of other proteins that have been reported to show the one-step irreversible thermal denaturation. Maltodextrin glucosidase had sufficiently high kinetic stability with a half-life of 68 days at a physiological temperature (37°C).     

High-resolution thermal denaturation of DNA. I. Theoretical and practical considerations for the resolution of thermal subtransitions.

The fidelity achieved in first derivative profiles of DNA thermal denaturation is shown to depend on a number of factors including the thermal increment of data gathering, the precision of absorbance readings, and the manner in which data are smoothed prior to calculating the derivative of hyperchromicity. The closeness with which thermal denaturation data can be fitted by a cubic polynomial is carefully considered, and a derivation is presented for the estimated error in calculated values of the derivative of hyperchromicity with respect to temperature. After reviewing both theoretical and experimental evidence for the expected minimum width of a thermal transition in DNA, we conclude that thermal increments of 0.05°C or less are required for an adequate representation of transitions in naturally occurring DNA's. Data gathered under conditions meeting the requirements suggested here for quantitative recording of thermal denaturation profiles (Vizard and Ansevin, submitted for publication) show that virtually all of the high-resolution thermal denaturation profile of a simple, naturally occuring DNA may consist of small subtransitions, which we call thermalites. The finding of substransitions is consistent with current theories of DNA melting. A particularly well-resolved thermalite of λ bacteriophage DNA has a breadth of only 0.30°C (2σ width), and thus is narrower than previously reported thermal transitions for DNA. For this thermalite, the combination of width, shape, and position in the profile suggests that the substransitions observed in accurately recorded DNA thermal denaturation profiles are not described satisfactorily by existing theories. Knowledge of the requirements for the quantitative recording of thermal denaturation profiles should greatly favor the usefulness of denaturation experiments for physical genomic analysis.     

Temperature-induced denaturation and renaturation of triosephosphate isomerase from Saccharomyces cerevisiae: evidence of dimerization coupled to refolding of the thermally unfolded protein

The thermal denaturation of the dimeric enzyme triosephosphate isomerase (TIM) from Saccharomyces cerevisiae was studied by spectroscopic and calorimetric methods. At low protein concentration the structural transition proved to be reversible in thermal scannings conducted at a rate greater than 1.0 degrees C min(-1). Under these conditions, however, the denaturation-renaturation cycle exhibited marked hysteresis. The use of lower scanning rates lead to pronounced irreversibility. Kinetic studies indicated that denaturation of the enzyme likely consists of an initial first-order reaction that forms thermally unfolded (U) TIM, followed by irreversibility-inducing reactions which are probably linked to aggregation of the unfolded protein. As judged from CD measurements, U possesses residual secondary structure but lacks most of the tertiary interactions present in native TIM. Furthermore, the large increment in heat capacity upon denaturation suggests that extensive exposure of surface area occurs when U is formed. Above 63 degrees C, reactions leading to irreversibility were much slower than the unfolding process; as a result, U was sufficiently long-lived as to allow an investigation of its refolding kinetics. We found that U transforms into nativelike TIM through a second-order reaction in which association is coupled to the regain of secondary structure. The rate constants for unfolding and refolding of TIM displayed temperature dependences resembling those reported for monomeric proteins but with considerably larger activation enthalpies. Such large temperature dependences seem to be determinant for the occurrence of kinetically controlled transitions and thus constitute a simple explanation for the hysteresis observed in thermal scannings.