Quantitative analysis of conformational equilibrium using capillary electrophoresis: applications to protein folding.
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Electrophoresis↗
Biomedical subjects
E Freire
Publications and source records attributed to E Freire.
A calorimetric characterization of the salt dependence of the stability of the GCN4 leucine zipper.
The effects of different salts (LiCl, NaCl, ChoCl, KF, KCl, and KBr) on the structural stability of a 33-residue peptide corresponding to the leucine zipper region of GCN4 have been studied by high-sensitivity differential scanning calorimetry. These experiments have allowed an estimation of the salt dependence of the thermodynamic parameters that define the stability of the coiled coil. Independent of the nature of the salt, a destabilization of the coiled coil is always observed upon increasing salt concentration up to a maximum of approximately 0.5 M, depending on the specific cation or anion. At higher salt concentrations, this effect is reversed and a stabilization of the leucine zipper is observed. The effect of salt concentration is primarily entropic, judging from the lack of a significant salt dependence of the transition enthalpy. The salt dependence of the stability of the peptide is complex, suggesting the presence of specific salt effects at high salt concentrations in addition to the nonspecific electrostatic effects that are prevalent at lower salt concentrations. The data is consistent with the existence of specific interactions between anions and peptide with an affinity that follows a reverse size order (F- > Cl- > Br-). Under all conditions studied, the coiled coil undergoes reversible thermal unfolding that can be well represented by a reaction of the form N2<==>2U, indicating that the unfolding is a two-state process in which the helices are only stable when they are in the coiled coil conformation.
Anions↗
Configurational effects in antibody-antigen interactions studied by microcalorimetry.
In this paper we study the binding of two monoclonal antibodies, E3 and E8, to cytochrome c using high-sensitivity isothermal titration calorimetry. We combine the calorimetric results with empirical calculations which relate changes in heat capacity to changes in entropy which arise from the hydrophobic effect. The change in heat capacity for binding E3 is -350 +/- 60 cal K-1 mol-1 while for E8 it is -165 +/- 40 cal K-1 mol-1. This result indicates that the hydrophobic effect makes a much larger contribution for E3 than for E8. Since the total entropy change at 25 degrees C is very similar for both antibodies, it follows that the configurational entropy cost for binding E3 is much larger than for binding E8 (-77 +/- 15 vs. -34 +/- 11 cal K-1 mol-1). These results illustrate a case of entropy compensation in which the cost of restricting conformational degrees of freedom is to a large extent compensated by solvent release. We also show that the thermodynamic data can be used to make estimates of the surface area changes that occur upon binding. The results of the present study are consistent with previous hydrogen-deuterium exchange data, detected using 2D NMR, on the two antibody-antigen interactions. The NMR study indicated that protection from exchange is limited to the binding epitope for E8, but extends beyond the epitope for E3.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
The heat capacity of proteins.
The heat capacity plays a major role in the determination of the energetics of protein folding and molecular recognition. As such, a better understanding of this thermodynamic parameter and its structural origin will provide new insights for the development of better molecular design strategies. In this paper we have analyzed the absolute heat capacity of proteins in different conformations. The results of these studies indicate that three major terms account for the absolute heat capacity of a protein: (1) one term that depends only on the primary or covalent structure of a protein and contains contributions from vibrational frequencies arising from the stretching and bending modes of each valence bond and internal rotations; (2) a term that contains the contributions of noncovalent interactions arising from secondary and tertiary structure; and (3) a term that contains the contributions of hydration. For a typical globular protein in solution the bulk of the heat capacity at 25 degrees C is given by the covalent structure term (close to 85% of the total). The hydration term contributes about 15 and 40% to the total heat capacity of the native and unfolded states, respectively. The contribution of non-covalent structure to the total heat capacity of the native state is positive but very small and does not amount to more than 3% at 25 degrees C. The change in heat capacity upon unfolding is primarily given by the increase in the hydration term (about 95%) and to a much lesser extent by the loss of noncovalent interactions (up to approximately 5%).(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acids↗
Thermodynamic strategies for rational protein and drug design.
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Amino Acid Sequence↗
Thermal denaturation methods in the study of protein folding.
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Calorimetry, Differential Scanning↗
Thermodynamics of partly folded intermediates in proteins.
Until recently, the energetics of protein-folding intermediates eluded direct measurement by high-sensitivity microcalorimetric techniques. But during the past year, the direct measurement of thermodynamic parameters for folding intermediates of alpha-lactalbumin, apomyoglobin, cytochrome c, and staphylococcal nuclease has provided new insights on the nature of the forces involved in the stabilization of nascent protein structures. In this review, I summarize those results and discuss the structural implications of the observed thermodynamic behavior.
Algorithms↗
Differential scanning calorimetry.
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Calorimetry, Differential Scanning↗
[A paradoxical intraventricular gradient in a patient with atypical hypertrophic myocardiopathy].
A paradoxic diastolic intraventricular flow (directed from apex to base), caused by a pressure gradient between a small apical aneurysm and the main left ventricular cavity, connected by a narrow muscular tunnel, was detected by Doppler echocardiography in a 69 year old male with apical hypertrophic cardiomyopathy and effort angina. Coronary arteriography was normal and the clinical, electrocardiographic, echocardiographic and angiocardiographic features of the patient were otherwise typical of apical hypertrophic cardiomyopathy. Paradoxic diastolic gradients have been occasionally described in patients with hypertrophic cardiomyopathy, and indicate the existence of a sequestered apical cavity, which is frequently not detected by echocardiography. Identification of these gradients may have important clinical implications, since they have been related with a higher incidence of embolic events, severe ventricular arrhythmias and myocardial ischaemia and necrosis.
Aged↗
Structure based prediction of protein folding intermediates.
The complete unfolding of a protein involves the disruption of non-covalent intramolecular interactions within the protein and the subsequent hydration of the backbone and amino acid side-chains. The magnitude of the thermodynamic parameters associated with this process is known accurately for a growing number of globular proteins for which high-resolution structures are also available. The existence of this database of structural and thermodynamic information has facilitated the development of statistical procedures aimed at quantifying the relationships existing between protein structure and the thermodynamic parameters of folding/unfolding. Under some conditions proteins do not unfold completely, giving rise to states (commonly known as molten globules) in which the molecule retains some secondary structure and remains in a compact configuration after denaturation. This phenomenon is reflected in the thermodynamics of the process. Depending on the nature of the residual structure that exists after denaturation, the observed enthalpy, entropy and heat capacity changes will deviate in a particular and predictable way from the values expected for complete unfolding. For several proteins, these deviations have been shown to exhibit similar characteristics, suggesting that their equilibrium folding intermediates exhibit some common structural features. Employing empirically derived structure-energetic relationships, it is possible to identify in the native structure of the protein those regions with the higher probability of being structured in equilibrium partly folded states. In this work, a thermodynamic search algorithm aimed at identifying the structural determinants of the molten globule state has been applied to six globular proteins; alpha-lactalbumin, barnase, IIIGlc, interleukin-1 beta, phage T4 lysozyme and phage 434 repressor. Remarkably, the structural features of the predicted equilibrium intermediates coincide to a large extent with the known structural features of the corresponding intermediates determined by NMR hydrogen-exchange experiments.
Bacterial Proteins↗
Thermodynamic and structural stability of cytochrome c oxidase from Paracoccus denitrificans.
The structural stability of the integral membrane protein cytochrome c oxidase from Paracoccus denitrificans has been measured by high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. Contrary to the mammalian enzyme or the yeast enzyme, which are composed of 13 subunits, the bacterial enzyme has only three or four subunits, thus providing a unique opportunity to examine the magnitude of the forces that stabilize this enzyme and to establish accurate structural assignments of events observed calorimetrically. In this paper, experiments have been performed with the wild-type enzyme and with a mutant enzyme lacking subunit III. Our results show that subunits I and II form a highly cooperative complex which denatures as a single cooperative unit at 67 degrees C, while subunit III is less stable and denatures 20 degrees C earlier. Reduction of the enzyme causes a large increase in the stability of subunits I and II but has absolutely no effect on subunit III. Despite the lack of a strong interaction between subunit III and the catalytic subunits, the absence of subunit III leads to a turnover-induced loss of electron-transfer activity. The magnitude of the energetic parameters and the infrared spectroscopic experiments indicate that the enzyme does not completely unfold upon thermal denaturation and that significant degrees of structure are preserved. The amount of native alpha-helix structure, which is 45% in the native state, decreases only to 30% after thermal denaturation. Presumably, the residual helical structure existing after thermal denaturation belongs to the intramembranous portions of the protein. The calorimetric behavior of subunit III does not fully conform to that expected for a highly alpha-helical membrane protein. The picture that emerges from these experiments is that, in the temperature-denatured form of the enzyme, most of the extramembranous structural elements are denatured while most of the intramembranous secondary structure is maintained even though native tertiary interactions appear to be disrupted.
Calorimetry, Differential Scanning↗
Energetics of the alpha-lactalbumin states: a calorimetric and statistical thermodynamic study.
The temperature dependence of the heat capacity function of holo and apo alpha-lactalbumin has been studied by high sensitivity differential scanning microcalorimetry. The heat capacities of the holo and apo forms in the native state were found to be close to, but somewhat higher than, that of lysozyme, which has a similar structure. At pH values higher than 5, the heat-denatured state and the unfolded state are indistinguishable. At lower pH values, the heat capacity of the state obtained by heat or acid denaturation is lower than what is expected for the completely unfolded polypeptide chain, but it approaches that value at higher temperatures. The heat capacity increment of the denatured state correlates well with the amount of residual structure measured by ellipticity (i.e., the lower the residual structure, the higher the heat capacity). The extent of residual structure in the denatured state, which is exceptionally high in alpha-lactalbumin, decreases upon increasing temperature and at approximately 110 degrees C becomes close to that observed in 6 M GdmCl. Above 110 degrees C, the denatured state of alpha-lactalbumin is practically indistinguishable in heat capacity and ellipticity from the fully unfolded state. The calorimetric data have been analyzed quantitatively using a statistically thermodynamic formalism. This analysis indicates that the long-range or global cooperativity of the protein is lost after heat denaturation of the native state, causing the remaining elements of residual structure to behave in a more or less independent fashion. At pH values close to neutral, heat denaturation occurs at high temperature and yields a totally unfolded polypeptide with no measurable population of partly folded intermediates. At lower pH values, denaturation occurs at lower temperatures and a progressively higher population of intermediates is observed. At pH 4.2, about 50% of the molecules is in compact intermediate states immediately after heat denaturation; however, at pH 3.5, this percentage is close to 80% and at pH 3.0 it reaches about 100% of the protein molecules. Upon heating, the unfolded state progressively becomes the predominant species. The analysis of the heat capacity data for alpha-lactalbumin indicates that the best model to account for the observed behavior is one in which the denatured state is represented as a distribution of substates with varying degrees of residual structure. At low temperatures, the distribution is centered around rather compact substates with significant residual structure. At higher temperatures, the distribution shifts toward states with less residual structure and eventually to the completely unfolded state.
Calorimetry, Differential Scanning↗
Thermodynamic strategies for stabilizing intermediate states of proteins.
This paper presents three theorems pertaining to thermodynamic properties of the intermediate (e.g., molten globule) state of proteins exhibiting such a conformation in the presence of GuHCl or urea. The theorems are proved for the three-state case using the denaturant binding model and the linear extrapolation model; their utility is illustrated via applications to examples in the literature. Theorem One states that the denaturant activity that maximizes the population of a partly folded conformation is at any temperature independent of the Gibbs free energy difference between the intermediate and native states. This result holds for both models of protein-denaturant interaction. The second theorem claims that the population maximum is independent of the denaturant association constant for the denaturant binding model. Theorem Three, which also applies to both models considered here, states that at the temperatures corresponding to the extrema in the population of the intermediate, the enthalpy change of the intermediate is equal to the excess enthalpy function, an experimentally accessible quantity. In the absence of denaturant, the enthalpy change of the intermediate state at the population extrema can be written as a function of the thermodynamic parameters of the unfolded state alone. These results, which can be applied to systems of any number of states under certain conditions, should aid in the optimization of conditions employed for experimental studies of partly organized states of proteins.
Drug Stability↗
Thermodynamic characterization of an equilibrium folding intermediate of staphylococcal nuclease.
High-sensitivity differential scanning calorimetry and CD spectroscopy have been used to probe the structural stability and measure the folding/unfolding thermodynamics of a Pro117-->Gly variant of staphylococcal nuclease. It is shown that at neutral pH the thermal denaturation of this protein is well accounted for by a 2-state mechanism and that the thermally denatured state is a fully hydrated unfolded polypeptide. At pH 3.5, thermal denaturation results in a compact denatured state in which most, if not all, of the helical structure is missing and the beta subdomain apparently remains largely intact. At pH 3.0, no thermal transition is observed and the molecule exists in the compact denatured state within the 0-100 degrees C temperature interval. At high salt concentration and pH 3.5, the thermal unfolding transition exhibits 2 cooperative peaks in the heat capacity function, the first one corresponding to the transition from the native to the intermediate state and the second one to the transition from the intermediate to the unfolded state. As is the case with other proteins, the enthalpy of the intermediate is higher than that of the unfolded state at low temperatures, indicating that, under those conditions, its stabilization must be of an entropic origin. The folding intermediate has been modeled by structural thermodynamic calculations. Structure-based thermodynamic calculations also predict that the most probable intermediate is one in which the beta subdomain is essentially intact and the rest of the molecule unfolded, in agreement with the experimental data. The structural features of the equilibrium intermediate are similar to those of a kinetic intermediate previously characterized by hydrogen exchange and NMR spectroscopy.
Calorimetry, Differential Scanning↗
Entropy in biological binding processes: estimation of translational entropy loss.
The loss of translational degrees of freedom makes an important, unfavorable contribution to the free energy of binding. Examination of experimental values suggest that calculation of this entropy using the Sackur-Tetrode equation produces largely overestimated values. Better agreement is obtained using the cratic entropy. Theoretical considerations suggest that the volumes available for the movement of a ligand in solution and in a complex are rather similar, suggesting also that the cratic entropy provides the best estimate of the loss of translational entropy.
Angiotensin II↗
Molecular basis of cooperativity in protein folding. V. Thermodynamic and structural conditions for the stabilization of compact denatured states.
The heat-denatured state of proteins has been usually assumed to be a fully hydrated random coil. It is now evident that under certain solvent conditions or after chemical or genetic modifications, the protein molecule may exhibit a hydrophobic core and residual secondary structure after thermal denaturation. This state of the protein has been called the "compact denatured" or "molten globule" state. Recently is has been shown that alpha-lactalbumin at pH < 5 denatures into a molten globule state upon increasing the temperature (Griko, Y., Freire, E., Privalov, P.L. Biochemistry 33:1889-1899, 1994). This state has a lower heat capacity and a higher enthalpy at low temperatures the stabilization of the molten globule state is of an entropic origin since the enthalpy contributes unfavorably to the Gibbs free energy. Since the molten globule is more structured than the unfolded state and, therefore, is expected to have a lower configurational entropy, the net entropic gain must originate primarily from solvent related entropy arising from the hydrophobic effect, and to a lesser extent from protonation or electrostatic effects. In this work, we have examined a large ensemble of partly folded states derived from the native structure of alpha-lactalbumin in order to identify those states that satisfy the energetic criteria of the molten globule. It was found that only few states satisfied the experimental constraints and that, furthermore, those states were part of the same structural family. In particular, the regions corresponding to the A, B, and C helices were found to be folded, while the beta sheet and the D helix were found to be unfolded. At temperatures below 45 degrees C the states exhibiting those structural characteristics are enthalpically higher than the unfolded state in agreement with the experimental data. Interestingly, those states have a heat capacity close to that observed for the acid pH compact denatured state of alpha-lactalbumin [980 cal (mol.K)-1]. In addition, the folded regions of these states include those residues found to be highly protected by NMR hydrogen exchange experiments. This work represents an initial attempt to model the structural origin of the thermodynamic properties of partly folded states. The results suggest a number of structural features that are consistent with experimental data.
Lactalbumin↗
Estimation of changes in side chain configurational entropy in binding and folding: general methods and application to helix formation.
Theoretical estimations of changes in side chain configurational entropy are essential for understanding the different contributions to the overall thermodynamic behavior of important biological processes like folding and binding. The configurational entropy of any given side chain in any particular protein can be evaluated from the complete energy profile of the side chain. Calculations of the energy profiles can be performed using the side chain single bond dihedrals as the only independent variables as long as the structures at each value of the dihedrals are allowed to relax through small changes in the valence bond angles. The probabilities of different side chain conformers obtained from these energy profiles are very similar to the conformer populations obtained by analysis of side chain preferences in the proteins of the Protein Data Bank. Also, side chain conformational entropies obtained from the energy profiles agree extremely well with those obtained from the Protein Data Bank conformer populations. Changes in side chain configurational entropy in binding and folding can be computed as differences in conformational entropy because, in most cases, the frequency of the rotational oscillation around the energy minimum of any given conformer does not appear to change significantly in the reactions. Changes of side chain conformational entropy calculated in this way were compared with experimental values. The only available experimental data--the effect of side chain substitution on the stability of alpha-helices--were used for this comparison. The experimental values were corrected to subtract the solvent contributions. This comparison yields an excellent agreement between calculated and experimental values, validating not only the theoretical estimates but also the separability of the entropic contributions into configurational terms and solvation related terms.
Amino Acids↗
Estimation of the folding/unfolding energetics of marginally stable proteins using differential scanning calorimetry.
We demonstrate a method for obtaining accurate estimates of the thermodynamic parameter values characteristic of a two-state folding/unfolding transition under conditions in which the onset of cold denaturation prevents the native state from being fully populated at any temperature. This situation occurs for proteins exhibiting low thermal stability, which may be intrinsic, the result of amino acid substitution, or the consequence of protein-solvent interactions (e.g., extremes of pH, the presence of denaturants, extremes of ionic strength). Conventional analysis of calorimetric scans obtained under such conditions yields erroneous values for the enthalpy, entropy, and heat capacity changes characteristic of the folding/unfolding transition of the protein. This paper describes for the first time the weighted average enthalpy function. In contrast to the van't Hoff and calorimetric enthalpies, the weighted average enthalpy yields thermodynamic parameter values which differ by less than 5% form the true value, even for situations in which the population of molecules in the native state at the start of the transition is half of the total.
Calorimetry, Differential Scanning↗