Phenomenological similarities between protein denaturation and small-molecule dissolution: Insights into the mechanism driving the thermal resistance of globular proteins

This article shows that the stability profiles of thermophilic proteins are significantly displaced toward higher temperatures as compared to those of mesophilic proteins. A similar trend characterizes the aqueous transfer of N-alkyl amides. In fact, as a general feature of transfer processes, liquid dissolution profiles are centered at temperatures higher than those of solid ones. This behavior is governed by packing contributions. A partition of the unfolding thermodynamics based on the analysis of phenomenological temperatures common to dissolution and unfolding phenomena provides a clue to understanding the mechanism of thermal stabilization. In fact, the position of stability profiles along the temperature axis does not appear to depend on solvation of internal residues. Instead, it is notably affected by solidlike components, whose progressive decrease appears to drive the heat denaturation temperature increase of most thermostable proteins. As a corollary, it is shown that there are actually two limiting mechanisms of thermal stabilization.     

Use of liquid hydrocarbon and amide transfer data to estimate contributions to thermodynamic functions of protein folding from the removal of nonpolar and polar surface from water.

This extension of the liquid hydrocarbon model seeks to quantify the thermodynamic contributions to protein stability from the removal of nonpolar and polar surface from water. Thermodynamic data for the transfer of hydrocarbons and organic amides from water to the pure liquid phase are analyzed to obtain contributions to the thermodynamics of folding from the reduction in water-accessible surface area. Although the removal of nonpolar surface makes the dominant contribution to the standard heat capacity change of folding (delta C0fold), here we show that inclusion of the contribution from removal of polar surface allows a quantitative prediction of delta C0fold within the uncertainty of the calorimetrically determined value. Moreover, analysis of the contribution of polar surface area to the enthalpy of transfer of liquid amides provides a means of estimating the contributions from changes in nonpolar and polar surface area as well as other factors to the enthalpy of folding (delta H0fold). In addition to estimates of delta H0fold, this extension of the liquid hydrocarbon model provides a thermodynamic explanation for the observation [Privalov, P. L., & Khechinashvili, N. N. (1974) J. Mol. Biol. 86, 665-684] that the specific enthalpy of folding (cal g-1) of a number of globular proteins converges to a common value at approximately 383 K. Because amounts of nonpolar and polar surface area buried by these proteins upon folding are found to be linear functions of molar mass, estimates of both delta C0fold and delta H0fold may be obtained given only the molar mass of the protein of interest.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrogen-bonding classes in proteins and their contribution to the unfolding reaction

This paper proposes to assess hydrogen-bonding contributions to the protein stability, using a set of model proteins for which both X-ray structures and calorimetric unfolding data are known. Pertinent thermodynamic quantities are first estimated according to a recent model of protein energetics based on the dissolution of alkyl amides. Then it is shown that the overall free energy of hydrogen-bond formation accounts for a hydrogen-bonding propensity close to helix-forming tendencies previously found for individual amino acids. This allows us to simulate the melting curve of an alanine-rich helical 50-mer with good precision. Thereafter, hydrogen-bonding enthalpies and entropies are expressed as linear combinations of backbone-backbone, backbone-side-chain, side-chain-backbone, and side-chain-side-chain donor-acceptor contributions. On this basis, each of the four components shows a different free energy versus temperature trend. It appears that structural preference for side-chain-side-chain hydrogen bonding plays a major role in stabilizing proteins at elevated temperatures.     

Water transfer energetics and solid-like packing of globular proteins

In this article we generalize the use of a relationship based on the occurrence of some characteristic temperatures in protein unfolding, which were originally high-lighted for proteins showing the unfolding enthalpy-entropy convergence. On this basis, we show how to dissect the unfolding Gibbs energy of globular proteins in terms of solid-like and liquid-like contributions, untangling the protein energetics by a scheme which does not suffer from excessive intricacy. We also provide an experimental estimate of unfavorable polar contributions to the protein stability, by which it seems possible to evaluate the number of buried residues in individual proteins. A comparison is assessed with the so-called hard sphere model of globular proteins. Results seem to reconcile the view that the protein interior is liquid-like with the observation that protein organization resembles an assembly of closely packed spheres.     

Solid model compounds and the thermodynamics of protein unfolding.

Analysis of thermodynamic data on the dissolution of solid cyclic dipeptides into water in terms of group additivity provides a rationale for the enthalpy and entropy convergence temperatures observed for small globular protein denaturation and the dissolution of model compounds into water. Convergence temperatures are temperatures at which the extrapolated enthalpy or entropy changes for a series of related compounds take on a common value. At these temperatures (TH* and TS*) the apolar contributions to the corresponding thermodynamic values (delta H degrees and delta S degrees) are shown to be zero. Other contributions such as hydrogen bonding and configurational effects can then be evaluated and their quantitative effects on the stability of globular proteins assessed. It is shown that the denaturational heat capacity is composed of a large positive contribution from the exposure of apolar groups and a significant negative contribution from the exposure of polar groups in agreement with previous results. The large apolar contribution suggests that a liquid hydrocarbon model of the hydrophobic effect does not accurately represent the apolar contribution to delta H degrees of denaturation. Rather, significant enthalpic stabilizing contributions are found to arise from peptide groups (hydrogen bonding). Combining the average structural features of globular proteins (i.e. number of residues, fraction of buried apolar groups and fraction of hydrogen bonds) with their specific group contributions permits a first-order prediction of the thermodynamic properties of proteins. The predicted values compare well with literature values for cytochrome c, myoglobin, ribonuclease A and lysozyme. The major thermodynamic features are described by the number of peptide and apolar groups in a given protein.     

Role of hydration water in protein unfolding

In this paper, following our work on the two-state outer neighbor mixed bonding model of water, it is proposed that polar groups promote the formation of the low density ice Ih-type bonding in their neighborhood, whereas nonpolar groups tend to promote the higher density ice II-type structure. In a protein, because of the large numbers of exposed polar and nonpolar groups, large changes in the neighboring water structure can occur. These changes, of course, depend on whether the protein is in its native or its unfolded state and will be shown here to have a direct impact on the thermodynamics of protein unfolding at both high and low temperatures. For example, it is known that the polar hydration entropies become rapidly more negative with increasing temperature. This very unusual behavior can be directly related to the promotion in the outer bulk liquid of the more stable Ih-type bonding at the expense of II-type bonding by polar groups of the protein. In contrast, nonpolar groups have an opposite effect on the thermodynamics. It is the delicate balance created by these outer hydration contributions, mixed with ordinary thermodynamic contributions from the inner hydration shell and those from hydrogen-bond and van der Waals forces within the protein molecule itself that is responsible for both heat and cold denaturation of proteins.     

Effect of redox state on unfolding energetics of heme proteins.

Both the enthalpic and entropic contributions to unfolding of three heme proteins, cytochrome b(562), cytochrome c and myoglobin, are larger for the reduced than for the oxidized form. Thus, the higher thermodynamic stability of a reduced, as compared to an oxidized, heme protein is the net result of a large increase of favorable enthalpy and a small increase in unfavorable entropy. Upon comparing the unfolding energetics of the heme proteins to those of other single-domain proteins I find that protein length is the primary determinant of the thermodynamics.     

Complex of human apolipoprotein C-1 with phospholipid: thermodynamic or kinetic stability?

Thermal unfolding of discoidal complexes of apolipoprotein (apo) C-1 with dimyristoyl phosphatidylcholine (DMPC) reveals a novel mechanism of lipoprotein stabilization that is based on kinetics rather than thermodynamics. Far-UV CD melting curves recorded at several heating/cooling rates from 0.047 to 1.34 K/min show hysteresis and scan rate dependence characteristic of slow nonequilibrium transitions. At slow heating rates, the apoC-1 unfolding in the complexes starts just above 25 degrees C and has an apparent melting temperature T(m) approximately 48 +/- 1.5 degrees C, close to T(m) = 51 +/- 1.5 degrees C of free protein. Thus, DMPC binding may not substantially increase the low apparent thermodynamic stability of apoC-1, DeltaG(25 degrees C) < 2 kcal/mol. The scan rate dependence of T(m) and Arrhenius analysis of the kinetic data suggest an activation enthalpy E(a) = 25 +/- 5 kcal/mol that provides the major contribution to the free energy barrier for the protein unfolding on the disk, DeltaG > or = 17 kcal/mol. Consequently, apoC-1/DMPC disks are kinetically but not thermodynamically stable. To explore the origins of this kinetic stability, we utilized dynode voltage measured in CD experiments that shows temperature-dependent contribution from UV light scattering of apoC-1/DMPC complexes (d approximately 20 nm). Correlation of CD and dynode voltage melting curves recorded at 222 nm indicates close coupling between protein unfolding and an increase in the complex size and/or lamellar structure, suggesting that the enthalpic barrier arises from transient disruption of lipid packing interactions upon disk-to-vesicle fusion. We hypothesize that a kinetic mechanism may provide a general strategy for lipoprotein stabilization that facilitates complex stability and compositional variability in the absence of high packing specificity.     

Studying pressure denaturation of a protein by molecular dynamics simulations

Many globular proteins unfold when subjected to several kilobars of hydrostatic pressure. This “unfolding‐up‐on‐squeezing” is counter‐intuitive in that one expects mechanical compression of proteins with increasing pressure. Molecular simulations have the potential to provide fundamental understanding of pressure effects on proteins. However, the slow kinetics of unfolding, especially at high pressures, eliminates the possibility of its direct observation by molecular dynamics (MD) simulations. Motivated by experimental results—that pressure denatured states are water‐swollen, and theoretical results—that water transfer into hydrophobic contacts becomes favorable with increasing pressure, we employ a water insertion method to generate unfolded states of the protein Staphylococcal Nuclease (Snase). Structural characteristics of these unfolded states—their water‐swollen nature, retention of secondary structure, and overall compactness—mimic those observed in experiments. Using conformations of folded and unfolded states, we calculate their partial molar volumes in MD simulations and estimate the pressure‐dependent free energy of unfolding. The volume of unfolding of Snase is negative (approximately ?60 mL/mol at 1 bar) and is relatively insensitive to pressure, leading to its unfolding in the pressure range of 1500–2000 bars. Interestingly, once the protein is sufficiently water swollen, the partial molar volume of the protein appears to be insensitive to further conformational expansion or unfolding. Specifically, water‐swollen structures with relatively low radii of gyration have partial molar volume that are similar to that of significantly more unfolded states. We find that the compressibility change on unfolding is negligible, consistent with experiments. We also analyze hydration shell fluctuations to comment on the hydration contributions to protein compressibility. Our study demonstrates the utility of molecular simulations in estimating volumetric properties and pressure stability of proteins, and can be potentially extended for applications to protein complexes and assemblies. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Protein reorientation and bound water molecules measured by 1H magnetic spin-lattice relaxation

The water-proton spin-lattice relaxation rate constant, 1/T(1), was measured as a function of magnetic field strength for several dilute protein solutions. By separating the intermolecular contributions from the intramolecular contributions to the water-proton spin-lattice relaxation, the number of water molecules that bind to the protein for a time long compared with the rotational correlation time may be measured. We find a good correlation between the number of long-lived water molecules and the predictions based on available free volume in the proteins studied. The rotational correlation times of these proteins are larger than predicted by the Stokes-Einstein-Debye (SED) model for a sphere reorienting in a viscous liquid. The discrepancy between experiment and theory is usually attributed to hydration effects increasing the effective radius of the particle. However, the average lifetime of water molecules at the protein interface is far too short to justify such a picture. We suggest that surface roughness may be responsible for the retardation of rotational mobility and find that the SED model provides a reasonable representation of experiment if the radius assumed for the reorienting particle is the arithmetic mean of the crystallographic packing radius and the radius deduced from the effective surface area of the protein.     

Analysis of the heat capacity dependence of protein folding.

This paper presents an analysis of plots of enthalpy versus heat capacity change at 25 degrees C for the unfolding of proteins and for the dissolution of gaseous, liquid and solid solutes, first reported by Murphy, Privalov & Gill. The negative slope in the enthalpy plot for proteins is interpreted as arising from a large penalty associated with burying polar groups in the protein interior. The small enthalpy changes that accompany protein unfolding at 25 degrees C are also discussed. It is argued that the combined effects of hydrogen bond formation and close packing predict a large positive enthalpy of unfolding. Electrostatic calculations indicate that the penalty associated with burying polar groups is large enough to effectively cancel these terms, leading to the small net enthalpy changes that are observed. The free energy changes associated with protein folding are also discussed. The free energy cost of burying polar groups largely compensates for the stabilizing contribution of the hydrophobic effect and would appear to account for the fact that proteins are marginally stable, independent of their size and of their relative hydrophobicities.     

Molecular-size corrections to the strength of the hydrophobic effect: a critical review

Large increases in the strength of the hydrophobic effect and, consequently, in the estimates of the hydrophobic contribution to the thermodynamics of protein folding (and other biologically-relevant processes), have been recently advocated on the basis of the application, to model transfer thermodynamic data, of corrections for the solute/solvent size disparity. In this work we first examine the effect of molecular-size corrections on the values calculated from several types of model transfer data. For the transfer of a solute from an organic solvent to water, the above increase is exclusively associated with the application of a solute/water molecular-size correction. Secondly, we critically review and assess the several theoretical arguments that lead to these corrections. In particular, we show that, contrary to previous claims in the literature, the analysis of dissolution processes in terms of ideal-gas, intermediate states does not lead to the molecular-size correction term, but only to expressions equivalent (although not strictly identical) to those derived from the well-known Ben-Naim's statistical-mechanical approach. In general, the several analyses offered or discussed in this work disfavor the application of the solute/water molecular-size corrections.     

Incorporation of cationic chains in the Dickerson-Drew dodecamer: correlation of energetics, structure, and ion and water binding

We have investigated the unfolding thermodynamics for incorporating cationic side chains in the Dickerson-Drew dodecamer duplex. Incorporation of two 3-aminopropyl-2'-deoxyuridine residues (one on each self-complementary strand) lowers the stability of the duplex. This reduction is driven by unfavorable heat contributions due to the removal of electrostricted water and higher exposure of polar and nonpolar atomic groups that immobilize structural water. These cationic chains effectively remove counterions from the major groove, neutralizing some negatively charged phosphates. The overall results are consistent with the NMR solution of the modified duplex that showed a small bend at each modified site.     

Heat capacity of hydrogen-bonded networks: an alternative view of protein folding thermodynamics

Large changes in heat capacity (deltaCp) have long been regarded as the characteristic thermodynamic signature of hydrophobic interactions. However, similar effects arise quite generally in order-disorder transitions in homogeneous systems, particularly those comprising hydrogen-bonded networks, and this may have significance for our understanding of protein folding and other biomolecular processes. The positive deltaCp associated with unfolding of globular proteins in water, thought to be due to hydrophobic interactions, is also typical of the values found for the melting of crystalline solids, where the effect is greatest for the melting of polar compounds, including pure water. This suggests an alternative model of protein folding based on the thermodynamics of phase transitions in hydrogen-bonded networks. Folded proteins may be viewed as islands of cooperatively-ordered hydrogen-bonded structure, floating in an aqueous network of less-well-ordered H-bonds in which the degree of hydrogen bonding decreases with increasing temperature. The enthalpy of melting of the protein consequently increases with temperature. A simple algebraic model, based on the overall number of protein and solvent hydrogen bonds in folded and unfolded states, shows how deltaCp from this source could match the hydrophobic contribution. This confirms the growing view that the thermodynamics of protein folding, and other interactions in aqueous systems, are best described in terms of a mixture of polar and non-polar effects in which no one contribution is necessarily dominant.     

Direct access to the cooperative substructure of proteins and the protein ensemble via cold denaturation

The modern view of protein thermodynamics predicts that proteins undergo cold-induced unfolding. Unfortunately, the properties of proteins and water conspire to prevent the detailed observation of this fundamental process. Here we use protein encapsulation to allow cold denaturation of the protein ubiquitin to be monitored by high-resolution NMR at temperatures approaching -35 degrees C. The cold-induced unfolding of ubiquitin is found to be highly noncooperative, in distinct contrast to the thermal melting of this and other proteins. These results demonstrate the potential of cold denaturation as a means to dissect the cooperative substructures of proteins and to provide a rigorous framework for testing statistical thermodynamic treatments of protein stability, dynamics and function.     

A convenient approach to synthesizing peptide C-terminal N-alkyl amides

Peptide C-terminal N-alkyl amides have gained more attention over the past decade due to their biological properties, including improved pharmacokinetic and pharmacodynamic profiles. However, the synthesis of this type of peptide on solid phase by current available methods can be challenging. Here we report a convenient method to synthesize peptide C-terminal N-alkyl amides using the well-known Fukuyama N-alkylation reaction on a standard resin commonly used for the synthesis of peptide C-terminal primary amides, the peptide amide linker-polyethylene glycol-polystyrene (PAL-PEG-PS) resin. The alkylation and oNBS deprotection were conducted under basic conditions and were therefore compatible with this acid labile resin. The alkylation reaction was very efficient on this resin with a number of different alkyl iodides or bromides, and the synthesis of model enkephalin N-alkyl amide analogs using this method gave consistently high yields and purities, demonstrating the applicability of this methodology. The synthesis of N-alkyl amides was more difficult on a Rink amide resin, especially the coupling of the first amino acid to the N-alkyl amine, resulting in lower yields for loading the first amino acid onto the resin. This method can be widely applied in the synthesis of peptide N-alkyl amides.     

Additive transfer free energies of the peptide backbone unit that are independent of the model compound and the choice of concentration scale

With knowledge of individual transfer free energies of chemical groups that become newly exposed on protein denaturation and assuming the group transfer free energy contributions are additive, it should be possible to predict the stability of a protein in the presence of denaturant. Unfortunately, several unresolved issues have seriously hampered quantitative development of this transfer model for protein folding/unfolding. These issues include the lack of adequate demonstration that group transfer free energies (DeltaG(tr)) are additive and independent of the choice of model compound, the problem arising from dependence of DeltaG(tr) on concentration scales, the lack of knowledge of activity coefficients, and the validity of the mathematical constructs used in obtaining DeltaG(tr) values. Regarding transfer from water to 1 M concentrations of the naturally occurring osmolytes, trimethylamine-N-oxide (TMAO), sarcosine, betaine, proline, glycerol, sorbitol, sucrose, trehalose, and urea, using cyclic glycylglycine, zwitterionic glycine peptides, and N-acetylglycine amide peptides as models for the peptide backbone of proteins, we set out to address these issues and obtain DeltaG(tr) values for the peptide backbone unit. We demonstrate experimental approaches that obviate the choice of concentration scale and demonstrate additivity in DeltaG(tr) of the peptide backbone unit for all solvent systems studied. Evidence is presented to show that the DeltaG(tr) values are independent of the chemical model studied, and experimental conditions are given to illustrate when the mathematical constructs are valid and when activity coefficients can be ignored. Resolution of the long-standing issues that have stymied development of the transfer model now make it possible to design transfer experiments that yield reliable and quantitative values for the interactions between osmolyte-containing solvents and native and unfolded protein.     

Between-species variation in the kinetic stability of TIM proteins linked to solvation-barrier free energies

Theoretical, computational, and experimental studies have suggested the existence of solvation barriers in protein unfolding and denaturation processes. These barriers are related to the finite size of water molecules and can be envisioned as arising from the asynchrony between water penetration and breakup of internal interactions. Solvation barriers have been proposed to play roles in protein cooperativity and kinetic stability; therefore, they may be expected to be subject to natural selection. We study the thermal denaturation, in the presence and in the absence of chemical denaturants, of triosephosphate isomerases (TIMs) from three different species: Trypanosoma cruzi, Trypanosoma brucei, and Leishmania mexicana. In all cases, denaturation was irreversible and kinetically controlled. Surprisingly, however, we found large differences between the kinetic denaturation parameters, with T. cruzi TIM showing a much larger activation energy value (and, consequently, much lower room-temperature, extrapolated denaturation rates). This disparity cannot be accounted for by variations in the degree of exposure to solvent in transition states (as measured by kinetic urea m values) and is, therefore, to be attributed mainly to differences in solvation-barrier contributions. This was supported by structure-energetics analyses of the transition states and by application of a novel procedure to estimate from experimental data the solvation-barrier impact at the entropy and free-energy levels. These analyses were actually performed with an extended protein set (including six small proteins plus seven variants of lipase from Thermomyces lanuginosus and spanning a wide range of activation parameters), allowing us to delineate the general trends of the solvation-barrier contributions. Overall, this work supports that proteins sharing the same structure and function but belonging to different organisms may show widely different solvation barriers, possibly as a result of different levels of the selection pressure associated with cooperativity, kinetic stability, and related factors.     

Empirical free energy calculation: comparison to calorimetric data.

An effective free energy potential, developed originally for binding free energy calculation, is compared to calorimetric data on protein unfolding, described by a linear combination of changes in polar and nonpolar surface areas. The potential consists of a molecular mechanics energy term calculated for a reference medium (vapor or nonpolar liquid), and empirical terms representing solvation and entropic effects. It is shown that, under suitable conditions, the free energy function agrees well with the calorimetric expression. An additional result of the comparison is an independent estimate of the side-chain entropy loss, which is shown to agree with a structure-based entropy scale. These findings confirm that simple functions can be used to estimate the free energy change in complex systems, and that a binding free energy evaluation model can describe the thermodynamics of protein unfolding correctly. Furthermore, it is shown that folding and binding leave the sum of solute-solute and solute-solvent van der Waals interactions nearly invariant and, due to this invariance, it may be advantageous to use a nonpolar liquid rather than vacuum as the reference medium.