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PubMed2002

Comparison of the stabilities and unfolding pathways of human apolipoprotein E isoforms by differential scanning calorimetry and circular dichroism.

Differential scanning calorimetry and circular dichroism experiments were performed to study structural differences among the common isoforms of human apolipoprotein E (apoE2, apoE3, and apoE4) and their N-terminal, 22-kDa fragments. Here, we examine thermodynamic properties that characterize the structural differences among isoforms, and also differences in their unfolding behavior. The 22-kDa fragments and their full-length counterparts were found to exhibit similar differences in thermal stability (apoE4<apoE3<apoE2; T(max) approximately 52<56<59 degrees C, respectively). Helical contents at the onset and completion of denaturation (as monitored calorimetrically) were about 51% and 35%, respectively, for 22-kDa apoE3. As much as 70% of the unfolding enthalpy for 22-kDa apoE3 could be due to disruption of alpha-helix. The monomeric 22-kDa fragments unfold differently, with both apoE3 and apoE4 exhibiting unfolding intermediates, whereas apoE2 unfolds according to a two-state mechanism. Interactions with the C-terminal domain appear to destabilize the N-terminal domains in full-length apoE2 and apoE3, but less so for full-length apoE4. Self-association of full-length apoE at higher concentrations (1.5-2.5 mg/ml), as well as interaction of full-length apoE with phospholipid, enhances thermal stability. Cooperative changes in secondary structure, as monitored by circular dichroism, begin near 37 degrees C for full-length apoE3 and apoE4, indicating that full-length apoE3 and apoE4, but not apoE2, may be partially unfolded in vivo. The differences in stability and unfolding behavior are likely to contribute to the molecular basis for defects in lipid transport and neurological function induced by apoE polymorphism.

Apolipoprotein E2↗
PubMedDate not supplied

Quantitative assays of the amount of diethylenetriaminepentaacetic acid conjugated to water-soluble polymers using isothermal titration calorimetry and colorimetry.

The level of conjugation of diethylenetriaminepentaacetic acid (DTPA) to the polysaccharide sodium hyaluronan (HA) has been measured by a colorimetric assay, isothermal titration calorimetry (ITC), and (1)H NMR spectroscopy. The colorimetric assay is based on the red shift, upon complexation with gadolinium ion (Gd3+), of the wavelength of maximum absorption of the dye arsenazo III. It can be performed in a few minutes using as little as 10 microg of polymer with a detection limit of approximately 0.03 mmol of DTPA (gram of polymer)-1. The ITC measurements yield values of the amount of DTPA linked to HA identical to those obtained by colorimetry. The levels of DTPA conjugation calculated by integration of signals at 3.1-3.2 ppm (DTPA protons) and at 2.0 ppm (HA acetamide protons) in the 1H NMR spectrum of HA-DTPA are consistently overestimated by a factor of approximately 2, compared to the data obtained by ITC and colorimetry. The longer relaxation times of protons of the polymer backbone, compared to those of protons attached to the freely moving DTPA side-chains may account for the discrepancy.

Calorimetry, Differential Scanning↗
PubMed2000

Binding of urate and caffeine to hemocyanin of the lobster Homarus vulgaris (E.) as studied by isothermal titration calorimetry.

Hemocyanin serves as an oxygen carrier in the hemolymph of the European lobster Homarus vulgaris. The oxygen binding behavior of the pigment is modulated by metabolic effectors such as lactate and urate. Urate and caffeine binding to 12-meric hemocyanin (H. vulgaris) was studied using isothermal titration calorimetry (ITC). Binding isotherms were determined for fully oxygenated hemocyanin between pH 7.55 and 8.15. No pH dependence of the binding parameters could be found for either effector. Since the magnitude of the Bohr effect depends on the urate concentration, the absence of any pH dependence of urate and caffeine binding to oxygenated hemocyanin suggests two conformations of the pigment under deoxygenated conditions. Urate binds to two identical binding sites (n = 2) each with a microscopic binding constant K of 8500 M(-1) and an enthalpy change DeltaH degrees of -32.3 kcal mol(-1). Caffeine binds cooperatively to hemocyanin with two microscopic binding constants: K(1) = 14 100 M(-1) and K(2) = 40 400 M(-1). The corresponding enthalpy changes in binding are as follows: DeltaH degrees (1) = -23.3 kcal mol(-1) and DeltaH degrees (2) = -27.1 kcal mol(-1). The comparison of urate and caffeine binding to the oxygenated pigment indicates the existence of two protein conformations for oxygen-saturated hemocyanin. Since effector binding is not influenced by protons, four different conformations are required to create a convincing explanation for caffeine and urate binding curves. This was predicted earlier on the basis of the analysis of oxygen binding to lobster hemocyanin, employing the nesting model.

Animals↗
PubMed1995

Comparison of Pf1 and Fd gene 5 proteins and their single-stranded DNA complexes by NMR spectroscopy and differential scanning calorimetry.

The Pf1 gene 5 protein forms a large helical nucleoprotein complex (Mr = 3.1 x 10(7)) with single-stranded viral DNA, from which a 32 amino acid sequence rich in alanine, proline, and glutamine residues can be removed from the C-terminus by limited proteolysis. Sharp resonances in the 1H NMR spectrum of the Pf1 nucleoprotein complex indicate that the C-terminal region of the protein subunits enjoys remarkable conformational flexibility in the complex. In contrast, the globular N-terminal domain of the protein subunits is rigidly held and does not contribute to the spectrum. The Fd gene 5 protein lacks this C-terminal flexible domain, and no distinct resonances can be observed in the 1H NMR spectrum when this protein is complexed to single-stranded viral DNA. Differential scanning calorimetry shows that the thermal stability of both the Pf1 and Fd gene 5 protein is increased by 8 degrees C in the complex with DNA, and the transition is highly cooperative. Removal of the C-terminal domain of the Pf1 gene 5 protein subunits has no appreciable effect either on the Tm of the DNA-protein complex or on the cooperative nature of the thermal transition. It is suggested that the C-terminal domain of the Pf1 gene 5 protein acts as a dynamic clamp which kinetically stabilizes the nucleoprotein complex.

Amino Acid Sequence↗
PubMed1995

Role of H1 in chromatin folding. A thermodynamic study of chromatin reconstitution by differential scanning calorimetry.

In a series of related papers, we have recently presented the results of a thermodynamic approach to the conformational transitions of bulk chromatin induced in vitro by different structure-perturbing agents, such as the intercalating dye ethidium bromide or the ionic strength. In all these studies, we took advantage of the capability of differential scanning calorimetry to detect the changes in the melting behavior of the structural domains of chromatin (the linker and the core particle) associated with the order-disorder transitions. This technique also revealed that the higher-order structure undergoes a catastrophic decondensation process in the course of the transformation of rat hepatocytes as well as of cultured cells. Therefore, several questions arose concerning the biological function (if any) of the changes in the degree of condensation of bulk chromatin, as well as the mechanism of transition and the nature of the modulating agents. In this paper, we report a thermodynamic analysis of the reconstitution of H1-depleted calf thymus chromatin with the purpose of establishing (1) the binding mode of H1 and (2) the energetics and cooperativity of the transition from the unfolded to the condensed state. When H1 is progressively extracted from calf thymus nuclei by high-salt treatment, the endotherm at 107 degrees C, characteristic of the core particles interacting within condensed domains, converts into the thermal transition at 90 degrees C, resulting from the denaturation of noninteracting core particles. Binding of H1 fully restores the thermal profile of native chromatin. Analysis of H1 association shows that binding occurs at independent sites with KA = (3.67 +/- 0.60) x 10(4) M-1 and each site comprising 180 +/- 10 bp. The experimental dependence of the fraction of condensed chromatin on R, the moles of bound H1 per nucleosome mole, was compared with a simple thermodynamic model for the conformational change. This analysis yields a value of -5 kcal per nucleosome mole for the interaction free energy of nucleosomes within the ordered state. The process of condensation, is not, however, a highly cooperative (all-or-none) one, as expected from a consideration of the solenoidal model for the 30 nm fiber. Rather, nucleation of the helical state involves the face-to-face interaction between consecutive core particles, and the growth is largely determined by the mergence and rearrangement of neighboring clusters of helically arrayed nucleosomes.

Animals↗
PubMed1995

Differences in the thermal stability of acclimation temperature-associated types of carp myosin and its rod on differential scanning calorimetry.

Differential scanning calorimetry (DSC) was employed for studying the thermal unfolding of myosin and its rod part prepared from carp acclimated to 10 and 30 degrees C. Differences in the thermal stability reflecting structural properties were clearly demonstrated by the DSC data obtained at pH 8.0 in 0.6 M KCl for the two types of carp myosin and rod. The transition temperatures on myosin and rod given by the major peaks for the 10 degrees C-acclimated carp were 33.9 and 47.4 degrees C and 33.0 and 44.0 degrees C, respectively, assuming two endotherms for this type. Since the shape of the first peaks at 33.9 and 33.0 degrees C was not symmetrical, two peaks having similar transition temperatures overlapped in this temperature range. When the data were analyzed using three endotherms, the three transition temperatures obtained for myosin and rod were 32.8, 34.9, and 47.4 degrees C and 32.9, 33.4, and 44.1 degrees C, respectively. Thus, the position of the first peak for the 10 degrees C-acclimated carp myosin did not change even after removal of the large subfragment-1 part, but the transition of the second peak shifted to a lower temperature by about 3 degrees C. The myosin and rod from carp acclimated to 30 degrees C showed three distinct peaks at 35.9, 39.7, and 49.1 degrees C and 34.5, 39.7, and 46.7 degrees C, respectively. The position of the largest peak for myosin remained unchanged, and the shift of the peak position of the highest temperature was about 3 degrees C, as obtained for the 10 degrees C-acclimated carp.(ABSTRACT TRUNCATED AT 250 WORDS)

Acclimatization↗
PubMed1995

A comparison of structure and thermal behavior in human plasma lipoprotein(a) and low-density lipoprotein. Calorimetry and small-angle X-ray scattering.

Differential scanning calorimetry (DSC) and small-angle X-ray scattering (SAXS) studies have been performed to investigate the structural properties of lipoprotein(a) [Lp(a)] and low-density lipoprotein (LDL) obtained from the same donor. In addition, a comparison was made between autologous LDL and the remnant particle Lp(a-) obtained by removal of apo(a) through chemical reduction. With Lp(a), three distinct thermal transitions have been observed: the first one around 20 degrees C, arising from the core-located apolar lipids, similar to LDL but with a significantly lower melting temperature as compared to LDL of the same donor. The second one, at 55.7 +/- 0.25 degrees C, can be attributed to apo(a), since it was found to be absent in Lp(a-) and LDL, whereas isolated apo(a) in aqueous solution exhibited a similar transition. The third transition, at 80.4 +/- 0.9 degrees C, corresponds to apo-B100 protein unfolding. The low melting temperature of the core lipids in Lp(a) is preserved in Lp(a-); this suggests that the apolar lipid interactions are unaffected by apo(a) binding, and that the difference in the core melting behavior between Lp(a) and LDL is due to a different stabilization through interaction between the apolar core and the surface monolayer lipids. SAXS curves exhibited qualitatively the same characteristic features for LDL, Lp(a), and Lp(a-). Thus, the SAXS results showed that no major deviations from spherical particle shape occur with Lp(a), indicating that apo(a) wraps around the particle surface without major globular protrusions into the aqueous surrounding.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Lung surfactant proteins, SP-B and SP-C, alter the thermodynamic properties of phospholipid membranes: a differential calorimetry study.

The ability of the low molecular weight lung surfactant-associated proteins, SP-B and SP-C, to alter the thermotropic properties of synthetic multilamellar vesicles was tested using differential scanning calorimetry (DSC). The presence of either SP-B or SP-C in dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) multilamellar vesicles broadened the DSC thermogram and reduced the enthalpy of transition in a concentration-dependent manner. With both proteins, the temperature at which the peak of the phase transition (Tm) was detected was shifted to a higher value. The increase in Tm caused by both proteins was greater with DPPG than DPPC. We have interpreted these results as implying the presence of a protein-perturbed domain of lipid. Both SP-B and SP-C were found to influence the surface activity of the phospholipids in a concentration-dependent fashion. We speculate that instability of lipid packing predicted to occur at protein-created lipid domain boundaries may be important for the expression of surface activity in pulmonary surfactant.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1993

Thermodynamics of binding of the CO2-competitive inhibitor imidazole and related compounds to human carbonic anhydrase I: an isothermal titration calorimetry approach to studying weak binding by displacement with strong inhibitors.

The visible spectrum of Co(II)-substituted human carbonic anhydrase I (HCA I) complexed with the unique CO2-competitive inhibitor imidazole undergoes a marked alkaline intensification, with a midpoint near pH 8 [Bauer, R., Limkilde, P., & Johansen, J. T. (1977) Carlsberg Res. Commun. 42, 325-339]. This change was first attributed to the ionization of a nondisplaced water ligand of the active-site metal in a five-coordinate complex. Later proposals favored assigning it to the deprotonation of the bound imidazole itself to give a tetrahedrally coordinated imidazolate anion at high pH. We have determined by isothermal titration calorimetry the pH dependence of the enthalpy of binding of imidazole and its analogues to HCA I and Co(II)HCA I. We devised an indirect strategy whereby the enthalpy of binding of the strong sulfonamide inhibitor methazolamide was determined in the absence and presence of a constant high concentration of the competing imidazole or its analogues. The standard enthalpy of binding of deprotonated methazolamide to the "acid" form of HCA I and Co(II)HCA I was found to be pH independent over the pH range of 6.5-9.5, as expected. It was also identical for both the zinc (-13.5 +/- 1.1 kcal M-1) and the cobalt (-13.7 +/- 0.4 kcal M-1) forms. The standard enthalpy of binding of neutral imidazole (average value -6.1 +/- 0.8 kcal M-1) surprisingly did not show any marked pH dependence, varying by about 1.1 and 2.6 kcal M-1 for the zinc and cobalt enzymes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗
PubMed1993

Differential scanning calorimetry of the irreversible denaturation of Escherichia coli glucosamine-6-phosphate deaminase.

The thermal denaturation of Escherichia coli glucosamine-6-phosphate deaminase (G6PD) at neutral pH was studied by means of differential scanning calorimetry (DSC). In the concentration range 0.6-7.3 mg mL-1, the denaturation of this hexameric enzyme was completely irreversible as judged by the absence of any endotherm on rescanning of previously scanned samples. However, the study of the effect of scanning rate on DSC curves indicated that the denaturation of G6PD is, most likely, a complex process which includes transitions in equilibrium as well as an irreversible step; in addition, it was found that application of the equilibrium formalism to the analysis of calorimetric data seems to be justified in this case, provided that scanning rates used are above 0.75 K min-1. The calorimetric and van't Hoff enthalpies for G6PD were 1260 +/- 118 and 160 +/- 27 kcal mol-1, respectively, indicating the presence of intermediates in the process. Accordingly, the DSC curves were adequately fitted to a model including six two-state sequential transitions. The observed protein-concentration dependence of the temperature at the maximum heat capacity, Tm, for each of the individual transitions suggests that G6PD dissociates to dimers in two consecutive steps. Using a model that includes dissociation explicitly, we calculated the thermodynamic parameters for each step. From this data, the enthalpy and free energy for the disruption of one dimer-dimer contact were roughly estimated, at pH 7.1 and 51 degrees C, as 57 and 2.1 kcal mol-1, respectively.

Aldose-Ketose Isomerases↗
PubMed1993

Conformational dynamics of cytochrome P-450cam as monitored by photoacoustic calorimetry.

Conformational transitions of cytochrome P-450cam following the dissociation of CO from the ferrous heme were investigated by using photoacoustic calorimetry. The effect of substrate association on the acoustic signal was also examined. Results show that the conformational dynamics of cytochrome P-450cam substrate-free protein occur faster than 10 ns, which is the time scale of the instrument response. The enthalpy and volume change for the dissociation reaction are 2.2 kcal mol-1 and 1.8 mL mol-1, respectively. Upon addition of camphor, the reaction is markedly slowed. An intermediate is formed whose lifetime is 130 ns at 17 degrees C. The overall enthalpy and volume changes are -15.9 kcal mol-1 and 10.3 mL mol-1, respectively. These results, together with published transient Raman spectra [Wells, A. V., Pusheng, L., Champion, P. M., Martinis, S. A., & Sligar, S. G. (1992) Biochemistry 31, 4384-4393] suggest that camphor leaves the heme pocket concomitant with the photoinduced expulsion of CO into the solvent and induces a considerable conformational change in the protein.

Calorimetry↗
PubMed1993

Characterization of hemopexin and its interaction with heme by differential scanning calorimetry and circular dichroism.

Hemopexin is a plasma glycoprotein that has two structural domains (I and II) and binds and transports heme particularly to liver cells. Differential scanning calorimetry (DSC) studies show that hemopexin is largely stabilized by heme, which binds exclusively to domain I. The melting temperature (Tm) of heme-hemopexin is 66.4 +/- 0.7 degrees C as compared with 53.9 +/- 0.3 degrees C for apohemopexin, and this Tm increase is accompanied by a 100 kcal increase in molar enthalpy. Heme stabilizes hemopexin by stabilizing domain I. This is demonstrated by the 26 degrees C increase in Tm from 51.9 +/- 0.3 to 77.6 +/- 0.6 degrees C and the over 3-fold increase in molar enthalpy when domain I associates with heme. A moderate change in domain I secondary structure is indicated by an increase in negative molar ellipticity at 206 nm. However, there is no net effect on the secondary structure of holo-hemopexin caused by heme binding as indicated by both far-UV circular dichroism (CD) and Fourier-transform infrared spectra. The characteristic positive ellipticity of hemopexin at 233 nm, ascribed to tryptophan residues in domain II, is dramatically increased, suggesting a change in teritary structure for domain II of hemopexin. DSC and CD results show that isolated domain I and domain II interact both in the presence and absence of heme. Moreover, domain II destabilizes heme-domain I, which may be an important factor in facilitating heme release to the hemopexin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2000

Binding of Nisin Z to bilayer vesicles as determined with isothermal titration calorimetry.

Nisin Z, a 34-residue lantibiotic, is secreted by some lactic acid bacteria and exerts its antibacterial activity against various Gram-positive bacteria by permeabilizing the cell membrane. It is a cationic amphiphilic peptide with several unusual dehydro residues and thioether-bridged lanthionines. Isothermal titration calorimetry was used to provide a quantitative thermodynamic description for nisin Z adsorption to and penetration into negatively charged and neutral lipid bilayers. The binding of the cationic peptide (electric charge z approximately 3.8) to anionic membranes was found to be dominated by electrostatic forces which could be described with the Gouy-Chapman theory. For biologically relevant conditions with a membrane surface potential of -40 mV, the peptide concentration near the membrane surface increases by about 2-3 orders of magnitude compared to the bulk concentration. The binding step proper, i.e., the transition from the lipid-water interface into the membrane, is almost exclusively driven by the high surface concentration. Binding can be described by a partition equilibrium of the form X(b) = KC(M) = KC(p,f) exp(-z(p)psi(0)F(0)/RT), where C(M) is the peptide surface concentration, C(p,f) the bulk concentration, and psi(0) the membrane surface potential. The intrinsic partition coefficient (K = 1.8 M(-)(1)) is remarkably small, indicating a correspondingly small hydrophobic energy contribution to the binding process. The electrostatic model was confirmed with nisin Z mutants in which valine-32 was replaced with either lysine (V32K) or glutamate (V32E), increasing or decreasing the electric charge by 1 unit. The extent of peptide binding increased for V32K and decreased for V32E as predicted by the electrostatic theory. In contrast, electrostatic effects were almost negligible for the binding of nisin Z to neutral membranes. However, the binding isotherms were characterized by a distinctly larger intrinsic binding constant K(0) of approximately 540 M(-)(1) and an enhanced hydrophobic free energy of binding. The binding of nisin Z to sonicated lipid vesicles is exothermic with a DeltaH degrees of ca. -9 and -3.4 kcal/mol for charged and neutral membranes, respectively.

Amino Acid Sequence↗
PubMed1993

Actin conformation is drastically altered by direct interaction with membrane lipids: a differential scanning calorimetry study.

One of the current dogmas in cytoskeleton research holds that actin filaments are attached to the cell membrane through integral membrane actin-binding proteins. We have challenged this concept, using an in vitro system composed of pure actin and liposomes, and have found that actin may also interact with membrane lipids. Differential scanning calorimetry (DSC) shows that when the actin molecule is in contact with such lipids, it undergoes a major conformational change which results in the complete disappearance of its phase transition. Conversely, DSC scans reveal that the phase transition of the membrane lipids is only weakly affected by the presence of actin. Indeed, the lipids' main transition shows only slight shifts in Tm, from 56.6 to 57 degrees C, and delta Hcal, from 10.1 to 8.8 kcal/mol. In the lipids' pretransition, Tp is shifted from 52.7 to 53.7 degrees C, and delta Hcal is shifted from 0.75 to 0.33 kcal/mol. This interaction between purified actin and membrane lipids is inhibited by high concentrations of KCl, thus indicating that the phenomenon is primarily electrostatic in nature. The ultrastructural consequences of this change in actin conformation were investigated by electron microscopy, which revealed the formation of paracrystalline arrays of actin filaments at the surface of the liposomes. We therefore propose a model in which a limited number of lipid molecules may interact with specific sites on the actin molecule, resulting in the protein's observed conformational change.

Actins↗
PubMed1991

Thermodynamics of condensation of nuclear chromatin. A differential scanning calorimetry study of the salt-dependent structural transitions.

We present a detailed thermodynamic investigation of the conformational transitions of chromatin in calf thymus nuclei. Differential scanning calorimetry was used as the leading method, in combination with infrared spectroscopy, electron microscopy, and techniques for the molecular characterization of chromatin components. The conformational transitions were induced by changes in the counterion concentration. In this way, it was possible to discriminate between the interactions responsible for the folding of the higher order structure and for the coiling of nucleosomal DNA. Our experiments confirm that the denaturation of nuclear chromatin at physiological ionic strength occurs at the level of discrete structural domains, the linker and the core particle, and we were able to rule out that the actual denaturation pattern might be determined by dissociation of the nucleohistone complex and successive migration of free histones toward native regions, as recently suggested. The sequence of the denaturation events is (1) the conformational change of the histone complement at 66 degrees C, (2) the unstacking of the linker DNA at 74 degrees C, and (3) the unstacking of the core particle DNA, that can be observed either at 90 or at 107 degrees C, depending on the degree of condensation of chromatin. Nuclear chromatin unfolds in low-salt buffers, and can be refolded by increasing the ionic strength, in accordance with the well-known behavior of short fragments. The process is athermal, therefore showing that the stability of the higher order structure depends on electrostatic interactions. The transition between the folded conformation and the unfolded one proceeds through an intermediate condensation state, revealed by an endotherm at 101 degrees C. The analysis of the thermodynamic parameters of denaturation of the polynucleosomal chain demonstrates that the wrapping of the DNA around the histone octamer involves a large energy change. The most striking observation concerns the linker segment, which melts a few degrees below the peak temperature of naked DNA. This finding is in line with previous thermal denaturation investigations on isolated chromatin at low ionic strength, and suggests that a progressive destabilization of the linker occurs in the course of the salt-induced coiling of DNA in the nucleosome.

Animals↗
PubMed1991

Differential scanning calorimetry study of reversible, partial unfolding transitions in dodecameric glutamine synthetase from Escherichia coli.

Partial unfolding of dodecameric glutamine synthetase (GS) from Escherichia coli has been studied by differential scanning calorimetry (DSC). A single endotherm (tm = 51.6 +/- 0.1 degrees C and delta Hcal = 211 +/- 4 kcal/mol of enzyme) was observed in DSC experiments with Mn.GS in the presence of 1.0 mM free Mn2+ and 100 mM KCl at pH 7. The dodecameric structure of Mn.GS was retained throughout heating cycles, and thermal transitions were reversible as shown by rescans [with 6-18 mg of GS (Mr 622,000) from 15 to 68 degrees C at 20-60 degrees C/h] and by greater than 93% recovery of activity. A cooperative ratio delta Hcal/delta HvH of 1.6 +/- 0.1 and deconvolution analysis show two cooperative units (two-state transitions): t1 = 50.4 and t2 = 51.7 degrees C; the ratio of the relative sizes of thermally labile domains is approximately 1:2 as judged by delta H2/delta H1 approximately equal to 2. However, the thermally induced overall enthalpy change (0.34 cal/g) for GS dodecamer is only 5-10% of that for thermal unfolding of small globular proteins at 50 degrees C. The t1 and t2 values from deconvolutions of DSC data agree with t0.5 values previously calculated from spectral measurements of temperature-induced exposures of approximately 0.7 of 2 Trp and approximately 2 of 17 Tyr per subunit, respectively [Shrake et al. (1989) Biochemistry 28, 6281-6294], over a 14 degrees C temperature range using both stabilizing and destabilizing conditions for Mn.GS.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗
PubMed1992

Effects of salt concentration and H1 histone removal on the differential scanning calorimetry of nuclei.

The effects of increasing NaCl concentrations on the melting profiles of chromatin in isolated nuclei contradicted published claims that structural transitions near 76 degrees C (Tn-7), near 89 degrees C (Tn-8), and near 105 degrees C (Tn-10) were respectively the melting of linker DNA, the melting of extended nucleosomal strands, and the collapse of nucleosomes in the 300-A fiber. Contrary to expectations of such an interpretation, decreases in salt concentration stabilized Tn-7 and failed to eliminate Tn-10. Moreover, nuclei depleted of H1 histone, which is known to be essential for the formation of the 300-A fiber, gave the same melting profile as intact nuclei with regard to the relative magnitudes of Tn-8 and Tn-10. The effect of salt concentration on the melting profiles and the insensitivity of Tn-8 and Tn-10 to H1 histone removal supports the notion that Tn-7 is the collapse of the nucleosome while Tn-8 and Tn-10 are respectively the unstacking of nucleotide bases in relaxed chromatin and supercoiled chromatin. The identification of Tn-8 as the unstacking of bases in relaxed DNA, and Tn-10 as unstacking in supercoiled DNA, shows that scanning calorimetry can be used to measure the state of repair of DNA in the nucleus. The gain in Tn-8 at the expense of Tn-10 that is seen as the mitotic index drops and differentiation occurs suggests that nicks accumulate in the DNA, perhaps because the gross aggregation of the inactive majority of the chromatin makes it inaccessible to repair enzymes.

Calorimetry, Differential Scanning↗
PubMed1992

Spin-label electron paramagnetic resonance and differential scanning calorimetry studies of the interaction between mitochondrial cytochrome c oxidase and adenosine triphosphate synthase complex.

The interaction between cytochrome c oxidase complex and adenosine triphosphate synthase (F1F0) complex in the purified, dispersed state and embedded in phospholipid vesicles was studied by differential scanning calorimetry and by spin-label electron paramagnetic resonance. The detergent-dispersed cytochrome oxidase and F1F0 complexes undergo endothermic thermodenaturation. However, when these complexes are embedded in phospholipid vesicles, they undergo exothermic thermodenaturation. The energy released is believed to result from the collapse of a strained interaction between unsaturated fatty acyl groups of phospholipids and an exposed area of the complex formed by the removal of interacting proteins. The exothermic enthalpy change of thermodenaturation of a protein-phospholipid exothermic enthalpy change of thermodenaturation of a protein-phospholipid vesicle containing both cytochrome oxidase complex and F1F0 was smaller than that of a mixture of protein-phospholipid vesicles formed from each individual electron transfer complex. This suggests specific interaction between cytochrome oxidase complex and F1F0 in the membrane. Further evidence for interaction between these two complexes is provided by saturation transfer EPR studies in which the rotational correlation time of spin-labeled cytochrome oxidase increases significantly when the complex is mixed with F1F0 prior to being embedded in phospholipid vesicles. From these results, it is concluded that at least a part of cytochrome oxidase and a part of F1F0 form a supermacromolecular complex in the inner mitochondrial membrane. No such supermacromolecular complex is detected between F1F0 and ubiquinol--cytochrome c reductase.

Animals↗