Solubility of proteins

A theory is presented on the solubility of proteins, in the hydrated as well as in the dry state, and in water as well as in organic solvents. To this effect, colloidal stability is assimilated with the solubility of the proteins, considered as hydrated entities. By means of a surface thermodynamic approach it can be shown that an increase in size of a hydrated protein must lead to insolubility, even in the absence of any change in a protein's surface properties. This can be substantiated experimentally by comparing the surface properties of immune complexes with those of their constituent immunoglobulins, as well as by comparing some of the properties of intact tobacco mosaic virus with those of its monomeric capsid subunits. Insolubilization of proteins by means of charge interactions as well as by dehydration is studied; an explanation is given of why precipitation caused by charge interactions is more likely to lead to partial irreversible denaturation than precipitation caused by protein-protein interactions brought about by partial dehydration (e.g., by “salting-out”). A link is established between the smallness (or even the negative value) of the interfacial tension between given proteins and various solvents and their solubility in these solvents. The energy of hydration of proteins can also be measured, and the differences between the free energies of interaction of dried and hydrated proteins with water point toward the additional processes underlying the solubilization, i.e., toward the conformational change of a protein in the process of becoming hydrated. The parameter of conformational change of a protein, while becoming hydrated, appears to be more closely linked to its degree of hydration than to its hydration energy.     

Orientation of the water molecules of hydration of human serum albumin

Through contact-angle measurements with a number of liquids, on layers of hydrated human serum albumin (HSA), built on anisotropic ultrafilter membranes, the apolar, Lifshitz-van der Waals surface tension component, as well as the polar, electron-acceptor and electron-donor parameters of the hydrated layers could be determined. From these data, it was found that the degree of orientation of the water molecules of hydration of HSA is 98% in the first layer of hydration and 30% of the second layer. The water molecules of hydration are oriented with the H atoms closest to, and the O atoms farthest from, the protein surface.     

Surface thermodynamic properties and chromatographic and salting-out behavior of IgA and other serum proteins

By means of contact angle determinations with two liquids, on hydrated as well as on dried protein layers, the long-range and the short-range contributions to the protein surface tensions, and from these the protein (G ₁₃₁) and the protein-ligand (G ₁₃₂) free energies of interaction in aqueous media, were determined. For human serum albumin (HSA), human IgG, and human IgA, the differences between G ₁₃₁ ^HYDRATED and G ₁₃₁ ^DRY were connected with the behavior of these proteins in low concentrations of (NH₄)₂SO₄ versus saturated (NH₄)₂SO₄ solutions. By interpolation, intermediate points are found that correlate well with the known salting-out properties of these three proteins. On the basis of the data, it is predicted that the precipitation of IgG by 1/3 saturated (NH₄)₂SO₄ is preventable, or reversible, by the admixture of 15% dimethylsulfoxide; both predictions are confirmed experimentally. From the G ₁₃₂ values found, it is shown that HSA and IgG should attach to phenyl ligands under physiological conditions, but that IgA is so hydrophilic that it only can adhere to phenyl ligands after partial dehydration brought about when admixed with 1 M (NH₄)₂SO₄. Closer analysis of the values obtained for the long-range and short-range components of the surface tensions of HSA, IgG, and IgA allow deeper insight into their functional, chemical, and physicochemical properties.     

Induction of changes in the secondary structure of globular proteins by a hydrophobic surface

Circular dichroism, ellipsometry and radiolabeling techniques were employed to study the induction of changes in the secondary structure of BSA, myoglobin and cytochrome C by a hydrophobic surface. The results showed that adsorbed protein molecules lose their ordered native structure in the initial stage of adsorption and the structure appears to be a random or disordered conformation. Protein molecules adsorbed in later stages adopt a more ordered secondary structure ( helix and structure). The changes of secondary structure of globular proteins induced by a hydrophobic surface can be explained by the steric interaction between adsorbed proteins as well as by hydrophobic interactions during the adsorption process. In addition, there is obviously an intermediate stage in which the protein molecules are mainly in the structure, indicating that for certain proteins, the structure may be a more stable secondary structure than helix on the hydrophobic surface. Correspondence to: S.-F. Sui     

Protein precipitation and denaturation by dimethyl sulfoxide

Solvent conditions play a major role in a wide range of physical properties of proteins in solution. Organic solvents, including dimethyl sulfoxide (DMSO), have been used to precipitate, crystallize and denature proteins. We have studied here the interactions of DMSO with proteins by differential refractometry and amino acid solubility measurements. The proteins used, i.e., ribonuclease, lysozyme, beta-lactoglobulin and chymotrypsinogen, all showed negative preferential DMSO binding, or preferential hydration, at low DMSO concentrations, where they are in the native state. As the DMSO concentration was increased, the preferential interaction changed from preferential hydration to preferential DMSO binding, except for ribonuclease. The preferential DMSO binding correlated with structural changes and unfolding of these proteins observed at higher DMSO concentrations. Amino acid solubility measurements showed that the interactions between glycine and DMSO are highly unfavorable, while the interactions of DMSO with aromatic and hydrophobic side chains are favorable. The observed preferential hydration of the native protein may be explained from a combination of the excluded volume effects of DMSO and the unfavorable interaction of DMSO with a polar surface, as manifested by the unfavorable interactions of DMSO with the polar uncharged glycine molecule. Such an unfavorable interaction of DMSO with the native protein correlates with the enhanced self-association and precipitation of proteins by DMSO. Conversely, the observed conformational changes at higher DMSO concentration are due to increased binding of DMSO to hydrophobic and aromatic side chains, which had been newly exposed on protein unfolding.     

Temperature-induced complementarity as a mechanism for biomolecular assembly

Recent advances in the measurement and theory of “hydration” interactions between biomolecules provide a basis on which to formulate mechanisms of biomolecular recognition. In this paper we have developed a mathematical formalism for analyzing specificity encoded in dynamic distributions of surface polar groups, a formalism that incorporates newly recognized properties of directly measured “hydration” forces. As expected, attraction between surfaces requires complementary patterns of surface polar groups. In contrast to usual expectations, thermal motion can create these complementary surface configurations. We have demonstrated that assembly can occur with an increase in conformational entropy of polar residues. Elevated temperature then facilitates recognition rather than hinders it. This mechanism might underlie some temperature-favored assembly reactions common in biological systems that are usually associated with the “hydrophobic effect” only. © 1994 Wiley-Liss, Inc.     

Conformational properties of intrinsically disordered proteins bound to the surface of silica nanoparticles

Background

Protein-nanoparticle (NP) interactions dictate properties of nanoconjugates relevant to bionanotechnology. Non-covalent adsorption generates a protein corona (PC) formed by an inner and an outer layer, the hard and soft corona (HC, SC). Intrinsically disordered proteins (IDPs) exist in solution as conformational ensembles, whose response to the presence of NPs is not known.

Solution properties of γ‐crystallins: Hydration of fish and mammal γ‐crystallins

Lens γ crystallins are found at the highest protein concentration of any tissue, ranging from 300 mg/mL in some mammals to over 1000 mg/mL in fish. Such high concentrations are necessary for the refraction of light, but impose extreme requirements for protein stability and solubility. γ‐crystallins, small stable monomeric proteins, are particularly associated with the lowest hydration regions of the lens. Here, we examine the solvation of selected γ‐crystallins from mammals (human γD and mouse γS) and fish (zebrafish γM2b and γM7). The thermodynamic water binding coefficient B₁ could be probed by sucrose expulsion, and the hydrodynamic hydration shell of tightly bound water was probed by translational diffusion and structure‐based hydrodynamic boundary element modeling. While the amount of tightly bound water of human γD was consistent with that of average proteins, the water binding of mouse γS was found to be relatively low. γM2b and γM7 crystallins were found to exhibit extremely low degrees hydration, consistent with their role in the fish lens. γM crystallins have a very high methionine content, in some species up to 15%. Structure‐based modeling of hydration in γM7 crystallin suggests low hydration is associated with the large number of surface methionine residues, likely in adaptation to the extremely high concentration and low hydration environment in fish lenses. Overall, the degree of hydration appears to balance stability and tissue density requirements required to produce and maintain the optical properties of the lens in different vertebrate species.     

Effect of oxidative sulfitolysis of disulfide bonds of bovine serum albumin on its structural properties: A physicochemical study

The effect of S-S bond cleavage of bovine serum albumin (BSA) on some of its structural properties, including solubility, viscosity, and conformation, were investigated. Cleavage of S-S bonds decreased the solubility of serum albumin and also shifted its isoelectric point to lowerpH values. S-S bond cleavage resulted in changes in shape and hydrodynamic volume of the protein, increasing the specific viscosity, with cleavage of up to 14 S-S bonds, followed by a decrease with further cleavage. Both UV difference and fluorescence spectral measurements indicated that conformational flexibility increases with S-S bond cleavage. Secondary structure estimations by far UV-CD suggested a gradual decrease in -helical content of the protein with progressive cleavage of its S-S bonds. However, fully S-S bond cleaved protein maintained some -helical structure. Sulfitolysis of the protein also decreased its I, 8-anilino-naphthalene sulfonate-binding ability.     

Regulation of sodium channel function by bilayer elasticity: the importance of hydrophobic coupling. Effects of Micelle-forming amphiphiles and cholesterol

Membrane proteins are regulated by the lipid bilayer composition. Specific lipid-protein interactions rarely are involved, which suggests that the regulation is due to changes in some general bilayer property (or properties). The hydrophobic coupling between a membrane-spanning protein and the surrounding bilayer means that protein conformational changes may be associated with a reversible, local bilayer deformation. Lipid bilayers are elastic bodies, and the energetic cost of the bilayer deformation contributes to the total energetic cost of the protein conformational change. The energetics and kinetics of the protein conformational changes therefore will be regulated by the bilayer elasticity, which is determined by the lipid composition. This hydrophobic coupling mechanism has been studied extensively in gramicidin channels, where the channel-bilayer hydrophobic interactions link a "conformational" change (the monomer<-->dimer transition) to an elastic bilayer deformation. Gramicidin channels thus are regulated by the lipid bilayer elastic properties (thickness, monolayer equilibrium curvature, and compression and bending moduli). To investigate whether this hydrophobic coupling mechanism could be a general mechanism regulating membrane protein function, we examined whether voltage-dependent skeletal-muscle sodium channels, expressed in HEK293 cells, are regulated by bilayer elasticity, as monitored using gramicidin A (gA) channels. Nonphysiological amphiphiles (beta-octyl-glucoside, Genapol X-100, Triton X-100, and reduced Triton X-100) that make lipid bilayers less "stiff", as measured using gA channels, shift the voltage dependence of sodium channel inactivation toward more hyperpolarized potentials. At low amphiphile concentration, the magnitude of the shift is linearly correlated to the change in gA channel lifetime. Cholesterol-depletion, which also reduces bilayer stiffness, causes a similar shift in sodium channel inactivation. These results provide strong support for the notion that bilayer-protein hydrophobic coupling allows the bilayer elastic properties to regulate membrane protein function.     

Peculiar properties of the PsaF photosystem I protein from the green alga Chlamydomonas reinhardtii: presequence independent import of the PsaF protein into both chloroplasts and mitochondria

It has previously been shown that presequences of nuclear-encoded chloroplast proteins from the green alga Chlamydomonas reinhardtii contain a region that may form an amphiphilic -helix, a structure characteristic of mitochondrial presequences. We have tested two precursors of chloroplast proteins (the PsaF and PsaK photosystem I subunits) from C. reinhardtii for the ability to be imported into spinach leaf mitochondria in vitro. Both precursors bound to spinach mitochondria. The PsaF protein was converted into a protease-protected form with high efficiency in a membrane potential-dependent manner, indicating that the protein had been imported, whereas the PsaK protein was not protease protected. The protease protection of PsaF was not inhibited by a synthetic peptide derived from the presequence of the N. plumbaginifolia mitochondrial F₁ subunit. Furthermore, if the presequence of PsaF was truncated or deleted by in vitro mutagenesis, the protein was still protease-protected with approximately the same efficiency as the full-length precursor. These results indicate that PsaF can be imported by spinach mitochondria in a presequence-independent manner. However, even in the absence of the presequence, this process was membrane potential-dependent. Interestingly, the presequence-truncated PsaF proteins were also protease-protected upon incubation with C. reinhardtii chloroplasts. Our results indicate that the C. reinhardtii chloroplast PsaF protein has peculiar properties and may be imported not only into chloroplasts but also into higher-plant mitochondria. This finding indicates that additional control mechanisms in the cytosol that are independent of the presequence are required to achieve sorting between chloroplasts and mitochondria in vivo.Abbreviations cTP chloroplast transit peptide - mTP mitochondrial targeting peptide - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - pF₁(1,25) a synthetic peptide derived from the first 25 residues of the Nicotiana plumbaginifolia mitochondrial ATP synthase F₁ subunit - PsaF(2–30) and PsaF(2–61) mutant proteins lacking regions corresponding to residues 2–30 and 2–61 in the PsaF precursor protein, respectively     

Effects of hydrated water on protein unfolding

The conformational stability of a protein in aqueous solution is described in terms of the thermodynamic properties such as unfolding Gibbs free energy, which is the difference in the free energy (Gibbs function) between the native and random conformations in solution. The properties are composed of two contributions, one from enthalpy due to intramolecular interactions among constituent atoms and chain entropy of the backbone and side chains, and the other from the hydrated water around a protein molecule. The hydration free energy and enthalpy at a given temperature for a protein of known three-dimensional structure can be calculated from the accessible surface areas of constituent atoms according to a method developed recently. Since the hydration free energy and enthalpy for random conformations are computed from those for an extended conformation, the thermodynamic properties of unfolding are evaluated quantitatively. The evaluated hydration properties for proteins of known transition temperature (Tm) and unfolding enthalpy (delta Hm) show an approximately linear dependence on the number of constituent heavy atoms. Since the unfolding free energy is zero at Tm, the enthalpy originating from interatomic interactions of a polypeptide chain and the chain entropy are evaluated from an experimental value of delta Hm and computed properties due to the hydrated water around the molecule at Tm. The chain enthalpy and entropy thus estimated are largely compensated by the hydration enthalpy and entropy, respectively, making the unfolding free energy and enthalpy relatively small. The computed temperature dependences of the unfolding free energy and enthalpy for RNase A, T4 lysozyme, and myoglobin showed a good agreement with the experimental ones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hexahydrated Mg2+ Binding and Outer-Shell Dehydration on RNA Surface

The interaction between metal ions, especially Mg²⁺ ions, and RNA plays a critical role in RNA folding. Upon binding to RNA, a metal ion that is fully hydrated in bulk solvent can become dehydrated. Here we use molecular dynamics simulation to investigate the dehydration of bound hexahydrated Mg²⁺ ions. We find that a hydrated Mg²⁺ ion in the RNA groove region can involve significant dehydration in the outer hydration shell. The first or innermost hydration shell of the Mg²⁺ ion, however, is retained during the simulation because of the strong ion-water electrostatic attraction. As a result, water-mediated hydrogen bonding remains an important form for Mg²⁺-RNA interaction. Analysis for ions at different binding sites shows that the most pronounced water deficiency relative to the fully hydrated state occurs at a radial distance of around 11 Å from the center of the ion. Based on the independent 200 ns molecular dynamics simulations for three different RNA structures (Protein Data Bank: 1TRA, 2TPK, and 437D), we find that Mg²⁺ ions overwhelmingly dominate over monovalent ions such as Na⁺ and K⁺ in ion-RNA binding. Furthermore, application of the free energy perturbation method leads to a quantitative relationship between the Mg²⁺ dehydration free energy and the local structural environment. We find that $\text{ΔΔ}{G}_{\text{hyd}},$ the change of the Mg²⁺ hydration free energy upon binding to RNA, varies linearly with the inverse distance between the Mg²⁺ ion and the nearby nonbridging oxygen atoms of the phosphate groups, and $\text{ΔΔ}{G}_{\text{hyd}}$ can reach ?2.0 kcal/mol and ?3.0 kcal/mol for an Mg²⁺ ion bound to the surface and to the groove interior, respectively. In addition, the computation results in an analytical formula for the hydration ratio as a function of the average inverse Mg²⁺-O distance. The results here might be useful for further quantitative investigations of ion-RNA interactions in RNA folding.     

Integration of protein motions with molecular networks reveals different mechanisms for permanent and transient interactions

The integration of molecular networks with other types of data, such as changing levels of gene expression or protein-structural features, can provide richer information about interactions than the simple node-and-edge representations commonly used in the network community. For example, the mapping of 3D-structural data onto networks enables classification of proteins into singlish- or multi-interface hubs (depending on whether they have >2 interfaces). Similarly, interactions can be classified as permanent or transient, depending on whether their interface is used by only one or by multiple partners. Here, we incorporate an additional dimension into molecular networks: dynamic conformational changes. We parse the entire PDB structural databank for alternate conformations of proteins and map these onto the protein interaction network, to compile a first version of the Dynamic Structural Interaction Network (DynaSIN). We make this network available as a readily downloadable resource file, and we then use it to address a variety of downstream questions. In particular, we show that multi-interface hubs display a greater degree of conformational change than do singlish-interface ones; thus, they show more plasticity which perhaps enables them to utilize more interfaces for interactions. We also find that transient associations involve smaller conformational changes than permanent ones. Although this may appear counterintuitive, it is understandable in the following framework: as proteins involved in transient interactions shuttle between interchangeable associations, they interact with domains that are similar to each other and so do not require drastic structural changes for their activity. We provide evidence for this hypothesis through showing that interfaces involved in transient interactions bind fewer classes of domains than those in a control set.     

Hydration of protein-protein interfaces

We present an analysis of the water molecules immobilized at the protein-protein interfaces of 115 homodimeric proteins and 46 protein-protein complexes, and compare them with 173 large crystal packing interfaces representing nonspecific interactions. With an average of 15 waters per 1000 A2 of interface area, the crystal packing interfaces are more hydrated than the specific interfaces of homodimers and complexes, which have 10-11 waters per 1000 A2, reflecting the more hydrophilic composition of crystal packing interfaces. Very different patterns of hydration are observed: Water molecules may form a ring around interfaces that remain "dry," or they may permeate "wet" interfaces. A majority of the specific interfaces are dry and most of the crystal packing interfaces are wet, but counterexamples exist in both categories. Water molecules at interfaces form hydrogen bonds with protein groups, with a preference for the main-chain carbonyl and the charged side-chains of Glu, Asp, and Arg. These interactions are essentially the same in specific and nonspecific interfaces, and very similar to those observed elsewhere on the protein surface. Water-mediated polar interactions are as abundant at the interfaces as direct protein-protein hydrogen bonds, and they may contribute to the stability of the assembly.     

Thermodynamics of the temperature-induced unfolding of globular proteins.

The heat capacity, enthalpy, entropy, and Gibbs energy changes for the temperature-induced unfolding of 11 globular proteins of known three-dimensional structure have been obtained by microcalorimetric measurements. Their experimental values are compared to those we calculate from the change in solvent-accessible surface area between the native proteins and the extended polypeptide chain. We use proportionality coefficients for the transfer (hydration) of aliphatic, aromatic, and polar groups from gas phase to aqueous solution, we estimate vibrational effects, and we discuss the temperature dependence of each constituent of the thermodynamic functions. At 25 degrees C, stabilization of the native state of a globular protein is largely due to two favorable terms: the entropy of non-polar group hydration and the enthalpy of interactions within the protein. They compensate the unfavorable entropy change associated with these interactions (conformational entropy) and with vibrational effects. Due to the large heat capacity of nonpolar group hydration, its stabilizing contribution decreases quickly at higher temperatures, and the two unfavorable entropy terms take over, leading to temperature-induced unfolding.     

Influence of hydration on the conformational stability and formation of bends in terminally blocked dipeptides

The conformations of 23 terminally blocked dipeptide sequences were examined by conformational energy calculations that included the effects of the aqueous solvent. Starting structures were derived from combinations of minimum-energy conformations of hydrated single residues. Their conformational energies were then minimized using the ECEPP potential (Empirical Conformational Energy Program for Peptides) with hydration included. Short-range interactions dominate in stabilizing the conformations of the hydrated dipeptides. Differences between conformational stabilities of hydrated and unhydrated dipeptides in many cases are due to the competition of solute–water and intramolecular hydrogen bonds. In other cases, perturbation of the hydration shell of the solute by close approach of solute atoms alters conformational preferences. Probabilities of formation of bends were calculated and compared to the corresponding quantities for unhydrated dipeptides and to those calculated from x-ray structures. For bends in dipeptides containing two nonpolar amino acids, computations omitting hydration yield better results. However, better agreement with experimental (x-ray) bend probabilities for dipeptides containing glycine or polar amino acids is obtained only in some sequences when hydration is included. The results are rationalized by the observation that, in proteins, bends containing nonpolar sequences occur on the inside, shielded from the solvent. Bends containing glycine or polar amino acids occur frequently on the surface of the protein, but they are not completely hydrated.     

Influence of the spacer of cationic gemini amphiphiles on the hydration of lipoplexes

The impact of the length of gemini surfactant spacer on complexation and condensation of calf thymus DNA by cationic mixed phospholipid/gemini liposomes was investigated by monitoring the conformational changes of DNA by circular dichroism and the lipid hydration level by the emission characteristics of the fluorescent probe laurdan included in the lipid bilayer. The length of the spacer was shown to influence, on one hand, the hydration level and the organization of the corresponding liposomes and, on the other, the variation of lipid hydration level and the DNA conformation upon complexation. In fact, in correspondence with the longest spacer we observed more hydrated liposomes, probably organized in domains, a higher extent of dehydration promoted by the addition of DNA, and a minor extent of DNA conformational change. The physicochemical features of lipoplexes were shown to depend on the [cationic headgroup]/[DNA single base] ratio.     

Volume and hydration changes of DNA-ligand interactions

We report the volumetric and other thermodynamic properties of ethidium bromide (EB), propidium iodide (PI) and daunomycin (DAU) intercalating with poly(dA).poly(dT), poly[d(A-T)].poly[d(A-T)], and poly[d(G-C)].poly[d(G-C)], respectively, as well as minor groove binder Hoechst 33258 binding with poly[d(A-T)].poly[d(A-T)]. The data were obtained using fluorescence titration and hydrostatic pressure measurements. Our thermodynamic data are combined with enthalpies from literature reports to analyze the thermodynamic characteristics of the different interactions. The differences are interpreted based on three processes related to hydration: I. burial of non-polar hydrophobic solvent accessible surface, II. burial of polar surface and formation of solute-solute H-bonds, and III. disruption of "structural" hydration. Sequence dependent conformational changes may also be important when comparing ligand binding to different DNA sequences. We conclude that a combination of different thermodynamic parameters, especially volume change, is essential in order to understand the role of hydration in the energetics of DNA-ligand interactions.     

Steric exclusion is the principal source of the preferential hydration of proteins in the presence of polyethylene glycols.

The preferential interactions of bovine serum albumin, lysozyme, chymotrypsinogen, ribonuclease A, and beta-lactoglobulin with polyethylene glycols (PEGs) of molecular weight 200-6,000 have been measured by dialysis equilibrium coupled with high precision densimetry. All the proteins were found to be preferentially hydrated in all the PEGs, and the magnitude of the preferential hydration increased with increasing PEG size for each protein. The change in the chemical potentials of the proteins with the addition of the PEGs had highly positive values, indicating a strong thermodynamic destabilization of the system by the PEGs. A viscosity study of the PEGs showed them to be randomly coiled polymers, as their radii of gyration were related to the molecular weight by Rg = aM0.55. The thickness of the effective shell impenetrable to PEG around protein molecules, calculated from the preferential hydration, was found to vary with PEG molecular weight in similar fashion as the PEG radius of gyration, supporting the proposal (Arakawa, T. & Timasheff, S.N., 1985a, Biochemistry 24, 6756-6762) that the preferential exclusion of PEGs from proteins is due principally to the steric exclusion of PEG from the protein domain, although favorable interactions with protein surface residues, in particular nonpolar ones, may compete with the exclusion. These thermodynamically unfavorable preferential exclusion interactions lead to the action of PEGs as precipitants, although they may destabilize protein structure at higher temperatures.