Kinetics of adsorption of proteins at interfaces: role of protein conformation in diffusional adsorption

To elucidate the role of protein conformation in the kinetics of adsorption at interfaces, seven structural intermediates of bovine serum albumin were prepared and their adsorption at the air/water interface was studied. Molecular area calculations indicated two distinct molecular processes, the first being the creation of an area, delta A1, for anchoring the molecule during the initial phase of adsorption and the second being the delta A2 cleared during subsequent reorientation and rearrangement of adsorbed molecules at the interface. The delta A1 values for all the albumin intermediates were the same, indicating that the initial work pi delta A1 needed to anchor the molecule at the interface was independent of solution conformation of the protein. Unlike delta A1, delta A2 exhibited a bell-shaped relationship with the extent of refolded state of the intermediates. Calculation of diffusion coefficients indicated that greater the unfolded state of the albumin intermediate, the greater was the diffusion coefficient. It is shown that the simple diffusion theory is inadequate to explain quantitatively the kinetics of protein adsorption. Specific, conformation-dependent, solute-solvent and solute-interface interactions also seem to influence the kinetics of adsorption of proteins.     

Protein adsorption at solid-liquid interfaces: Part I--Affinities of proteins for alumina surface

Extent of adsorption (gamma pw) of bovine serum albumin, beta-lactoglobulin, gelatin and myosin at the alumina-water interface has been measured as function of protein concentration (Cp) at several temperatures, pH, and ionic strengths of the medium. gamma pw for proteins in most cases increases with increase of protein concentration but it attains maximum value gamma pw(m) when Cp is high. Values of maximum adsorption have been examined in terms of molecular orientation, molecular size and shape and unfolding of the packed proteins at the interface. In few cases, gamma pw increases with increase of Cp without reaching a real state of saturation as a result of aggregation of molecules or extensive unfolding of the protein at the interface. In the case of beta-lactoglobulin at pH 5.2 and ionic strength 0.05, gamma pw in high concentration region decreases to zero value when Cp increases. For myosin at 45 degrees C and pH 6.4, and also at 27 degrees and pH 7.8, the values of gamma pw are all negative and these negative values increase with increase of Cp. All these results have been explained in terms of significant competitions of water and protein for binding to the surface sites of the powdered alumina. Adsorption of myosin has also been found to be affected in the presence of NaCl, KCl, CaCl2, KI, Na2SO4, LiCl and urea. The relative affinities of the adsorption of various proteins for the surface of alumina at different physical conditions of the system have been compared in terms of maximum values of adsorption attained when gamma pw is varied with Cp. The affinities are shown to be compared more precisely in terms of the standard free energy decrease for the saturation of the surface by protein as a result of the change in its concentration from zero to unity in the mole fraction scale.     

The use of amphipathic maleimides to study membrane-associated proteins

A series of amphiphilic polymethylenecarboxymaleimides has been synthesized for use as sulfhydryl reagents applicable to membrane proteins. Physical properties of the compounds which are relevant to their proposed mode of action have been determined. By comparing rates of reaction in aqueous and aprotic solvents, the compounds have been shown to react exclusively with the thiolate ion. The effects of the reagents on three membrane-associated proteins are reported, and in two cases a comparative study has been made of the effects on the proteins in the absence of membranes. A mechanism is proposed whereby the reagents are anchored at the lipid/water interface by the negatively charged carboxyl group, thus siting the reactive maleimide in a plane whose depth is defined by the length of the reagent. Supporting evidence for this model is provided by the inability of the reagents to traverse membranes, and variation of their inhibitory potency with chain length when the proteins are embedded in the membrane, but not when extracted into solution. As examples of general use of the reagents to probe sulfhydryl groups in membrane proteins, the reagents have been used to (a) determine the depths in the membrane at which two populations of sulfhydryl groups occur in the mitochondrial phosphate transporter; (b) locate a single sulfhydryl associated with the active site ofD--hydroxybutyrate dehydrogenase in the inner mitochondrial membrane; (c) examine sulfhydryl groups in theD-3-glyceraldehyde phosphate dehydrogenase associated with the human red blood cell membrane.     

Interfacial properties of amphipathic beta strand consensus peptides of apolipoprotein B at oil/water interfaces

The region between residues 968 and 1882 of apolipoprotein B (apoB-21 to apoB-41) is rich in amphipathic beta strands (AbetaSs) and promotes the assembly of primordial triacylglyceride (TAG)-rich lipoproteins. To understand the importance of AbetaS in recruiting TAG, the interfacial properties of two AbetaS consensus peptides, P12 and P27, were studied at dodecane/water (DD/W) and triolein/water (TO/W) interfaces. P12 (acetyl-LSLSLNADLRLK-amide) and P27 (acetyl-LSLSLNADLRLKNGNLSLSLNADLRLK-amide), when added into the aqueous phase surrounding a suspended oil drop (dodecane or triolein), decreased the interfacial tension (gamma) in a concentration-dependent manner. At the DD/W interface, 1 x 10(-5) M P12 decreased gamma to approximately 20 mN/m and 6.6 x 10(-6) M P27 decreased gamma to approximately 13 mN/m. At the TO/W interface, 1.5 x 10(-5) M P12 decreased gamma to approximately 14 mN/m and 9.0 x 10(-6) M P27 decreased gamma to approximately 12 mN/m. The surface area of both peptides was between 11.2 and 15.1 angstroms2 per residue, consistent with beta sheets lying flat on DD/W and TO/W interfaces. P12 and P27 are almost purely elastic on DD/W, TO/W, and air/water interfaces. When P12 and P27 were compressed beyond the equilibrium gamma to as low as 4 mN/m, they could not be readily desorbed from either interface. These properties probably help in assembling nascent TAG-rich lipoproteins, and AbetaS may anchor apoB to beta lipoproteins.     

Interfacial properties of an amphipathic alpha-helix consensus peptide of exchangeable apolipoproteins at air/water and oil/water interfaces

Amphipathic alpha-helices are the main structure and the major lipid binding motif of exchangeable apolipoproteins. To understand how these apolipoproteins behave at an hydrophobic lipoprotein interface, the interfacial properties of a consensus sequence peptide (CSP) derived from three exchangeable apolipoproteins (A-I, A-IV, and E) were studied using an oil drop tensiometer at air/water (A/W) and dodecane/water (DD/W) interfaces. CSP ((PLAEELRARLRAQLEELRERLG)2-NH2) contains two 22-amino acid tandem repeat sequences that form amphipathic alpha-helices. CSP, when added into the aqueous phase, lowered the interfacial tension (gamma) of A/W and DD/W in a concentration-dependent fashion. The gammaA/W was lowered approximately 24 mn/m, and gammaDD/W approximately 31 mn/m, indicating a greater affinity of CSP for DD/W. Using the Gibbs equation for surface, the surface area per CSP molecule was estimated at approximately 702 A2 ( approximately 16 A2/amino acid) on A/W and approximately 622 A2 on DD/W ( approximately 14 A2/amino acid) suggesting that adsorbed CSP lies flat with alpha-helices in the plane of both interfaces. At equilibrium gamma, CSP desorbed from the interface when compressed and re-adsorbed when expanded. The adsorption rate was concentration-dependent, but the desorption rate was not. Less CSP desorbed from DD/W than A/W indicating that CSP has higher affinity for DD/W. Dynamic analysis of elasticity shows that the faster the oscillation period (4, 8 s) and the lower the oscillation amplitude the more elastic the surfaces. CSP can be compressed 6-12% while remaining on the surface, but large increases in pressure eject it from the surface. We suggest that surface pressure-mediated desorption and readsorption of amphipathic alpha-helices provide lipoprotein stability during remodeling reactions in plasma.     

Peripheral protein adsorption to lipid-water interfaces: the free area theory

In fluid monolayers approaching collapse, phospholipids and their complexes with diacylglycerols hinder adsorption to the monolayer of the amphipathic protein, colipase. Herein, a statistical, free-area model, analogous to that used to analyze two-dimensional lipid diffusion, is developed to describe regulation by lipids of the initial rate of protein adsorption from the bulk aqueous phase to the lipid-water interface. It is successfully applied to rate data for colipase adsorption to phospholipid alone and yields realistic values of the two model parameters; the phospholipid excluded area and the critical free surface area required to initiate adsorption. The model is further developed and applied to analyze colipase adsorption rates to mixed monolayers of phospholipid and phospholipid-diacylglycerol complexes. The results are consistent with complexes being stably associated over the physiologically relevant range of lipid packing densities and being randomly distributed with uncomplexed phospholipid molecules. Thus, complexes should form in fluid regions of cellular membranes at sites of diacylglycerol generation. If so, by analogy with the behavior of colipase, increasing diacylglycerol may not trigger translocation of some amphipathic peripheral proteins until its abundance locally exceeds its mole fraction in complexes with membrane phospholipids.     

Non-cooperative effects of lung surfactant proteins on early adsorption to an air/water interface

Two small hydrophobic proteins, SP-B and SP-C, are responsible for rapid adsorption of pulmonary surfactant to the air/water interface. Despite their physiological importance, the number of protein molecules required to trigger an absorption event remains unknown. To investigate this issue, we varied the protein content of calf lung surfactant extract (CLSE) by dilution with protein-depleted surfactant lipids (neutral and phospholipids, N&PL). Vesicles of a constant size and of composition ranging between 100% N&PL and 100% CLSE were generated by probe sonication. Their adsorption kinetics to an air/water interface were monitored at different temperatures using a Wilhelmy plate to measure surface tension. When plotted versus protein concentration, the adsorption rates during the initial change in surface tension exhibit a diphasic behavior, first increasing rapidly and linearly between 0% and 25% CLSE, and then more slowly at higher concentrations. Direct linearity at low protein content (0-5% CLSE ratio) was confirmed at 37 degrees C. These observations argue against cooperative behavior, for which the adsorption rate would first rise slowly with the protein content, and then increase suddenly once the critical number of proteins on each vesicle is reached. The apparent activation energy E(a) and the free energy of activation DeltaG(0)*, calculated from the temperature dependence of adsorption, further support the view that at least the early stages of protein-induced surfactant adsorption proceeds through a sequence of events involving not several, but a single surfactant protein.     

Insertion and pore formation driven by adsorption of proteins onto lipid bilayer membrane-water interfaces

We describe the binding of proteins to lipid bilayers in the case for which binding can occur either by adsorption to the lipid bilayer membrane-water interface or by direct insertion into the bilayer itself. We examine in particular the case when the insertion and pore formation are driven by the adsorption process using scaled particle theory. The adsorbed proteins form a two-dimensional "surface gas" at the lipid bilayer membrane-water interface that exerts a lateral pressure on the lipid bilayer membrane. Under conditions of strong intrinsic binding and a high degree of interfacial converge, this pressure can become high enough to overcome the energy barrier for protein insertion. Under these conditions, a subtle equilibrium exists between the adsorbed and inserted proteins. We propose that this provides a control mechanism for reversible insertion and pore formation of proteins such as melittin and magainin. Next, we discuss experimental data for the binding isotherms of cytochrome c to charged lipid membranes in the light of our theory and predict that cytochrome c inserts into charged lipid bilayers at low ionic strength. This prediction is supported by titration calorimetry results that are reported here. We were furthermore able to describe the observed binding isotherms of the pore-forming peptides endotoxin (alpha 5-helix) and of pardaxin to zwitterionic vesicles from our theory by assuming adsorption/insertion equilibrium.     

Non-cooperative effects of lung surfactant proteins on early adsorption to an air/water interface

Two small hydrophobic proteins, SP-B and SP-C, are responsible for rapid adsorption of pulmonary surfactant to the air/water interface. Despite their physiological importance, the number of protein molecules required to trigger an absorption event remains unknown. To investigate this issue, we varied the protein content of calf lung surfactant extract (CLSE) by dilution with protein-depleted surfactant lipids (neutral and phospholipids, N&PL). Vesicles of a constant size and of composition ranging between 100% N&PL and 100% CLSE were generated by probe sonication. Their adsorption kinetics to an air/water interface were monitored at different temperatures using a Wilhelmy plate to measure surface tension. When plotted versus protein concentration, the adsorption rates during the initial change in surface tension exhibit a diphasic behavior, first increasing rapidly and linearly between 0% and 25% CLSE, and then more slowly at higher concentrations. Direct linearity at low protein content (0-5% CLSE ratio) was confirmed at 37 °C. These observations argue against cooperative behavior, for which the adsorption rate would first rise slowly with the protein content, and then increase suddenly once the critical number of proteins on each vesicle is reached. The apparent activation energy E_a and the free energy of activation ΔG₀*, calculated from the temperature dependence of adsorption, further support the view that at least the early stages of protein-induced surfactant adsorption proceeds through a sequence of events involving not several, but a single surfactant protein.     

A theoretical approach to the binding of amphipathic molecules to globular proteins.

1. A theory based on a multiple equilibrium model with stoicheiometric binding constants has been formulated. It is applicable to the interaction of amphipathic molecules with charged macromolecules. 2. The theory has been applied to the binding of sodium n-dodecyl sulphate to ribonuclease A, beta-lactoglobulin and bovine serum albumin. 3. Over the ranges of surfactant concentration where binding is non-co-operative and co-operative, the experimental data can be satisfactorily fitted with energy and co-operatively parameters which are of comparable magnitude for the three globular proteins. 4. The results imply that the energy of interaction of sodium n-dodecyl sulphate with the proteins is equivalent to the formation of approximately four CH2 hydrophobic bonds.     

Effect of proteins on reovirus adsorption to clay minerals

Organic matter in sewage, soil, and aquatic systems may enhance or inhibit the infectivity of viruses associated with particulates (e.g., clay minerals, sediments). The purpose of this investigation was to identify the mechanisms whereby organic matter, in the form of defined proteins, affects the adsorption of reovirus to the clay minerals kaolinite and montmorillonite and its subsequent infectivity. Chymotrypsin and ovalbumin reduced the adsorption of reovirus to kaolinite and montmorillonite homoionic to sodium. Lysozyme did not reduce the adsorption of the virus to kaolinite, but it did reduce adsorption to montmorillonite. The proteins apparently competed with the reovirus for sites on the clay. As lysozyme does not adsorb to kaolinite by cation exchange, it did not inhibit the adsorption of reovirus to this clay. The amount of reovirus desorbed from lysozyme-coated montmorillonite was approximately 38% less (compared with the input population) than that from uncoated or chymotrypsin-coated montmorillonite after six washings with sterile distilled water. Chymotrypsin and lysozyme markedly decreased reovirus infectivity in distilled water, whereas infectivity of the virus was enhanced after recovery from an ovalbumin-distilled water-reovirus suspension (i.e., from the immiscible pelleted fraction plus supernatant). The results of these studies indicate that the persistence of reovirus in terrestrial and aquatic environments may vary with the type of organic matter and clay mineral with which the virus comes in contact.     

Protein adsorption at air-water interfaces: a combination of details

Using a variety of spectroscopic techniques, a number of molecular functionalities have been studied in relation to the adsorption process of proteins to air-water interfaces. While ellipsometry and drop tensiometry are used to derive information on adsorbed amount and exerted surface pressure, external reflection circular dichroism, infrared, and fluorescence spectroscopy provide, next to insight in layer thickness and surface layer concentration, molecular details like structural (un)folding, local mobility, and degree of protonation of carboxylates. It is shown that the exposed hydrophobicity of the protein or chemical reactivity of solvent-exposed groups may accelerate adsorption, while increased electrostatic repulsion slows down the process. Also aggregate formation enhances the fast development of a surface pressure. A more bulky appearance of proteins lowers the collision intensity in the surface layer, and thereby the surface pressure, while it is shown to be difficult to affect protein interactions within the surface layer on basis of electrostatic interactions. This work illustrates that the adsorption properties of a protein are a combination of molecular details, rather than determined by a single one.     

Refolding SDS-denatured proteins by the addition of amphipathic cosolvents

Sodium dodecyl sulfate (SDS) is a highly effective and widely used protein denaturant. We show that certain amphipathic cosolvents such as 2-methyl-2,4-pentanediol (MPD) can protect proteins from SDS denaturation, and in several cases can refold proteins from the SDS-denatured state. This cosolvent effect is observed with integral membrane proteins and soluble proteins from either the α-helical or the β-sheet structural classes. The SDS/MPD system can be used to study processes involving native protein states, and we demonstrate the reversible thermal denaturation of the outer membrane protein PagP in an SDS/MPD buffer. MPD and related cosolvents can modulate the denaturing properties of SDS, and we describe a simple and effective method to recover refolded, active protein from the SDS-denatured state.     

The amphipathic membrane proteins associated with gangliosides: The Paul-Bunnell antigen is one of the gangliophilic proteins

Two amphipathic protein fractions soluble in organic solvents as well as in water have been isolated from the ganglioside fraction of bovine erythrocyte membranes by successive chromatography in chloroform-methanol mixture on DEAE-Sephadex, silicic acid, and α-hydroxypropylated Sephadex G50 (LH60) columns. These two fractions contained a similar low molecular weight protein but with distinctively different amino acid composition. One of these proteins has been characterized by having a strong Paul-Bunnell antigen activity and had a binding affinity to ganglioside. A similar protein without Paul-Bunnell antigen activity was isolated as the major ganglioside-associated protein.     

Effect of proteins on reovirus adsorption to clay minerals.

Organic matter in sewage, soil, and aquatic systems may enhance or inhibit the infectivity of viruses associated with particulates (e.g., clay minerals, sediments). The purpose of this investigation was to identify the mechanisms whereby organic matter, in the form of defined proteins, affects the adsorption of reovirus to the clay minerals kaolinite and montmorillonite and its subsequent infectivity. Chymotrypsin and ovalbumin reduced the adsorption of reovirus to kaolinite and montmorillonite homoionic to sodium. Lysozyme did not reduce the adsorption of the virus to kaolinite, but it did reduce adsorption to montmorillonite. The proteins apparently competed with the reovirus for sites on the clay. As lysozyme does not adsorb to kaolinite by cation exchange, it did not inhibit the adsorption of reovirus to this clay. The amount of reovirus desorbed from lysozyme-coated montmorillonite was approximately 38% less (compared with the input population) than that from uncoated or chymotrypsin-coated montmorillonite after six washings with sterile distilled water. Chymotrypsin and lysozyme markedly decreased reovirus infectivity in distilled water, whereas infectivity of the virus was enhanced after recovery from an ovalbumin-distilled water-reovirus suspension (i.e., from the immiscible pelleted fraction plus supernatant). The results of these studies indicate that the persistence of reovirus in terrestrial and aquatic environments may vary with the type of organic matter and clay mineral with which the virus comes in contact.     

Mechanism of membrane curvature sensing by amphipathic helix containing proteins

There are several examples of membrane-associated protein domains that target curved membranes. This behavior is believed to have functional significance in a number of essential pathways, such as clathrin-mediated endocytosis, which involve dramatic membrane remodeling and require the recruitment of various cofactors at different stages of the process. This work is motivated in part by recent experiments that demonstrated that the amphipathic N-terminal helix of endophilin (H0) targets curved membranes by binding to hydrophobic lipid bilayer packing defects which increase in number with increasing membrane curvature. Here we use state-of-the-art atomistic simulation to explore the packing defect structure of curved membranes, and the effect of this structure on the folding of H0. We find that not only are packing defects increased in number with increasing membrane curvature, but also that their size distribution depends nontrivially on the curvature, falling off exponentially with a decay constant that depends on the curvature, and crucially that even on highly curved membranes defects large enough to accommodate the hydrophobic face of H0 are never observed. We furthermore find that a percolation model for the defects explains the defect size distribution, which implies that larger defects are formed by coalescence of noninteracting smaller defects. We also use the recently developed metadynamics algorithm to study in detail the effect of such defects on H0 folding. It is found that the comparatively larger defects found on a convex membrane promote H0 folding by several kcal/mol, while the smaller defects found on flat and concave membrane surfaces inhibit folding by kinetically trapping the peptide. Together, these observations suggest H0 folding is a cooperative process in which the folding peptide changes the defect structure relative to an unperturbed membrane.     

Studies on the amphipathic nature of the membrane proteins in Semliki Forest virus

The membrane glycoproteins E₁ and E₂ of Semliki Forest virus form spikes protruding from the external surface of the virion. They have been cleaved off by thermolysin or subtilisin leaving peptide segments in the membrane of the spikeless virus particles with a molecular weight of about 5000 enriched in hydrophobic amino acids. These peptides are soluble in chloroform/methanol and are solubilized into mixed micelles with Triton X100, with sodium dodecyl sulphate and with sodium deoxycholate. Peptide mapping studies show that each membrane glycoprotein has its own lipophilic peptide segment which presumably serves to anchor these proteins to the lipid membrane. The hydrophobic segments of the glycoproteins appear to be shielded from proteolysis not only by the lipids in the intact membrane but also by Triton X100 in the detergent-protein complexes obtained when this detergent is used to remove the lipid and solubilize the proteins.     

The adsorption of lysine vasopressin at ionized interfaces

It has been shown by surface potential measurements that lysine vasopressin and oxytocin may be bound by ionic surfaces to very varied extents. To dodecyl sulphate and phosphatidylserine monolayers the binding is very strong and is comparable to that for biological receptors such as those in toad bladder. For dioleyl phosphate and the carboxyl group of the polypeptide alamethicin, the binding is rather weaker while, for the zwitterionic lipids phosphatidylcholine and phosphatidylethanolamine, and for the erythrocyte surface, which contains two varieties of carboxylic acid group, no interaction seems to take place.In no system does the lysyl amino group of the vasopressin appear necessary for adsorption and, in the dodecyl sulphate monolayers, the interaction is strong even when the ionization of the terminal α-amino group is suppressed.     

Purification of antifreeze proteins by adsorption to ice

Antifreeze proteins (AFPs) can protect organisms from freezing injury by adsorbing to ice and inhibiting its growth. We describe here a method where ice, grown on a cold finger, is used to selectively adsorb and purify these ice-binding proteins from a crude mixture. Type III recombinant AFP was enriched approximately 50-fold after one round of partitioning into ice and purified to homogeneity by a second round. This method can also be used to purify non-ice-binding proteins by linkage to AFP domains as demonstrated by the recovery of a 50 kDa maltose-binding protein-AFP fusion from a crude lysate of Escherichia coli.