Search

We have recorded X-ray diffraction patterns at 3.1 A resolution from magnetically aligned fibres of the Pf3 strain of filamentous bacteriophage (Inovirus). The patterns are similar to patterns from the higher-temperature form of the Pf1 strain, indicating that the Pf3 and Pf1 virions have the same helix symmetry and similar protein subunit shape. This is of particular interest, given that the primary structures of the two protein subunits are quite different; and the nucleotide/protein subunit ratio in the Pf3 virion is more than twice that in Pf1, indicating important differences in DNA packaging. We have built a molecular model of the Pf3 protein capsid based on the model of Pf1, and refined it against the diffraction data using simulated annealing. The refinement confirms that the two structures are similar, which may reflect a fundamental motif of α-helix packing. However, there are some differences between the structures: the Pf3 subunit appears to be completely α-helical, beginning at the N terminus, whereas the first few residues of the Pf1 subunit are not helical; and the structure of the C-terminal region of the Pf3 subunit at the inner surface of the tubular capsid indicates that DNA/protein interactions in this virion may involve both aromatic side-chains and positively charged side-chains, whereas those in the Pf1 virion involve predominantly only the latter. In the course of this work, we have developed new approaches to refinement and validation of helical structures with respect to continuous transform fibre diffraction data.

Differential scanning calorimetry is employed at a scan rate of 2 K h−1 to study the effects of sodium chloride and calcium chloride on the main phase transition of dilute aqueous suspensions of dimyristoylphosphatidylcholine. The primary effect of sodium chloride over the concentration range studied, 0–5 molar, is to raise the transition temperature. In the case of calcium chloride, studied over the range 0–2 molar, the increase in the transition temperature is much larger, and the transitions become unsymmetrical and much broadened at concentrations above 0.75 M.

Cytochrome b562 is a four-helix-bundle protein containing a non-covalently bound b-type heme prosthetic group. In the absence of heme, cytochrome b562 remains highly structured under native conditions. Here we report thermodynamic data for the thermal denaturation of the holo- and apoproteins as determined by differential scanning calorimetry. Thermal denaturation of holocytochrome b562 is a highly reversible process, and unexpectedly does not involve dissociation of the heme prosthetic group. Thermal denaturation of the corresponding apoprotein, with the heme group chemically removed, remains a cooperative, reversible process. Apocytochrome b562 is substantially destabilized relative to the holoprotein: the t1/2 is more than ten degrees lower, and enthalpy and heat capacity changes are about one-half of the holoprotein values. However, the energetic parameters of apocytochrome b562 denaturation are within the range of observed values for small proteins.

The adaptability of Escherichia coli thioredoxin to the substitution of a series of non-natural amino acids has been investigated. Different thiosulfonated alkyl groups were inserted into the hydrophobic core of the protein in position 78 via disulfide bonding with a buried cysteine residue as previously described (Wynn R, Richards FM. 1993. Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques. Protein Sci 2:395-403). The side chains added to the cysteine included methyl, ethyl, n-propyl, n-butyl, n-pentyl, and cyclo-pentyl derivatives. The side chains appear to exploit the presence of the large cavities to incorporate these variant forms, enabling the protein to fold and have some activity. Solution structural and kinetic data suggested that these substitutions had little effect on the overall fold of the protein. Thermodynamic data revealed that the entropic effect of restricting the side chains in the folded protein has an effect on the stability. The variant forms of thioredoxin have different propensities to form dimers despite the limited structural perturbations. Molecular modeling studies allow the conformation of the side chains to be assessed.

The study of a wide variety of reversible reactions in solution indicates that the enthalpy, DeltaH(vH), which controls the temperature variation of the equilibrium constant for a reaction, can seldom, if ever, be taken to be independent of the temperature. It is also found in most cases that the values for DeltaH(vH), properly evaluated as varying with the temperature, differ significantly from the values for the enthalpy, DeltaH(cal), determined by direct calorimetry under the same experimental conditions. In a continuing search for reactions which show agreement between DeltaH(vH) and DeltaH(cal), we have studied by isothermal titration calorimetry the reactions of heptylamine with heptanoic acid in dodecane solution and of alpha-cyclodextrin with sodium heptanoate in aqueous solution.

Here we describe how the systematic redesign of a protein's hydrophobic core alters its structure and stability. We have repacked the hydrophobic core of the four-helix-bundle protein, Rop, with altered packing patterns and various side chain shapes and sizes. Several designs reproduce the structure and native-like properties of the wild-type, while increasing the thermal stability. Other designs, either with similar sizes but different shapes, or with decreased sizes of the packing residues, destabilize the protein. Finally, overpacking the core with the larger side chains causes a loss of native-like structure. These results allow us to further define the roles of tight residue packing and the burial of hydrophobic surface area in the construction of native-like proteins.

The N-terminal 200 amino acids of SHC constitute a unique phosphotyrosine (Tyr(P)) interaction (PI) domain that shows no significant sequence similarity to the other Tyr(P)-recognizing module, the SH2 domain. We describe the thermodynamic parameters characterizing PI domain binding to various tyrosyl phosphopeptides, using isothermal titration calorimetry. The PI domain forms 1:1 complexes of similar affinity with a 12-mer peptide (ISLDNPDpYQQDF) derived from Tyr-1148 of the epidermal growth factor receptor (EGFR) (KD = 28 nm) and an 18-mer (LQGHIIENPQpYFSDACVH) derived from Tyr-490 of Trk (KD = 42 nM). Binding of the EGFR-derived peptide was largely enthalpy-driven at 25 degrees C, while Trk490 peptide binding was entropy-driven. Based on the change in heat capacity upon binding, approximately 700 A2 of nonpolar surface was estimated to be buried upon interaction. Alteration of the Asn or Pro to Ala in the NPXpY motif of the EGFR Tyr-1148 peptide increased the KD of PI domain interactions to 238 and 370 nM, respectively. Alteration of a Leu at position -5 (with respect to Tyr(P)) in the EGFR peptide to Gly also reduced the binding affinity (KD = 580 nM). It is proposed that the PI domain recognizes the beta1 turn that is found in NPXpY-containing peptides and also interacts with a larger segment of the peptide than seen for SH2 domains.

The thermal unfolding of eglin c, a small proteinase inhibitor of molecular weight 8.1 kDa, is studied by means of high sensitivity scanning calorimetry over a wide pH range in dilute buffer solutions, and in the presence of varying concentrations of guanidinium chloride at pH 7.00 and 10.55. The temperature of half-completion of the unfolding transition, t1/2, in dilute buffer varies from 41 degrees C at pH 1.1 to 86 degrees C at pH 7.0 to 10.55, with corresponding enthalpy changes of approximately 40 kcal mol-1 and 71 kcal mol-1. This latter enthalpy change, amounting to 8.7 cal g-1, is unusually large for a protein, especially for one of unusually small molecular weight. Addition of 3.3 M guanidinium chloride at pH 10.55 lowered t1/2 from 86 degrees C to 40 degrees C and decreased the enthalpy change from approximately 71 kcal mol-1 to 25 kcal mol-1.

Amino acid replacements of Met73, which is hyper-exposed in the native structure, with Asp, Glu, Lys, Gly, Ala, Val, Leu or Ile changed the stability of Streptomyces subtilism inhibitor by +1.60 to -0.94 kcal/mol-1 in free energy at 82.21 degrees C at pH 7.0, with higher hydrophobicity of the replacing amino acid side-chain showing a correlation with lower stability. Thermodynamic parameters obtained from detailed calorimetric analyses gave a nearly proportional relation between the entropy change and the enthalpy change of denaturation, i.e. T delta delta S degrees = -0.03 (+/- 0.11) + 1.14 (+/- 0.03) delta delta H, with the linear correlation coefficient 0.996 for 18 data points. This proportionality observed uniquely for the substitutions at position 73 was caused primarily by the water/side-chain interaction, or hydration effect, which can account for the majority of the changes in enthalpy and entropy induced by the mutations.

The thermodynamic effects of replacing the Met residue at amino acid position 103 ofStreptomycessubtilisin inhibitor with other non-polar aliphatic residues were studied by means of differential scanning calorimetry. All but the Leu mutant, which is as stable as the wild-type but has different cooperative units in the course of unfolding, showed destabilization in terms of free energy. Similar losses in free energy, however, were caused by different reasons, i.e. by increased entropy for the Ala mutant and by decreased enthalpy for the Ile mutant, with a tendency that increases in entropy are accompanied by increases in enthalpy. The gain in entropy that caused the largest loss in free energy for the Gly mutant was unexpectedly smaller than that for the Ala mutant. The changes in enthalpy and entropy induced by the mutations exhibited some correlations with hydrophobicity, while no clear correlation was found between the changes in free energy and hydrophobicity.

We have measured the heats of formation of the trp repressor/operator complex by direct titration calorimetry over the temperature range 10 degrees C to 40 degrees C. A primary strong mode of binding displays the characteristic large negative heat capacity change observed by other methods in the formation of specific protein/DNA complexes. Unlike most such reactions, however, the formation of the trp repressor/operator complex is enthalpically driven throughout the physiological temperature range. After saturation of this principal mode, we also detected a secondary weaker binding mode, which we ascribe to a now well documented interaction called "half-site" binding. Although weak, this mode also exhibits an unusually large negative heat capacity change. Since the interface of the proposed secondary half-site binding mode has the same complementary stereochemistry as the primary one (due to internal symmetry), we correlate the negative heat capacity change with the formation of a stereospecific interface and not with high affinity. As in similar cases, the empirical correlation between buried non-polar surfaces and reduction of heat capacity does not account for the large negative delta Cp, nor do crystal structures reveal any further reduction in solvent excluded surfaces within the reactants upon complex formation. We attribute the "unaccounted for" decrement in the heat capacity of the complex to the stereospecific restriction of the hydrated polar elements that form the specific interface. We suggest that the "tightening of soft internal modes" at and near the polar interface of the complex is more important than previously recognized because previous considerations did not take into account the highly hydrated nature of these polar elements and the concomitant reduction in the degrees of freedom of the water structure.

The denaturation of Escherichia coli acyl carrier protein (ACP) in buffers containing both monovalent and divalent cations was followed by variable-temperature NMR and differential scanning calorimetry. Both high concentrations of monovalent salts (Na+) and moderate concentrations of divalent salts (Ca2+) raise the denaturation temperature, but calorimetry indicates that a significant increase in the enthalpy of denaturation is obtained only with the addition of a divalent salt. NMR experiments in both low ionic strength monovalent buffers and low ionic strength monovalent buffers containing calcium ions show exchange between native and denatured forms to be slow on the NMR time scale. However, in high ionic strength monovalent buffers, where the temperature of denaturation is elevated as it is in the presence of Ca2+, the transition is fast on the NMR time scale. These results suggest that monovalent and divalent cations may act to stabilize ACP in different ways. Monovalent ions may nonspecifically balance the intrinsic negative charge of this protein in a way that is similar for native, denatured, and intermediate forms. Divalent cations provide stability by binding to specific sites present only in the native state.

The development of cloning procedures has made possible a rapid increase in the appearance of reports on the thermodynamic effects of mutations in proteins. It will be helpful if the availability of excellent calorimetric instrumentation leads to an increase in the fraction of these studies directed toward the determination of enthalpies and entropies as well as free energies.

The effects of eight mutations on the thermodynamics of the reversible thermal unfolding of staphylococcal nuclease have been determined over a range of pH and protein concentration by means of differential scanning calorimetry. Variation of the protein concentration was included in our study because we found a significant dependence of the thermodynamics of protein unfolding on concentration. Values for the change in the standard free energy of unfolding, delta delta G0d, produced by the mutations in the pH range 5.0-7.0 varied from 1.9 kcal mol-1 (apparent stabilization) for H124L to -2.8 kcal mol-1 (apparent destabilization) for L25A. As has been observed in numerous other cases, there is no correlation in magnitude or sign between delta delta G0d and the corresponding values for delta delta Hd and T delta delta S0d, the latter quantities being in most cases much larger in magnitude than delta delta G0d. This fact emphasizes the difficulty in attempting to correlate the thermodynamic changes with structural changes observed by X-ray crystallography.

Results obtained by means of differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) with several proteins and mutant forms thereof are reported. DSC has been employed primarily to determine the thermodynamic changes accompanying the thermally induced unfolding of proteins, and ITC to measure the thermodynamics of the interactions of proteins with various ligands. It has become inreasingly evident that we are dealing with very complicated systems in which a single amino acid replacement probably causes numerous small effects which are widely distributed in the molecule, and which add up to an observed effect that is obviously difficult to rationalize in terms of molecular structure. It is nevertheless important to continue to broaden the base of quantitative information in the field.

Aspartate transcarbamylase (EC 2.1.3.2) from E. coli is a multimeric enzyme consisting of two catalytic subunits and three regulatory subunits whose activity is regulated by subunit interactions. Differential scanning calorimetric (DSC) scans of the wild-type enzyme consist of two peaks, each comprised of at least two components, corresponding to denaturation of the catalytic and regulatory subunits within the intact holoenzyme (Vickers et al., J. Biol. Chem. 253 (1978) 8493; Edge et al., Biochemistry 27 (1988) 8081). We have examined the effects of nine single-site mutations in the catalytic chains. Three of the mutations (Asp-100-Gly, Glu-86-Gln, and Arg-269-Gly) are at sites at the C1: C2 interface between c chains within the catalytic subunit. These mutations disrupt salt linkages present in both the T and R states of the molecule (Honzatko et al., J. Mol. Biol. 160 (1982) 219; Krause et al., J. Mol. Biol. 193 (1987) 527). The remainder (Lys-164-Ile, Tyr-165-Phe, Glu-239-Gln, Glu-239-Ala, Tyr-240-Phe and Asp-271-Ser) are at the C1: C4 interface between catalytic subunits and are involved in interactions which stabilize either the T or R state. DSC scans of all of the mutants except Asp-100-Gly and Arg-269-Gly consisted of two peaks. At intermediate concentrations, Asp-100-Gly and Arg-269-Gly had only a single peak near the Tm of the regulatory subunit transition in the holoenzyme, although their denaturational profiles were more complex at high and low protein concentrations. The catalytic subunits of Glu-86-Gln, Lys-164-Ile and Asp-271-Ser appear to be significantly destabilized relative to wild-type protein while Tyr-165-Phe and Tyr-240-Phe appear to be stabilized. Values of delta delta G degree cr, the difference between the subunit interaction energy of wild-type and mutant proteins, evaluated as suggested by Brandts et al. (Biochemistry 28 (1989) 8588) range from -3.7 kcal mol-1 for Glu-86-Gln to 2.4 kcal mol-1 for Tyr-165-Phe.

In this paper we show that the usual assumption in studies of the temperature variation of equilibrium constants for equilibria of the form A+B AB that a plot of ln K vs. 1/T (K = equilibrium constant, T = temperature in degrees kelvin) is a straight line with slope equal to -delta HvH/R (delta HvH = van't Hoff or apparent enthalpy, R = gas constant) is not valid in many cases. In all the cases considered here, delta HvH is temperature dependent and is significantly different from the true or calorimetrically measured enthalpy, and the respective values for delta Cp are also significantly different.

Isothermal titration calorimetric (ITC) studies over a range of temperatures of the binding of maltose, maltotriose, maltotetraose and beta-cyclodextrin to the maltodextrin-binding protein (MBP) of Escherichia coli are reported. The binding constants of maltose, maltotriose and beta-cyclodextrin are not very different, namely 8.7 x 10(5), 13.0 x 10(5) and 2.55 x 10(5) M-1, respectively at 25 degrees C. The calorimetric data obtained with maltotetraose cannot be interpreted in terms of a definite binding constant. The binding of maltose and maltotriose is endothermic with a large entropy increase while that of beta-cyclodextrin is exothermic, with a smaller entropy increase. The binding of maltotetraose was endothermic or exothermic depending on the temperature.

Significantly different m values (1.9-2.7 kcal mol-1 M-1) were observed for point mutations at a single, solvent-exposed site (T53) in a variant of the B1 domain of streptococcal Protein G using guanidine hydrochloride (GuHCl) as a denaturant. This report focuses on elucidating the energetic and structural implications of these m-value differences in two Protein G mutants, containing Ala and Thr at position 53. These two proteins are representative of the high (m+) and low (m-) m-value mutants studied. Differential scanning calorimetry revealed no evidence of equilibrium intermediates. A comparison of GuHCl denaturation monitored by fluorescence and circular dichroism showed that secondary and tertiary structure denatured concomitantly. The rates of folding (286 S-1 for the m+ mutant and 952 S-1 for the m- mutant) and the rates of unfolding (11 S-1 for m+ mutant and 3 S-1 for the m- mutant) were significantly different, as determined by stopped-flow fluorescence. The relative solvation free energies of the transition states were identical for the two proteins (alpha ++ = 0.3). Small-angle X-ray scattering showed that the radius of gyration of the denatured state (Rgd) of the m+ mutant did not change with increasing denaturant concentrations (Rgd approximately 23 A); whereas, the Rgd of the m- mutant increased from approximately 17 A to 23 A with increasing denaturant concentration. The results indicate that the mutations exert significant effects in both the native and GuHCl-induced denatured state of these two proteins.

Isothermal calorimetric titration of 18-crown-6 ether with BaCl2 in pure aqueous solution over the temperature range 7-40 degrees C gives precise binding constants and enthalpy changes. Nonlinear least-squares fitting of the binding constants to the integrated van't Hoff equation, including a temperature-independent change in heat capacity, leads to van't Hoff enthalpies that differ significantly from the observed calorimetric enthalpies. This perplexing discrepancy appears at present to be very widely occurring.

Amino acid replacements in a homodimeric protein, Streptomyces subtilisin inhibitor, at the position of Val13, which is located at the center of the beta-sheet interface of the domain-domain interaction, changed both the overall stability and the denaturational scheme. All the mutant forms lost stability, losses in free energy at 82.21 degrees C at pH 7.0 obtained by calorimetric measurements ranging from 10.3 kcal (mol dimer)-1 for the Gly mutant, which involved a loss in enthalpy without a compensating loss in entropy, to 0.84 kcal (mol dimer)-1 for the Ile mutant, which involved comparable losses in enthalpy and entropy. A gain in enthalpy for the Ala mutant was overcome by a gain in entropy, resulting in a loss in free energy by 6.78 kcal (mol dimer)-1. It was found that decreases in enthalpy and entropy showed good correlation with increasing side-chain hydrophobicity, while no such correlation was present for free energy. Replacements with Gly, Ala, Met and Phe altered the thermal denaturational scheme from N2 reversible 2D, which was the case for the Leu and Ile mutants as well as for the wild-type, to N2 reversible 2N* reversible 2D, where the first step can be regarded as a dissociation of the native dimer followed by a major unfolding in the second step. The free energy acquired by forming the dimer was estimated to be 2.03 kcal mol-1 for the Ala mutant at 66.95 degrees C at pH 9.5 and lower for the other three mutants.

Rationally redesigned variants of the 4-helix-bundle protein Rop are described. The novel proteins have simplified, repacked, hydrophobic cores and yet reproduce the structure and native-like physical properties of the wild-type protein. The repacked proteins have been characterized thermodynamically and their equilibrium and kinetic thermal and chemical unfolding properties are compared with those of wild-type Rop. The equilibrium stability of the repacked proteins to thermal denaturation is enhanced relative to that of the wild-type protein. The rate of chemically induced folding and unfolding of wild-type Rop is extremely slow when compared with other small proteins. Interestingly, although the repacked proteins are more thermally stable than the wild type, their rates of chemically induced folding and unfolding are greatly increased in comparison to wild type. Perhaps as a consequence of this, their equilibrium stabilities to chemical denaturants are slightly reduced in comparison to the wild type.

Differential scanning calorimetry (DSC) is employed in a study of the effects of the p -nitrophenyl esters of the even numbered fatty acids from C 6 (caproic acid) to C 18 (stearic acid) on the main phase transition of multilamellar suspensions of dimyristoylphosphatidylcholine (DMPC). Mole fractions in the range of 0.02–0.12 were used. Within this concentration range the observed transitions could be well fit on the basis of a model assuming ideal behavior of the esters in both the gel and liquid crystal phases of the lipid, and independently additive van't Hoff and impurity broadening. As expected on the basis of this model, the transition temperatures decreased linearly with increasing ester concentration and the transition enthalpies were independent of ester concentration. Significant differences between the effects of the various esters were observed, but these did not change with changing ester chain length in any regular fashion.

Amino acid substitutions at a site in the center of the bacteriophage protein P22 tailspike polypeptide chain suppress temperature-sensitive folding mutations at many sites throughout the chain. Characterization of the intracellular folding and chain assembly process reveals that the suppressors act in the folding pathway, inhibiting the aggregation of an early folding intermediate into the kinetically trapped inclusion body state. The suppressors alone increase the folding efficiency of the otherwise wild-type polypeptide chain without altering the stability or activity of the native state. These amino acid substitutions identify an unexpected aspect of the protein folding grammar--sequences within the chain that carry information inhibiting unproductive off-pathway conformations. Such mutations may serve to increase the recovery of protein products of cloned genes.

The main phase transitions of aqueous dispersions of four synthetic phosphatidylcholines (PCs) containing sulfur atoms in thioester and thioether linkages have been studied by high sensitivity differential scanning calorimetry (DSC). The transition enthalpies ranged from 7.4 to 10.3 kcal mol-1, with values for tm, the temperature of maximal excess heat capacity, in the range 38.0 to 40.8 degrees C. The corresponding values for dipalmitoyl PC (DPPC) are 8.5 kcal mol-1 and 41.7 degrees C. Curve fitting required the sum of from one to four two-state components to give an accurate representation of the observed DSC curves. Comparison of the results given here with those reported by B.Z. Chowdhry, G. Lipka, J. Hajdu and J.M. Sturtevant, (1984) Biochemistry 23, 2044-2049, for PCs containing amide, ether or carbamoyl linkages in place of the usual ester bonds shows that small changes in organic structure can result in large changes in thermotropic behavior. The complexity in the cases showing more than a single two-state component is presumably due to a series of sequential cooperative transitions the character of which is at present unknown.

Mixed lipid samples containing dimyristoylglycerophosphocholine and small amounts of myristic acid were examined calorimetrically. Examination of multilamellar and small vesicle samples indicated that upon heating small vesicles combine to form more extended structures. An exothermic peak (at 19.5°C) can be associated with the structural transformation. The enthalpy for this process, which may be interpreted as vesicle-vesicle fusion, is found to be approx. -2 kcal/mol.

In the concentration range of 10 −5 to 10 −1 M Ca 2+ modulates the thermotropic properties of several neutral and anionic glycosphingolipids (galactosylceramide, asialo-G M1 , sulfatide, G M1 , G D1a , G T1b ) and of their mixtures with dipalmitoylphosphatidylcholine. The transition temperature of gangliosides is not appreciably changed while the transition enthalpy increases by 20% in the presence of Ca 2+ . The more marked effect of Ca 2+ is on the thermotropic behavior of systems containing sulfatide. Increasing concentrations of Ca 2+ between 10 −5 and 10 −3 M (up to a molar ratio of Ca 2+ /sulfatide 1:2) induce a progressive increase of both the transition temperature and enthalpy. Further increases up to 10 −1 M Ca 2+ induce a new phase transition at a lower temperature. No evidence is found for induction of phase separation of pure glycosphingolipid-Ca 2+ domains in mixtures of any of the glycosphingolipids with dipalmitoylphosphatidylcholine. The modification of the phase behavior of anionic glycosphingolipids by Ca 2+ does not involve detectable variations of the intermolecular packing but is accompanied by marked modifications of the dipolar properties of the polar head group region.

The thermotropic behavior of the natural glycosphingolipids galactosylceramide, asialo-Gal beta 1-3GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-Cer (GM1), sulfatide, GM1, NeuAc alpha 2-3Gal beta 1-3GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1Cer (GD1a), and NeuAc alpha 2-3Gal beta 1-3GalNAc beta 1-4Gal(3-2 alpha NeuAc8-2 alpha NeuAc)beta 1-4Glc beta 1-1 Cer (GT1b), and their mixtures with dipalmitoylphosphatidylcholine (DPPC) in the presence of myelin basic protein (MBP) was studied by high sensitivity differential scanning calorimetry. The transition temperature of DPPC, galactosylceramide, and asialo-GM1 is affected little by MBP while their transition enthalpy is decreased in proportion to the amount of protein in the mixture. The thermotropic behavior of anionic glycosphingolipids is considerably perturbed by MBP. The transition temperature of gangliosides increases in the presence of MBP, whereas that of sulfatide decreases. The enthalpy of the transition of anionic glycosphingolipids increases markedly in the presence of MBP. The excess heat capacity function of these systems can be resolved into two independent phase transitions. Phase separation of enriched lipid/protein domains occurs in a magnitude that depends on the amount of MBP; the rest of the lipid phase exhibits some altered thermodynamic properties. In mixtures of glycosphingolipids with DPPC, phase separation is also present but no phase transition with the characteristic of pure DPPC is found. MBP is changing the properties of the lipid mixture as a whole and does not interact exclusively with the glycosphingolipids. The proportion of MBP required to produce the maximal changes is greater the greater the complexity of the glycosphingolipids polar head group. Relatively small variations of the amount of MBP induce large shifts in the proportion of the different phases present.

Temperature-sensitive folding mutations (tsf) of the thermostable P22 tailspike protein prevent the mutant polypeptide chain from reaching the native state at the higher end of the temperature range of bacterial growth (37–42 °C). At lower temperatures the mutant polypeptide chains fold and associate into native proteins. The melting temperatures of the purified native forms of seven different tsf mutant proteins have been determined by differential scanning calorimetry. Under conditions in which the wild type protein had a melting temperature of 88.4 °C, the melting temperatures of the mutant proteins were all above 82 °C, more than 40 °C higher than the temperature for expression of the folding defect. Because the folding defects were observed in vivo,the thermostability of the native protein was also examined with infected cells. Once matured at 28 °C, intracellular tsf mutant tailspikes remained native when the cells were transferred to 42 °C, a temperature that prevents newly synthesized tsf chains from folding correctly. These results confirm that the failure of tsf polypeptide chains to reach their native state is not due to a lowered stability of the native state. Such mutants differ from the class of ts mutations which render the native state thermolabile. The intracellular folding defects must reflect decreased stabilities of folding intermediates or alteration in the off-pathway steps leading to aggregation and inclusion body formation. These results indicate that the stability of a native protein within the cells is not sufficient to insure the successful folding of the newly synthesized chains into the native state.

Heats of thermal denaturation of chromatin core particles and core particles with covalently crosslinked histones were measured by differential scanning calorimetry. The additional stabilization of the nucleoprotein complex by crosslinking is not reflected in the transition enthalpy. The contribution of protein denaturation to the total heat was estimated by comparison of core particles with core particle DNA in a high-salt solution. By taking into account the temperature dependence of the transition enthalpy of DNA, we conclude that the enthalpy change for denaturation of DNA in core particles is nearly the same as that for naked DNA in solution.

Differential scanning calorimetry (DSC) and the closely related differential thermal analysis (DTA) have been widely employed during the last several decades in the thermodynamic study of processes that are initiated by either an increase or a decrease in temperature. This review focuses on biochemical applications of DSC. Macromolecular and polymolecular structures stabilized by the cooperation of numerous weak forces are important to most biochemical processes. Since such highly cooperative structures undergo conformational or phase transitions upon being heated, significant information concerning these structures can be obtained by DSC. Small molecules cannot be studied by DSC unless they form aggregates showing intermolecular cooperation, as in crystals. This is illustrated in Table 1. Since the enthalpies of chemical processes rarely are as large as 20 cal g-l, it is evident that molecules having molecular weights, or molecular aggregates having aggregate weights, in the thousands of daltons are required to give transitions sufficiently sharp for useful DSC observation. In a scanning calorimeter, one measures the specific heat of a system as a function of the temperature. For a solution, the apparent specific heat of the solute, c2, is given by the expression

The thermotropic behavior of three fluorinated phospholipids has been studied by means of high-sensitivity differential scanning calorimetry. The three lipids are 1,2-di(X,X-difluoromyristovyl)phosphatidylcholine with X = 4,8, or 12. The compound with X = 4 has a higher transition temperature than dimyristoylphosphatidylcholine whereas the other two have lower values. All three lipids have transition enthalpies approximately twice that of the unsubstituted lipid. It was found the substituted lipids form strikingly nonideal mixtures with the parent compound. In view of these observations it is evident that results obtained using these or related compounds as probes of bilayer or membrane behavior must be interpreted with due caution. Some of the properties exhibited by geminal fluorine atoms in fluorinated phospholipids appear to be similar to those of cis double bonds in lipids.

The thermotropic behavior of mixtures of dipalmitoylphosphatidylcholine (DPPC) with natural glycosphingolipids (galactosylceramide, phrenosine, kerasine, glucosylceramide, lactosylceramide, asialo-G M1 , sulfatide, G M3 , G M1 , G D1a , G T1b ) in dilute aqueous dispersions were studied by high sensitivity differential scanning calorimetry over the entire composition range. The pretransition of DPPC is abolished and the cooperativity of the main transition decreases sharply at mole fractions of glycosphingolipids below 0.2. All systems exhibit non-ideal temperature-composition phase diagrams. The mono- and di-hexosylceramides are easily miscible with DPPC when the proportion of glycosphingolipids in the system is high. A limited quantity (1–6 molecules of DPPC per molecule of glycosphingolipid (GSL) can be incorporated into a homogeneously mixed lipid phase. Domains of DPPC, immiscible with the rest of a mixed GSL-DPPC phase that shows no cooperative phase transition, are established as DPPC exceeds a certain proportion in the system. One negative charge (sulfatide) or four neutral carbohydrate residues (asialo-G M1 ) in the oligosaccharide chain of the glycosphingolipids results in phase diagrams exhibiting coexistence of gel and liquid phases over a broad temperature-composition range. Systems containing gangliosides show complex phase diagrams, with more than one phase transition. However, no evidence for phase-separated domains of pure ganglioside species is found. The thermotropic behavior of systems containing DPPC and glycosphingolipids correlates well with their interactions in mixed monolayers at the air/water interface.

The thermodynamics of the binding of L-arabinose and of D-galactose to the L-arabinose-binding protein of Escherichia coli have been studied by isothermal and scanning calorimetry. The binding reaction with arabinose is characterized by an enthalpy change of -15.3 +/- 0.5 kcal mol-1 at 25 degrees C, and a large decrease in apparent heat capacity, amounting to -0.44 +/- 0.05 kcal K-1 mol-1, which is constant over the temperature range 8 to 30 degrees C. Very similar results were obtained with D-galactose. These calorimetric results have been combined with binding constants determined by equilibrium dialysis (Clark, A. F., Gerken, T. A., and Hogg, R. W. (1982) Biochemistry 21, 2227-2233) to obtain free energy and entropy changes over the range 5 to 30 degrees C, and by extrapolation to 60 degrees C. The protein undergoes reversible unfolding on being heated with an increase in enthalpy at 53.5 degrees C of 151.8 +/- 1.1 kcal mol-1 (169.2 +/- 1.2 kcal mol-1 at 59.0 degrees C) and in apparent heat capacity of 3.16 +/- 0.07 kcal K-1 mol-1. In the presence of arabinose, the unfolding enthalpy is increased to 200.7 +/- 1.8 kcal mol-1 at 59.0 degrees C, the increase being due to the enthalpy of dissociation of the ligand which amounts to 31 kcal mol-1 at the unfolding temperature. The unfolding temperature is increased by the presence of excess arabinose or galactose, an effect which is due solely to displacement by the added ligand of the unfolding-dissociation equilibrium. The thermodynamic data are discussed in connection with the detailed structural information available for this system from x-ray crystallography (Newcomer, M. E., Gilliland, G. L. and Quiocho, F. A. (1981) J. Biol. Chem. 256, 13213-13217, and references cited therein).

The phase transition properties of aqueous suspensions of a series of nonhydrated (not heated above room temperature) and hydrated 1,2 diacylphosphatidylethanolamines (PE's) have been examined by high sensitivity differential scanning calorimetry at scan rates of 0.02–1.0 K min-1. At all scan rates nonhydrated PE's show a single asymmetric transition curve of excess heat capacity as a function of temperature. Multilamellar dispersions of hydrated PE's, however, exhibit transitions with fine structure, which can be fitted as the sum of three two-state component transitions, at scan rates of 0.02–0.1 K min-1, but give only a single asymmetric transition at 1.0 K min-1. At all scan rates the transition(s) of hydrated samples occur at lower temperatures than those of nonhydrated samples. One of the component transitions of hydrated PE's may be analogous to the pretransition that occurs in 1,2 diacylphosphatidylcholines.

Differential scanning calorimetry, temperature-dependent absorbance, and circular dichroic spectroscopy were used to study the thermodynamics of the B-Z transition in poly(m5dG-dC). In sodium phosphate buffer (pH 7.0) containing 50 mM NaCl and 1.0 mM MgCl2, the reversible B-Z transition of the polymer is centered at 38.2 degrees C and is characterized by delta Hcal = 0.61 +/- 0.07 kcal/(mol X base pair) (1 cal = 4.184 J), delta HvH = 68 +/- 7 kcal/mol, and a cooperative unit of length 110 +/- 20 base pairs. A second transition centered at 53.6 degrees C is observed and represents a conformational change of poly(m5dG-dC) from the Z to an alternate left-handed form of unknown structure. This transition does not arise from aggregation to the Z form, or from the helix-coil transition. The left-handed helix-coil transition is observed at 120.9 +/- 0.3 degrees C and is characterized by delta Hcal = 9.1 +/- 1.0 kcal/(mol X base pair). These results indicate a substantial enthalpic contribution to the B-Z transition, in contrast to previous assertions that the process is entropically driven.

High sensitivity differential scanning calorimetry was employed to study the thermotropic behavior of multilamellar vesicles of neutral and acidic phospholipids and binary mixtures thereof in the presence of anthracycline antibiotics. Adriamycin and its lipophilic analogue, N-trifluoroacetyladriamycin-14-valerate (AD32) were investigated and compared to chlorpromazine and quinidine with respect to their ability to affect the pretransition and the main transition of the phospholipids suspended in physiological buffer. With liposomes of neutral dipalmitoylphosphatidylcholine the observed effects paralleled to some extent the corresponding octanol/buffer partition coefficients, with adriamycin being the least effective. Calorimetric measurements on liposomes prepared from pure dipalmitoylphosphatidylglycerol or from binary mixtures of dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine showed that modulation of bilayer properties by adriamycin was greatly enhanced in the presence of negatively charged lipid headgroups presumably as a result of electrostatic interactions. AD32 interacted differently from adriamycin with the acidic bilayers at low drug concentrations, in a manner similar to that of its interaction with neutral bilayers. At high drug concentrations both adriamycin and AD32 produced transitions with multiple peaks not exhibited by chlorpromazine and quinidine which may be the result of a specific association of the anthracyclines with dipalmitoylphosphatidylglycerol. All four drugs produced only minor changes in the enthalpy of the main transition of the investigated lipids. The present findings are discussed in terms of their possible physiological relevance.

High sensitivity differential scanning calorimetry is applied to the study of the thermotropic behavior of mixtures of synthetic phospholipids in multilamellar aqueous suspensions. The systems dimyristoylphosphatidylcholine dipalmitoylphosphatidylcholine, and dimyristoylphosphatidylethanolamine-distearoylphosphatidylcholine, although definitely nonideal, exhibit essentially complete miscibility in both gel and liquid crystalline states, while the system dilauroylphosphatidylcholine-distearoylphosphatidylcholine is monotectic with lateral phase separation in the gel state. Comparison of the observed transition curves with theoretical curves calculated from the calorimetrically determined phase diagrams supports a literal interpretation of the phase diagrams.

A flow modification of the Beckman Model 190 Microcalorimeter is described with which the enthalpies of mixing of pairs of liquids can be very conveniently and accurately measured. The enthalpies of mixing of ten pairs of liquids at 25 °C are reported, composition ranges at least as wide as volume fraction 0.05 to 0.95 of one component being covered. Of the systems studied, only cyclohexane + carbon tetrachloride and n-octane + iso-octane were found to conform within experimental error to the strict requirements of Scatchard's description of regular solutions. The systems cyclohexane + cis-decalin and cyclohexane + trans-decalin are endothermic over only part of the composition range, changing to exothermic in decalin-rich mixtures; the two decalins mix exothermically throughout the composition range. Cyclohexane + n-hexane and cyclohexane + n-heptane show rather marked deviations from symmetric behavior. The remaining systems, benzene + toluene, toluene + o-xylene and o-xylene + p-xylene, though differing widely in enthalpies of mixing, exhibit only slight deviations from regular behavior. The results confirm the Scatchard-Hildebrand equation in suggesting that enthalpies of mixing in cases such as those studied here are best presented in the form of values of ΔQ >φ 1 >φ 2 given as a function of >φ1. In this expression, ΔQ is the enthalpy of mixing per unit volume and >φ1 and >φ2 are the volume fractions of the components.

Use of a very sensitive differential calorimeter has revealed two major regions of heat absorption in purified plasma membranes of Mycoplasma laidlawii. These consist of a rapidly increasing rate of heat absorption showing an inflection point at approx. 40°; this is followed at higher temperatures by a slightly decreased rate of heat uptake. Within this latter region it is barely possible to distinguish a second inflection point. These transitions were found also to be properties of whole cells. Experimental results presented in this paper support the conclusion that the first region of heat absorption is due to a reversible transition in the lipid component of the membrane, and that the second region is due to an irreversible denaturation of the protein component. These results also provide evidence that the lipid transition is essentially unaffected by the protein component of the membrane. The calorimetric transition curves indicate that the apparent heat capacity of the membrane lipids increases during the transition. This most likely arises from new modes of freedom available to the fatty acid hydrocarbon chains in the melted state. By means of a viable cell titer taken before and after cells were submitted to a calorimetric run extending to 40°, the lipid transition was demonstrated unambiguously to be a property of living cells. The Tm of the lipid transition was found to be a function of the conditions of growth of the cells. In all cases studied the temperature at which the cells grew lies within the range of the lipid transition. Results suggest that the cells attempt to regulate their position in the lipid transition.

The gel-liquid crystal transitions in lipid bilayers formed from synthetic dimyristoyl, dipalmitoyl and distearoyl l-α-lecithins have been studied by high sensitivity differential scanning calorimetry in dilute aqueous suspensions ranging in concentration from 0.4 to 6.6 mg ml-1. Each lipid shows two endothermic transitions, an extremely sharp main transition and a broader transition accompanied by a smaller heat absorption at a temperature 5–10° below the main transition. The enthalpy increases in the main transition are respectively 6.3, 9.7, and 10.8 kcal per mole of monomeric lipid. The main transition widths, which are not very reproducible, reach a minimum in the case of dimyristoyl lecithin of as little as 0.2° for 10% to 90% conversion, suggesting that these transitions would be truly isothermal with completely pure lipids. The apparent heat capacities of the lipids in the liquid crystal state are, with the possible exception of dipalmitoyl lecithin, no more than 5 cal deg-1 (mole of lipid)-1 larger than in the gel state, indicating that the hydrocarbon chains have much less mobility in the liquid crystalline state than in the corresponding liquid normal paraffins. This conclusion was also reached by Phillips et al. (Phillips, M. C., Williams, R. M., and Chapman, D. (1969) Chem Phys. Lipids 3, 234) on the basis of entropy comparisons.

The circular dichroism spectra of oxidized and reduced cytochrome b2, the l(+)-lactate dehydrogenase (EC 1.1.2.3) of bakers' yeast, are observed in the wave length range 188 to 600 mµ. The spectrum at short wave lengths, which is unaffected by change in the oxidation state of the enzyme, is similar in character to that observed with α-helical polypeptides, the amplitudes of the extrema indicating an apparent helical content of roughly 25%. Between 250 and 600 mµ numerous dichroic bands are observed. Type I (DNA-containing) enzyme has a large positive band at 282 mµ because of the DNA component, and Type II (DNA-free) enzyme has a prominent negative band at 270 mµ which appears to result from an interaction of the flavin prosthetic group with 1 or more aromatic residues since it is entirely missing from FMN-free apoenzyme. Neither of these bands is much affected by the oxidation state of the enzyme. All the bands above 300 mµ are strongly dependent on the oxidation state of the enzyme, but are affected very little by removal of the DNA. These bands can be ascribed to transitions in the flavin and heme prosthetic groups, and many of them appear to be strongly affected by the flavin-heme interaction which also appears to be essential for enzymic activity. Numerical integration over the circular dichroism spectrum according to the Kronig-Kramers equation gives a calculated optical rotatory dispersion spectrum which reproduces all the features of the observed spectrum but falls below it in amplitude. A Lowry plot of the difference between the observed and calculated spectra suggests that a dichroic band or bands centered at 150 mµ, the result of peptide units in α-helical conformation, contributes significantly to the dispersion over the entire wave length range studied. The effects of urea and changes of pH on the circular dichroism and activity of the enzyme have been studied. A remarkable parallelism between the amplitude of the dichroic band in the region of the heme Soret band and the activity of the enzyme is observed. At a urea concentration of approximately 3 m (neutral pH) and a pH of 9.5 (no urea present), the dichroic spectrum of the enzyme is similar to that observed for the FMN-free apoenzyme and the enzyme is inactive. These observations give further support to the view that interaction of some sort between the FMN and heme groups is responsible for both the character of the circular dichroism spectrum and the catalytic activity. The circular dichroism of a heme peptide, with a molecular weight of about 12,000, obtained by tryptic digestion of cytochrome b2, is found to be similar to that observed for the FMN-free apoenzyme in the wave length region below 250 mµ and in the region of the Soret band. It is interesting that the apparent α-helical content is roughly the same as for the native enzyme. The cytochrome b2 obtained from the yeast Hansenula anomala, which has an activity approximately 6 times that of the enzyme from Saccharomyces cerevisiae, is found to have circular dichroism spectra similar to those observed with the Type II enzyme below 300 mµ, but differing markedly at higher wave lengths, especially in the Soret region. The difference dichroic spectra of Hansenula enzyme minus Saccharomyces enzyme consist largely of a symmetrical positive band at 423 mµ for the reduced enzymes and one at 413 mµ for the oxidized enzymes. It appears that these bands do not involve the FMN-heme interaction responsible for enzyme activity.

Cytochrome b2, the l-(+)lactate dehydrogenase of bakers' yeast (EC 1.1.2.3), contains both protoheme and flavin mononucleotide prosthetic groups. The heme protein obtained by dissociating the flavin with (NH4)2SO4 at low pH is unable to accept electrons directly from l-lactate, and is a much less effective electron acceptor for the active enzyme than is ferricytochrome c. Cytochrome b2 reacts with p-chloromercuribenzenesulfonate with eventual loss of activity and dissociation of the flavin mononucleotide prosthetic group. In the reduced form of the enzyme, approximately 6 thiol groups per heme unit bind the mercurial, while in the oxidized form approximately 5 groups react. In the presence of 0.2% dodecyl sulfate, an additional thiol group becomes available in the oxidized enzyme. All available —SH groups can be reacted without complete loss of activity or immediate dissociation of the FMN; however, the enzyme is much less stable after treatment with the mercurial and soon becomes completely inactive, and the binding of the flavin group is very much weakened. If the enzyme is allowed to react with excess mercurial, the FMN can be removed by Sephadex filtration; it is possible to secure nearly complete recovery of activity by treatment with excess mercaptoethanol and FMN with Sephadex filtration to remove the excess of the reagents. The reaction of cytochrome b2 with p-chloromercuribenzenesulfonate follows complex kinetics which indicates that the reactive —SH groups are not all of equal reactivity. The initial rate is proportional to the p-chloromercuribenzenesulfonate concentration up to about 200 µm and then appears to reach a saturation plateau at about 1000 µm. It is concluded that the —SH groups which can react with p-chloromercuribenzenesulfonate are not directly involved in the catalytic mechanism or in binding the FMN groups. Introduction of the mercurial groups appears to cause a conformational change which labilizes the enzyme, decreases the strength of binding of the FMN groups, and interferes with the heme-FMN interaction which is necessary for catalytic activity.

The α-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate has been studied at pH 7 and 25° in isopropyl alcoholwater and dioxane-water mixtures. It was found that the initial reaction in which the enzyme is acylated follows second order kinetics rather than Michaelis-Menten kinetics as previously reported. The second order rate constant for this reaction is markedly decreased by addition of either isopropyl alcohol or dioxane in a manner which can be formally attributed to either competitive or noncompetitive inhibition. The rate of the steady state reaction increases with substrate concentration in a manner which can be formally described in terms of substrate activation. After correction for this effect, the rate at substrate saturation is increased by organic solvents (by a factor of 3.5 in 20% (v/v) dioxane); the apparent Michaelis-Menten constant is also greatly increased (by a factor of 150 in 20% (v/v) dioxane). The primary amine tris(hydroxymethyl)aminomethane, which was used as buffer, is shown to have a strong inhibitory effect on the pre-steady state reaction, and to be very much more effective than water as an acyl acceptor. Although several of these effects can be expressed in reasonably quantitative terms, it is not possible to offer convincing explanations for them. Organic solvents such as dioxane may produce more or less extensive changes in the enzyme, perhaps conformational in character, which drastically alter its kinetic properties; they may also exert an influence by disrupting the water structure in the vicinity of the active site.

The kinetics of the deamination of 5'-adenosine monophosphate by rabbit muscle adenylate deaminase has been studied over a wide range of substrate and enzyme concentrations at pH 6.5. The kinetics in the presence of concentrations of the cationic activator K+ of 0.1 m or higher appears to be of normal Michaelis-Menten character, except that the apparent enthalpy of activation has the unusually high value of 19 kcal mole-1. At low K+ concentrations, the apparent specific activity of the enzyme depends on the concentrations of substrate, K+, and enzyme. In the absence of K+, a lag phase in the deamination reaction, observable by the stopped flow technique, indicates activation by substrate with an apparent second order rate constant of 7000 m-1 sec-1 at 25°. It is shown that a quantitatively consistent interpretation of all the data presented in this paper can be based on the allosteric model of Monod, Wyman, and Changeux (Monod, J., Wyman, J., and Changeux, J.-P., J. Mol. Biol., 12, 88 (1965)), with the assumption that the enzyme is composed of six or more identical subunits. Equally satisfactory interpretations could undoubtedly be based on other models for apparent allosteric effects which have been proposed by other authors. A decrease in specific activity observed at very low enzyme concentrations, also in the absence of K+, suggests that the enzyme undergoes dissociation, with only the intact oligomer having catalytic activity.