Role of hydrophobic and polar interactions for BSA-amphiphile composites

To evaluate the role of hydrophobic and electrostatic or other polar interactions for protein–ligand binding, we have studied the interactions of bovine serum albumin (BSA) with 2-alkylmalonic acid and 2-alkylbenzimidazole amphiphiles having different head group and alkyl chain length. The binding affinity for the protein–amphiphile interactions is found to depend predominantly on the length of hydrocarbon chain, suggesting the crucial role of hydrophobic forces, supported by polar interactions at the protein surface. The BSA fluorescence exhibits appreciable hypsochromic shift along with a reduction in fluorescence intensity and mean lifetime upon binding with 2-alkylmalonic acid. UV–visible, steady state and time-resolved fluorescence measurements were performed to compare the effects of amphiphiles on BSA as a function of the amphiphiles head group and alkyl chain length.     

Crystallographic studies of protein denaturation and renaturation. 1. Effects of denaturants on volume and X-ray pattern of cross-linked triclinic lysozyme crystals

Triclinic crystals of hen egg-white lysozyme cross-linked with glutaraldehyde have been treated with various denaturants and found to be susceptible to x-ray structure analysis even after major conformational changes in the protein. Cross-linked crystals were isomorphous with the native form, and electron density difference maps indicated the locations of intermolecular corss-links, but showed no appreciable differences in the protein conformation. Soaking of the cross-linked crystals in danaturant solutions of increasing concentrations caused corresponding increases in crystal volume and decreases in minimum observable x-ray spacings. These changes proved partly reversible on diluting the solutions, and measurements of crystal volume and minimums x-ray spacing were used to follow denaturation and renaturation as a function of concentration for several denaturants. Some of these, including bromoethanol and sodium dodecyl sulfate, had little effect on the crystals below critical concentrations at which there was a sharp volume increase and loss of x-ray pattern, which could, however, be regenerated to about 3.2-A resolution. Others, including KCNS and urea, caused more gradual changes, but with a smaller degree of recovery. It is suggested that at least two different denaturation mechanisms are involved with detergent-like reagents disrupting the hydrophobic interactions joining the two wings of the lysozyme molecule and hydrophilic denaturants interacting primarily with polar groups on the molecular surface.     

Interaction stabilizing tertiary structure of bacteriorhodopsin studied by denaturation experiments

The structural stability of bacteriorhodopsin was studied by denaturation experiments, using aliphatic alcohol as denaturants. The disappearance of a positive peak at 285 nm of the circular dichroism spectra, the change in the intrinsic fluorescence decay time, and the decrease of the regeneration activity bacteriorhodopsin indicated the denaturation of the tertiary structure of this protein at a methanol concentration of about 3 M. The circular dichroism band at 222 nm was unchanged by the denaturation. It was concluded that the alcohol-denatured state in water was similar to the molten globule state of soluble proteins, in which only the tertiary structure was destroyed. Solvent substitution from water to hexane did not cause denaturation of bacteriorhodopsin. However, further addition of alcohol destroyed the secondary as well as the tertiary structures. Comparing the alcohol effects on bacteriorhodopsin in water to that in hexane, the dominant interactions for the structure formation of this protein could be revealed: the hydrophobic interaction that arose from the structure of water is essential for the stability of membrane spanning helices, while the interaction which binds the helices is polar in nature. © 1995 Wiley-Liss, Inc.     

Denaturation of bacteriorhodopsin by organic solvents

The denaturation of bacteriorhodopsin by various organic solvents was studied using absorption, circular dichroism (CD) and fluorescence measurements. Organic solvents with a hydrogen-bonding group caused the release of retinal. The CD measurements showed that the helical structure was maintained even in the denatured state, whereas its tertiary structure was destroyed. The change in fluorescence intensity of tryptophan and fluorescent retinal also confirmed that the tertiary structure was destroyed. Comparison of the denaturation efficiency of various organic solvents showed that the concentration at denaturation was inversely proportional to the partition coefficient of the denaturant. This inverse proportionality clearly indicated that denaturation was determined by the concentration of denaturants which partitioned into the hydrophobic region of the membrane. It was discussed from the experimental results that the tertiary structure of bacteriorhodopsin was stabilized by the hydrogen-bonding networks between side chains of the helices. The results obtained from analysis of the amino acid sequence were also consistent with the hydrogen-bonding mechanism for the formation of the tertiary structure.     

Recent Applications of Kirkwood–Buff Theory to Biological Systems

The effect of cosolvents on biomolecular equilibria has traditionally been rationalized using simple binding models. More recently, a renewed interest in the use of Kirkwood–Buff (KB) theory to analyze solution mixtures has provided new information on the effects of osmolytes and denaturants and their interactions with biomolecules. Here we review the status of KB theory as applied to biological systems. In particular, the existing models of denaturation are analyzed in terms of KB theory, and the use of KB theory to interpret computer simulation data for these systems is discussed.     

Matrix proteins from insect pliable cuticles: are they flexible and easily deformed?

Proteins from pliable cuticle of locusts, Schistocerca gregaria, and silk moth larvae, Hyalophora cecropia, were studied in solution by means of a fluorescent probe, 8-anilinonaphthalene-1-sulphonic acid (ANS), which is much more fluorescent in non-polar media than in polar media. An intense ANS-fluorescence was observed in the presence of the cuticular proteins at pH-values close to their acidic isoelectric points, and the fluorescence decreased markedly when pH was increased to neutrality or when small amounts of denaturants were added. Aggregation and eventual precipitation of both H. cecropia and locust proteins were obtained by addition of neutral salts, and the aggregation was accompanied by an increased ANS-fluorescence intensity. A decreased ANS-fluorescence was observed at salt concentrations too low to cause visible aggregation of the H. cecropia proteins, probably due to weakened electrostatic interactions between chain segments, but such a decrease was not observed for the locust proteins. The changes in intensity of ANS-fluorescence induced by addition of small amounts of denaturants or salts to solutions of the proteins indicate that more hydrophobic residues are exposed to the solvent, when either hydrophobic interactions or electrostatic attractions between chain segments are weakened. The result is a less compact protein structure, where fewer and smaller hydrophobic clusters are available for protecting ANS-molecules from the quenching effects of water. The effects of denaturants on ANS-fluorescence in the presence of the cuticular proteins are different from those observed for globular proteins, such as hen egg albumen, and the differences can be explained by the suggestion that the cuticular proteins do not have a precisely folded and densely packed hydrophobic core comparable to that present in native globular proteins, and that accordingly they do not undergo a process of denaturation corresponding to that of globular proteins. The behaviour of the cuticular proteins resembles that described for unordered, randomly coiled, thermally agitated polymer chains, whose hydrodynamic volumes depend upon the composition of the medium. It is proposed that the major part of the peptide chains of the cuticular proteins are in an unordered, random structure both when the proteins are in solution and when present in the intact cuticle; probably only the chain regions involved in binding the proteins to chitin will have a well-defined spatial organisation.     

Thermodynamic stability of ribonuclease A in alkylurea solutions and preferential solvation changes accompanying its thermal denaturation: a calorimetric and spectroscopic study

The effect of methylurea, N,N'-dimethylurea, ethylurea, and butylurea as well as guanidine hydrochloride (GuHCl), urea and pH on the thermal stability, structural properties, and preferential solvation changes accompanying the thermal unfolding of ribonuclease A (RNase A) has been investigated by differential scanning calorimetry (DSC), UV, and circular dichroism (CD) spectroscopy. The results show that the thermal stability of RNase A decreases with increasing concentration of denaturants and the size of the hydrophobic group substituted on the urea molecule. From CD measurements in the near- and far-UV range, it has been observed that the tertiary structure of RNase A melts at about 3 degrees C lower temperature than its secondary structure, which means that the hierarchy in structural building blocks exists for RNase A even at conditions at which according to DSC and UV measurements the RNase A unfolding can be interpreted in terms of a two-state approximation. The far-UV CD spectra also show that the final denatured states of RNase A at high temperatures in the presence of different denaturants including 4.5 M GuHCl are similar to each other but different from the one obtained in 4.5 M GuHCl at 25 degrees C. The concentration dependence of the preferential solvation change delta r23, expressed as the number of cosolvent molecules entering or leaving the solvation shell of the protein upon denaturation and calculated from DSC data, shows the same relative denaturation efficiency of alkylureas as other methods.     

Pseudomonas aeruginosa cytochrome c551 denaturation by five systematic urea derivatives that differ in the alkyl chain length

Reversible denaturation of Pseudomonas aeruginosa cytochrome c₅₅₁ (PAc₅₅₁) could be followed using five systematic urea derivatives that differ in the alkyl chain length, i.e. urea, N-methylurea (MU), N-ethylurea (EU), N-propylurea (PU), and N-butylurea (BU). The BU concentration was the lowest required for the PAc₅₅₁ denaturation, those of PU, EU, MU, and urea being gradually higher. Furthermore, the accessible surface area difference upon PAc₅₅₁ denaturation caused by BU was found to be the highest, those by PU, EU, MU, and urea being gradually lower. These findings indicate that urea derivatives with longer alkyl chains are stronger denaturants. In this study, as many as five systematic urea derivatives could be applied for the reversible denaturation of a single protein, PAc₅₅₁, for the first time, and the effects of the alkyl chain length on protein denaturation were systematically verified by means of thermodynamic parameters.     

Structural changes and fluctuations of proteins. II. Analysis of the denaturation of globular proteins.

The statistical thermodynamic model of protein structure proposed in paper I is developed with special attention to the hydrophobic interaction. Calorimetric measurements of the thermal denaturation of five globular proteins, ribonuclease A, lysozyme, alpha-chymotrypsin, cytochrome c, and myoglobin, are quantitatively analyzed using the model. The thermodynamic parameters obtained by the least squares method reflect the global, average properties of proteins and are in good agreement with the expected values estimated from experimental and theoretical studies for model peptides. The average bond energy epsilon is well related to the tertiary structure of each protein. However, the difference in the parameters between different proteins is not observed for the cooperative energy ZJ and the chain entropy alpha. The individuality of a protein as far as its structural stability is concerned, is mainly reflected by the parameter gamma specifying the hydrophobic nature of a protein. The model is further applied in the analysis of several aspects of the structural stability of globular proteins. Denaturation induced by denaturants, salts, and pH are also explained by the model in a unified manner.     

Acceleration of phospholipid flip-flop in the erythrocyte membrane by detergents differing in polar head group and alkyl chain length

The detergents, alkyltrimethylammonium bromide, N-alkyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (zwittergent), alkane sulfonate, alkylsulfate, alkyl-beta-D-glucopyranoside, alkyl-beta-D-maltoside, dodecanoyl-N-methylglucamide, polyethylene glycol monoalkyl ether and Triton X-100, all produce a concentration-dependent acceleration of the slow passive transbilayer movement of NBD-labeled phosphatidylcholine in the human erythrocyte membrane. Above a threshold concentration, which was well below the CMC and characteristic for each detergent, the flip rate increases exponentially upon an increase of the detergent concentration in the medium. The detergent-induced flip correlates with reported membrane-expanding effects of the detergents at antihemolytic concentrations. From the dependence of the detergent concentration required for a defined flip acceleration on the estimated membrane volume, membrane/water partition coefficients for the detergents could be determined and effective detergent concentrations in the membrane calculated. The effective membrane concentrations are similar for most types of detergents but are 10-fold lower for octaethylene glycol monoalkyl ether and Triton X-100. The effectiveness of a given type of detergent is rather independent of its alkyl chain length. Since detergents do not reduce the high temperature dependence of the flip process the detergent-induced flip is proposed to be due to an enhanced probability of formation of transient hydrophobic structural defects in the membrane barrier which may result from perturbation of the interfacial region of the bilayer by inserted detergent molecules.     

Partitioning behavior of indocarbocyanine probes between coexisting gel and fluid phases in model membranes

Gel-fluid partition coefficients, Kp, were measured for a series of indocarbocyanine dyes in multilamellar lipid vesicles. The dyes examined had alkyl chain lengths from 12 to 22 carbons. Fluorescence quenching by a spin-labeled phosphatidylcholine-enriched fluid phase created a large difference in quantum yield for indocarbocyanine fluorescence between fluid and gel phases, enabling reliable Kp determinations. The values range from Kp = 8 for the 12-carbon chain, favoring a fluid phase over a Ca2-phosphatidylserine rigid phase, to Kp = 0.02 for the 20-carbon chain dye, favoring a distearoylphosphatidylcholine-rich gel phase over the fluid phase.     

A new method for determining the constant-pressure heat capacity change associated with the protein denaturation induced by guanidinium chloride (or urea)

Differential scanning calorimetry (DSC) provides authentic and accurate value of DeltaC(p)(X), the constant-pressure heat capacity change associated with the N (native state)<-->X (heat denatured state), the heat-induced denaturation equilibrium of the protein in the absence of a chemical denaturant. If X retains native-like buried hydrophobic interaction, DeltaC(p)(X) must be less than DeltaC(p)(D), the constant-pressure heat capacity change associated with the transition, N<-->D, where the state D is not only more unfolded than X but it also has its all groups exposed to water. One problem is that for most proteins D is observed only in the presence of chemical denaturants such as guanidinium chloride (GdmCl) and urea. Another problem is that DSC cannot yield authentic DeltaC(p)(D), for its measurement invokes the existence of putative specific binding sites for the chemical denaturants on N and D. We have developed a non-calorimetric method for the measurements of DeltaC(p)(D), which uses thermodynamic data obtained from the isothermal GdmCl (or urea)-induced denaturation and heat-induced denaturation in the presence of the chemical denaturant concentration at which significant concentrations of both N and D exist. We show that for each of the proteins (ribonuclease-A, lysozyme, alpha-lactalbumin and chymotrypsinogen) DeltaC(p)(D) is significantly higher than DeltaC(p)(X). DeltaC(p)(D) of the protein is also compared with that estimated using the known heat capacities of amino acid residues and their fractional area exposed on denaturation.     

Free energy changes in denaturation of ribonuclease A by mixed denaturants. Effects of combinations of guanidine hydrochloride and one of the denaturants lithium bromide, lithium chloride, and sodium bromide

The denaturation of ribonuclease A by guanidine hydrochloride, lithium bromide, and lithium chloride and by mixed denaturants consisting of guanidine hydrochloride and one of the denaturants lithium chloride, lithium bromide, and sodium bromide was followed by difference spectral measurements at pH 4.8 and 25 degrees C. Both components of mixed denaturant systems enhance each other's effect in unfolding the protein. The effect of lithium bromide on the midpoint of guanidine hydrochloride denaturation transition is approximately the sum of the effects of the constituent ions. For all the mixed denaturants tested, the dependence of the free energy change on denaturation is linear. The conformational free energy associated with the guanidine hydrochloride denaturation transition in water is 7.5 +/- 0.1 kcal mol-1, and it is unchanged in the presence of low concentrations of lithium bromide, lithium chloride, and sodium bromide which by themselves are not concentrated enough to unfold the protein. The conformational free energy associated with the lithium bromide denaturation transition in water is 11.7 +/- 0.3 kcal mol-1, and it is not affected by the presence of low concentrations of guanidine hydrochloride which by themselves do not disrupt the structure of native ribonuclease A.     

Induction of protein-like molecular architecture by monoalkyl hydrocarbon chains

Numerous approaches have been described for creating relatively small folded biomolecular structures. "Peptide-amphiphiles," whereby monoalkyl or dialkyl hydrocarbon chains are covalently linked to peptide sequences, have been shown previously to form specific molecular architecture of enhanced stability. The present study has examined the use of monoalkyl hydrocarbon chains as a more general method for inducing protein-like structures. Peptide and peptide-amphiphiles have been characterized by CD and one- and two-dimensional nmr spectroscopic techniques. We have examined two structural elements: alpha-helices and collagen-like triple helices. The alpha-helical propensity of a 16-residue peptide either unmodified or acylated with a C(6) or C(16) monoalkyl hydrocarbon chain has been examined initially. The 16-residue peptide alone does not form a distinct structure in solution, whereas the 16-residue peptide adopts predominantly an alpha-helical structure in solution when a C(6) or C(16) monoalkyl hydrocarbon chain is N-terminally acylated. The thermal stability of the alpha-helix is greater upon addition of the C(16) compared with the C(6) chain, which correlates to the extent of aggregation induced by the respective hydrocarbon chains. Very similar results are seen using a 39-residue triple-helical model peptide, in that structural thermal stability (a) is increasingly enhanced as alkyl chain length is increased and (b) correlates to the extent of peptide-amphiphile aggregation. Overall, structures as diverse as alpha-helices, triple helices, and turns/loops have been shown to be induced and/or stabilized by alkyl chains. Increasing alkyl chain length enhances stability of the structural element and induces aggregates of defined sizes. Hydrocarbon chains may be useful as general tools for protein-like structure initiation and stabilization as well as biomaterial modification.     

Synthesis of benzopyran derivatives as PPARα and/or PPARγ activators

We describe the synthesis of 26 compounds, small polycerasoidol analogs, that are Lipinski’s rule-of-five compliant. In order to confirm key structural features to activate PPARα and/or PPARγ, we have adopted structural modifications in the following parts: (i) the benzopyran core (hydrophobic nucleus) by benzopyran-4-one, dihydrobenzopyran or benzopyran-4-ol; (ii) the side chain at 2-position by shortening to C3, C4 and C5-carbons versus C-9-carbons of polycerasoidol; (iii) the carboxylic group (polar head) by oxygenated groups (hydroxyl, acetoxy, epoxide, ester, aldehyde) or non-oxygenated motifs (allyl and alkyl). Benzopyran-4-ones 6, 12, 13 and 17 as well as dihydrobenzopyrans 22, 24 and 25 were able to activate hPPARα, whereas benzopyran-4-one (7) with C5-carbons in the side chain exhibited hPPARγ agonism. According to our previous docking studies, SAR confirm that the hydrophobic nucleus (benzopyran-4-one or dihydrobenzopyran) is essential to activate PPARα and/or PPARγ, and the flexible linker (side alkyl chain) should containg at least C5-carbon atoms to activate PPARγ. By contrast, the polar head (“carboxylic group”) tolerated several oxygenated groups but also non-oxygenated motifs. Taking into account these key structural features, small polycerasoidol analogs might provide potential active molecules useful in the treatment of dyslipidemia and/or type 2 diabetes.     

Conformational and thermodynamic characterization of the molten globule state occurring during unfolding of cytochromes-c by weak salt denaturants

The denaturation of bovine and horse cytochromes-c by weak salt denaturants (LiCl and CaCl(2)) was measured at 25 degrees C by observing changes in molar absorbance at 400 nm (Delta epsilon(400)) and circular dichroism (CD) at 222 and 409 nm. Measurements of Delta epsilon(400) and mean residue ellipticity at 409 nm ([theta](409)) gave a biphasic transition for both modes of denaturation of cytochromes-c. It has been observed that the first denaturation phase, N (native) conformation <--> X (intermediate) conformation and the second denaturation phase, X conformation <--> D (denatured) conformation are reversible. Conformational characterization of the X state by the far-UV CD, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements led us to conclude that the X state is a molten globule state. Analysis of denaturation transition curves for the stability of different states in terms of Gibbs energy change at pH 6.0 and 25 degrees C led us to conclude that the N state is more stable than the X state by 9.55 +/- 0.32 kcal mol(-1), whereas the X state is more stable than the D state by only 1.40 +/- 0.25 kcal mol(-1). We have also studied the effect of temperature on the equilibria, N conformation <--> X conformation and X conformation <--> D conformation in the presence of different denaturant concentrations using two different optical probes, namely, [theta](222) and Delta epsilon(400). These measurements yielded T(m), (midpoint of denaturation) and Delta H(m) (enthalpy change) at T(m) as a function of denaturant concentration. A plot of Delta H(m) versus corresponding T(m) was used to determine the constant-pressure heat capacity change, Delta C(p) (= ( partial differential Delta H(m)/ partial differential T(m))(p)). Values of Delta C(p) for N conformation <--> X conformation and X conformation <--> D conformation is 0.92 +/- 0.02 kcal mol(-1) K(-1) and 0.41 +/- 0.01 kcal mol(-1) K(-1), respectively. These measurements suggested that about 30% of the hydrophobic groups in the molten globule state are not accessible to the water.     

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.     

On the edge of the denaturation process: application of X-ray diffraction to barnase and lysozyme cross-linked crystals with denaturants in molar concentrations

Structural data about the early step of protein denaturation were obtained from cross-linked crystals for two small proteins: barnase and lysozyme. Several denaturant agents like urea, bromoethanol or thiourea were used at increasing concentrations up to a limit leading to crystal disruption (>or=2 to 6 M). Before the complete destruction of the crystal order started, specific binding sites were observed at the protein surfaces, an indication that the preliminary step of denaturation is the disproportion of intermolecular polar bonds to the benefit of the agent "parasiting" the surface. The analysis of the thermal factors first agree with a stabilization effect at low or moderate concentration of denaturants rapidly followed by a destabilization at specific weak points when the number of sites increase (overflooding effect).     

Mutant forms of staphylococcal nuclease with altered patterns of guanidine hydrochloride and urea denaturation

Eleven mutant forms of staphylococcal nuclease with one or more defined amino acid substitutions have been analyzed by solvent denaturation by using intrinsic fluorescence to follow the denaturation reaction. On the basis of patterns observed in the value of m--the rate of change of log Kapp (the apparent equilibrium constant between the native and denatured states) with denaturant concentration--these proteins can be grouped into two classes. For class I mutants, the value of m with guanidine hydrochloride is less than the wild-type value and is either constant or increases slightly with increasing denaturant; the value of m with urea is also less than wild type but shows a marked increase with increasing denaturant concentration, often approaching but never exceeding the wild-type value. For class II mutants, m is constant and is greater than wild type in both denaturants, with the increase being consistently larger in guanidine hydrochloride than in urea. When double or triple mutants are constructed from members of the same mutant class, the change in m is usually the sum of the changes produced by each mutation in isolation. One plausible explanation for these altered patterns of denaturation is that chain-chain or chain-solvent interactions in the denatured state have been modified--interactions which appear to involve hydrophobic groups.