Protein Interactions In Solution Research Articles

A patch–antipatch representation of specific protein interactions

Protein interactions in solution are manifested in macroscopically measurable thermodynamic properties such as the osmotic second virial coefficient. These interactions are typically characterized by a sensitivity to electrostatic parameters such as pH and ionic strength, and by strong short-range attraction with a pronounced orientational dependence. We describe here a model that embodies all these features, yet is sufficiently compact to allow its use in simulations of dynamic and equilibrium properties. The model treats protein molecules as spheres with relatively weak isotropic contributions to intermolecular interactions. In addition, a discrete number of “patch–antipatch” pairs on the surfaces account for highly specific interactions that can arise from geometric complementarity of corresponding areas on the actual molecular surface. Implementation of the model is illustrated for interactions in solution of bovine chymotrypsinogen, based on structural information from crystallographic data. The general trends seen in virial coefficient measurements are captured by the model, despite anomalous features that are thought to result from the importance of the specific interactions.

Temperature-independent solubility and interactions between apoferritin monomers and dimers in solution

We used chromatographic, static and dynamic light scattering techniques, and atomic force microscopy (AFM) to study the structure of the protein species and the protein–protein interactions in solutions containing two apoferritin molecular forms, monomers and dimers, in the presence of NaAc buffer and CdSO 4. The sizes and shapes of the monomers and dimers, separated by size-exclusion chromatography, were determined by dynamic light scattering and AFM. While the monomer is an apparent sphere with a diameter corresponding to previous X-ray crystallography determinations, the dimer shape corresponds to two, bound monomer spheres. Static light scattering was used to characterize the interactions between solute molecules of monomers and dimers in terms of the second osmotic virial coefficients. The addition of even small amounts of Cd 2+ causes attraction between the monomer molecules. Furthermore, we found that the second virial coefficient and the protein solubility do not noticeably depend on temperature in the range from 0°C to 40°C. This suggests that the enthalpy for crystallization of apoferritin is close to zero, and the gain of entropy is essentially constant in this temperature range. We also found that in solutions of the apoferritin dimer, the molecules attract even in the presence of acetate buffer only, indicating a change in the surface of the apoferritin molecule. In view of the repulsion between the monomers at the same conditions, this suggests that the dimers and higher oligomers form only after partial unfolding of some of the apoferritin subunits. These observations suggest that aggregation and self-assembly of protein molecules or molecular subunits may be driven by forces other than those responsible for crystallization in the protein solution.

Interactions and Aggregation of Apoferritin Molecules in Solution: Effects of Added Electrolytes

We have studied the structure of the protein species and the protein–protein interactions in solutions containing two apoferritin molecular forms, monomers and dimers, in the presence of Na + and Cd 2+ ions. We used chromatographic, and static and dynamic light scattering techniques, and atomic force microscopy (AFM). Size-exclusion chromatography was used to isolate these two protein fractions. The sizes and shapes of the monomers and dimers were determined by dynamic light scattering and AFM. Although the monomer is an apparent sphere with a diameter corresponding to previous x-ray crystallography determinations, the dimer shape corresponds to two, bound monomer spheres. Static light scattering was applied to characterize the interactions between solute molecules of monomers and dimers in terms of the second osmotic virial coefficients. The results for the monomers indicate that Na + ions cause strong intermolecular repulsion even at concentrations higher than 0.15 M, contrary to the predictions of the commonly applied Derjaguin–Landau–Verwey–Overbeek theory. We argue that the reason for such behavior is hydration force due to the formation of a water shell around the protein molecules with the help of the sodium ions. The addition of even small amounts of Cd 2+ changes the repulsive interactions to attractive but does not lead to oligomer formation, at least at the protein concentrations used. Thus, the two ions provide examples of strong specificity of their interactions with the protein molecules. In solutions of the apoferritin dimer, the molecules attract even in the presence of Na + only, indicating a change in the surface of the apoferritin molecule. In view of the strong repulsion between the monomers, this indicates that the dimers and higher oligomers form only after partial denaturation of some of the apoferritin monomers. These observations suggest that aggregation and self-assembly of protein molecules or molecular subunits may be driven by forces other than those responsible for crystallization and other phase transitions in the protein solution.

Effect of the Microenvironment on the Recognition of Immobilized Cytochromes by Soluble Redox Proteins

This work examines the effect of the electrostatic properties of an immobilization substrate on the apparent affinity of immobilized receptors for their soluble ligands. In particular, we report measurements of the interaction between cytochrome c or cytochrome b5 bound to an oriented streptavidin monolayer and its soluble reaction partner. Surface plasmon resonance measurements demonstrate that the charge on the underlying streptavidin monolayer has a pronounced impact on the measured equilibrium constants for the heterologous interaction between cyt c and cyt b5. The pH dependence of the equilibrium binding constant is similar qualitatively to that reported for the protein interaction in solution. However, the pH optima of the affinities measured with the immobilized proteins are shifted by up to 1.0 pH units. Whether the shift is to acidic or basic pH depends on which of the two proteins is immobilized. The observed changes are consistent with the long-range repulsion or attraction between the soluble ...

Measured and calculated effects of mutations in bacteriophage T4 lysozyme on interactions in solution

Understanding the molecular determinants of protein interactions in solution has fundamental implications for understanding protein solution thermodynamics and, hence, processes as diverse as separations performance and cellular self-organization. Our earlier theoretical calculations indicate that the protein-protein interactions are dominated by a small number of configurations in which highly complementary surface regions are apposed, rather than by the overall colloidal interactions. To examine this paradigm more explicitly, we investigated the effects of protein structural modifications on protein-protein interactions. Experimental measurements are presented of B(22)(') values of a set of mutants of Ser44 in bacteriophage T4 lysozyme. Effects are seen with both charged and uncharged substitutions. The results with the charged substitutions follow the expected trends, whereas those with the uncharged substitutions may be explained by the impact of the mutations on the local protein geometry, which directly affects the complementarity of protein interactions. These effects are also captured well by molecular calculations that account for the mutations. The interaction energetics between protein pairs could provide information on the propensity for adventitious interactions, which can have important implications for separations and for normal and pathological self-assembly. Thus, protein structural data implicit in genomic information, coupled with appropriate calculational and experimental tools, can ultimately provide insights into protein interactions in vivo and in bioprocessing.

Interactions in protein solutions near crystallisation: a colloid physics approach

I present a review of current ideas from colloid physics which might be relevant in order to understand the onset of crystallisation and amorphous aggregation processes in protein solution. In particular, the inadequacy of DLVO theory to account for all phenomenological aspects of crystallisation, such as salt-specificity, and for the basic features of the phase diagram, such as the presence of a metastable fluid–fluid separation, is discussed. The fundamental role of additional short-range attractive forces, microscopically orginating from the salting-out effect, is conversely stressed. In order to establish a simple model of protein interparticle interactions near crystallisation, I discuss some recent results obtained by our group for the osmotic compressibility of the metastable fluid phase of hen egg-white lysozyme. Light scattering measurements were performed in an extended volume fraction range at pH=4.7 as a function of temperature, adding NaC1 to screen the electrostatic interactions. The experimental compressibility up to particle volume fractions Φ≈0.23 is very successfully compared to the theoretical expression for a model of adhesive (“sticky”) hard spheres. This surprising quantitative agreement, obtained using a very simple form for the effective interparticle force, suggests that the thermodynamics of the system in the fluid phase is mainly determined by the very short-range nature of the interactions, and is rather insensitive to the detailed form of the potential.

MIAX: A Novel System for Assessment of Macromolecular Interaction in Condensed Phases. 1) Description of the Interaction Model and Simulation Algorithm.

We describe a novel computer system directed to evaluate protein complex formation in a liquid environment. The relevant feature of the system is a potential function expressing the main thermodynamic and kinetic factors leading to protein interaction in solution. The protein interaction model expresses the interaction energy as basically composed of three forces: electrostatic (hydrogen bond), van der Waals, and hydrophobic. The latter is defined in function of the forces that the solvent molecules exert on the surface of the complex, and the van der Waals forces between the monomers and the solvent. The interaction model implemented in the system has proven a high discrimination ability between different protein dockings, scoring high those close to the observed crystal structures. These results have led to the establishment of the basic principles underlying protein interaction, which constitutes the main way of expression of the biological function of these macromolecules.

Interactions in solution of a large oligomeric protein

Up to now, systematic studies on protein interactions in solution have been (mostly) restricted to small molecular weight model proteins like lysozyme, BPTI or γ-crystallins. Those studies involving a combination of techniques (osmotic pressure, light scattering, small-angle X-ray scattering, etc.) led to an interpretation of the results in terms of interaction potentials, the parameters of which can be related in a semi-quantitative way to van der Waals forces and particle charges. We have undertaken an X-ray scattering study to extend the interaction potential analysis to the case of a large size oligomeric protein, aspartate transcarbamylase from E. coli. This heterododecamer comprises two trimers of catalytic chains and three dimers of regulatory chains for a total molecular weight of 306 kDA. It is a slightly acidic protein (pI=5.9). The main thermodynamic and chemical parameters were varied: temperature, protein concentration, pH, salt nature and concentration. Moreover, we took advantage of the large molecular weight of ATCase to study the effect of polyethylene glycols. The results are compared to those reached in the case of small proteins.

Measurements Using the Quartz Crystal Microbalance Technique of Ferritin Monolayers on Methyl-Thiolated Gold: Dependence of Energy Dissipation and Saturation Coverage on Salt Concentration

The adsorption kinetics of ferritin as a function of ionic strength has been studied with a new quartz crystal microbalance technique, allowing simultaneous measurement of the frequency shift (proportional to the mass uptake under certain conditions) and of changes in the energy dissipation caused by the adlayer. The measurements were performed with methyl-terminated (hydrophobic) thiol-covered gold surfaces, at pH 7.0 and ionic strengths in the range 1–200 mM KCl. The saturation uptake increases rapidly with increasing ionic strength in the range 20–50 mM and is then independent of ionic strength at >100 mM. The dissipation factor reveals, in the low coverage regime, distinct differences in the adlayer properties at low and high ionic strength, respectively. These results are briefly discussed in terms of the screening properties of the solvent and its influence on the protein–protein interaction in solution and on the surface.

Protein Interactions in Solution Characterized by Light and Neutron Scattering: Comparison of Lysozyme and Chymotrypsinogen

The effects of pH and electrolyte concentration on protein-protein interactions in lysozyme and chymotrypsinogen solutions were investigated by static light scattering (SLS) and small-angle neutron scattering (SANS). Very good agreement between the values of the virial coefficients measured by SLS and SANS was obtained without use of adjustable parameters. At low electrolyte concentration, the virial coefficients depend strongly on pH and change from positive to negative as the pH increases. All coefficients at high salt concentration are slightly negative and depend weakly on pH. For lysozyme, the coefficients always decrease with increasing electrolyte concentration. However, for chymotrypsinogen there is a cross-over point around pH 5.2, above which the virial coefficients decrease with increasing ionic strength, indicating the presence of attractive electrostatic interactions. The data are in agreement with Derjaguin-Landau-Verwey-Overbeek (DLVO)-type modeling, accounting for the repulsive and attractive electrostatic, van der Waals, and excluded volume interactions of equivalent colloid spheres. This model, however, is unable to resolve the complex short-ranged orientational interactions. The results of protein precipitation and crystallization experiments are in qualitative correlation with the patterns of the virial coefficients and demonstrate that interaction mapping could help outline new crystallization regions.

Freeze-thaw immobilization of liposomes in chromatographic gel beads: evaluation by confocal microscopy and effects of freezing rate.

Biological membranes immobilized in chromatographic gel beads constitute a multifunctional affinity matrix. Membrane protein-solute interactions and drug partitioning into the lipid bilayers can conveniently be studied. By the use of confocal laser-scanning microscopy (CLSM) the distribution of immobilized model membranes in the beads has been visualized for the first time. Freeze-thaw-immobilized liposomes in Superdex 200 gel beads were situated in a thick shell surrounding a liposome-free core. The amount of phospholipids immobilized by freeze-thawing was dependent on the temperature in the cooling bath and the type of test tube used. A bath temperature of -25 degrees C gave higher immobilization yield than freezing at -75 or -8 degrees C did. Freeze-thawing in the presence of liposomes did not affect the gel bead shape or the refractive index homogeneity of the agarose network of the beads, as shown by confocal microscopy.

NMR studies of carbohydrate–protein interactions in solution

This Reviews provides an overview of the current methods of NMR spectroscopy that may be used to obtain information about the conformational aspects, forces, and structural motifs that govern the interactions between proteins and carbohydrates in solution.

Interactions in undersaturated and supersaturated lysozyme solutions: Static and dynamic light scattering results

We have performed multiangle static and dynamic light scattering studies of lysozyme solutions at pH=4.7. The Rayleigh ratio Rθ and the collective diffusion coefficient Dc were determined as function of both protein concentration cp and salt concentration cs with two different salts. At low salt concentrations, the scattering ratio Kcp/Rθ and diffusivity increased with protein concentration above the values for a monomeric, ideal solution. With increasing salt concentration this trend was eventually reversed. The hydrodynamic interactions of lysozyme in solution, extracted from the combination of static and dynamic scattering data, decreased significantly with increasing salt concentration. These observations reflect changes in protein interactions, in response to increased salt screening, from net repulsion to net attraction. Both salts had the same qualitative effect, but the quantitative behavior did not scale with the ionic strength of the solution. This indicates the presence of salt specific effects. At low protein concentrations, the slopes of Kcp/Rθ and Dc vs cp were obtained. The dependence of the slopes on ionic strength was modeled using a DLVO potential for colloidal interactions of two spheres, with the net protein charge Ze and Hamaker constant AH as fitting parameters. The model reproduces the observed variations with ionic strength quite well. Independent fits to the static and dynamic data, however, led to different values of the fitting parameters. These and other shortcomings suggest that colloidal interaction models alone are insufficient to explain protein interactions in solutions.

A Continuum Model for Protein–Protein Interactions: Application to the Docking Problem

The prediction of protein–protein interactions in solution is a major goal of theoretical structural biology. Here, we implement a continuum description of the thermodynamic processes involved. The model differs considerably from previous models in its use of ‘ molecular surface’' area to describe the hydrophobic component to the free energy of conformational change in solution. We have applied this model to a data set of alternative docked conformations of protein–protein complexes which were generated independently of this work. It was found previously that commonly used energy evaluation techniques fail to distinguish between near-native and certain non-native complexes in this data set. Here, we found that an energy function that takes into account (1) total electrostatic free energy, (2) hydrophobic free energy and (3) loss in side-chain conformational energy was able to reliably discriminate between near-native and non-native configurations but only when molecular surface is used as a descriptor of the hydrophobic effect. It is shown that the molecular surface and the more conventional surface descriptor ‘ solvent accessible surface’' give very different quantitative measures of hydrophobicity. In terms of the contribution of different energy components to the free energy of complex formation it was found that loss in side-chain conformational entropy is a second order effect. Electrostatic interaction energy (which is commonly used to score docked conformations) was a poor indicator of complementarity when starting from unbound conformations. It was found that electrostatic desolvation energy and the hydrophobic contribution (based on a molecular surface area descriptor) are much less sensitive to local fluctuations in atomic structure than point-to-point interaction energies and thus may be more suited for use as a scoring function when docking unbound conformations, where atomic complementarity is much less apparent. Whilst a combined energy function was able to distinguish near-native from non-native energy function was able to distinguish near-native from non-native conformations in the six systems studied here, it remains to be determined to what extent more sizeable conformational changes would influence the results. f2 f2 Abbreviations used: vdW, van der Waals; MS, molecular surface; SAS, solvent accessible surface; r.m.s.d., root-mean-square deviation; BPTI, bovine pancreatic trypsin inhibitor.

Intermolecular protein interactions in solutions of bovine lens beta L-crystallin. Results from 1/T1 nuclear magnetic relaxation dispersion profiles

We report the magnetic field dependence of 1/T1 of solvent water protons and deuterons (nuclear magnetic relaxation dispersion, or NMRD, profiles) for solutions of steer lens beta L-crystallin. Such data allow the study of intermolecular protein interactions over a wide concentration range, here 1-34% vol/vol, by providing a measure of the rotational relaxation time of solute macromolecules. We conclude that, for approximately less than 5% protein, the solute particles are noncompact, with a rotationally averaged volume approximately three times that of a compact 60-kD sphere. (Earlier results for alpha-crystallin, approximately 1,000 kD, from optical and osmotic measurements (Vérétout and Tardieu, 1989. J. Mol. Biol. 205:713-728), show a similar, approximately twofold, effect). At intermediate concentrations, to approximately 20% protein, there is evidence for limited association or oligomerization, as found for the structurally related gamma II-crystallin (Koenig et al. 1990. Biophys. J. 57:461-469), to a limiting size about two-thirds that of alpha-crystallin. The difference in NMRD behavior of the three classes of crystallins is consonant with their differing osmotic properties (Vérétout and Tardieu. J. Mol. Biol. 1989, 205:713-728; Kenworthy, McIntosh, and Magid. Biophys. J. 1992. 61:A477; Tardieu et al. 1992. Eur. Biophys. J. 21:1-12). We indicate how the unusual structures and interactions of these three classes of proteins can be combined to optimize transparency and minimize colloid osmotic difficulties in eye lens.

Intermolecular protein interactions in solutions of calf lens alpha-crystallin. Results from 1/T1 nuclear magnetic relaxation dispersion profiles

From analyses of the magnetic field dependence of 1/T1 (NMRD profiles) of water protons in solutions of calf lens alpha-crystallin at several concentrations, we find two regimes of solute behavior in both cortical and nuclear preparations. Below approximately 15% vol/vol protein concentration, the solute molecules appear as compact globular proteins of approximately 1,350 (cortical) and approximately 1,700 (nuclear) kD. At higher concentrations, the effective solute particle size increases, reversibly, as evidenced by the appearance of spectra-like 14N peaks in the NMRD profiles and a change in the field and temperature dependence of 1/T1. At these higher concentrations, the profiles are very similar to those of calf gamma II-crystallin, a crystallin that undergoes an analogous transition near approximately 15% protein (Koenig, S. H., C.F. Beaulieu, R. D. Brown III, and M. Spiller, 1990. Biophys. J. 57:461-469). By comparison with recent analyses of NMRD results for solutions of immobilized proteins as models for the transition from protein solutions to tissue (Koenig, S. H., and R. D. Brown III. 1991. Prog. NMR Spectr. 22:487-567), we argue that alpha-crystallin solute behaves as aggregates approximately greater than 50,000 kD as protein concentration is progressively increased above 15%. Finally, the concentration dependence of the NMRD profiles of alpha- and gamma II-crystallin can readily explain recent osmotic pressure data, in particular the intersection of the respective pressure curves at approximately 23% vol/vol (Vérétout, F., and A. Tardieu. 1989. Eur. Biophys. J. 17:61-68).

7] Structure and interactions of proteins in solution studied by small-angle neutron scattering

The use of the small-angle neutron scattering (SANS) technique as an analytical tool for the study of global structures of macromolecules in solution follows naturally from the earlier development of the small-angle X-ray scattering (SAXS) technique. This chapter presents the method of SANS as applied to soluble globular proteins in solution. Its advantages over the SAXS technique are also brought out in the discussion. The emphasis is put on analysis of scattering data from systems at both low and moderately high concentrations, which better simulate physiological conditions. Owing to the considerable number of charges on proteins in solution at pH 7, there exists a long range electrostatic interaction between the molecules even at the 1% (g/dl) concentration level. It should be emphasized that the dilute regime where interactions can be neglected is an impractical regime in which to perform experiments because of the severe loss in scattered intensity for solutions with less than 0.1% protein. For globular proteins with known conformation, experiments performed at high concentrations provide additional information, such as the effective surface charge, the amount of bound water, and the local spatial ordering of the molecules in solution.

1] Characterization of macromolecules by sedimentation equilibrium in the air-turbine ultracentrifuge

Publisher Summary The development of sedimentation equilibrium in the air-driven preparative ultracentrifuge has provided an independent, nonempirical method to characterize partially purified macromolecules within heterogeneous preparations, based on the equilibrium distribution of their biochemical or other specific activities as a function of radial distance in a centrifugal field. Since this method is based on a theoretical thermodynamic description of the system in sedimentation-diffusion equilibrium and is independent of molecular weight standards and zonal transport, the difficulties outlined in this chapter are effectively avoided. In addition, the method has opened this area of investigation to the extensive existing literature concerning the use of sedimentation equilibrium to determine the molecular weights and interactions of proteins in solution, which had been previously restricted to highly purified protein preparations detected optically in the analytical ultracentrifuge. This chapter illustrates that as the time required to approach to within a specific deviation from true sedimentation equilibrium is proportional to the square of this radial distance, 17 short distances are necessary for achievement of experimental sedimentation equilibrium within a reasonable time. Furthermore, the nonsectorial geometry of the tube walls initially creates convective transport of solute 18 which contributes to achievement of experimental sedimentation equilibrium within 24 hr.

Solvation interactions of proteins in solution.

This lecture was to have been presented by Professor Rufus Lumry but complications resulting from a recent operation prevented his presence at the meeting, nor was he able to prepare a full-scale manuscript. He suggested that Professor Franks should prepare and deliver the lecture in his stead and this course was adopted. Professor Lumry did, however, supply Professor Franks with detailed notes on several recent developments which he felt should be dealt with in the presentation and these are also included in the following extended summary of the lecture delivered by Professor Franks and they are indicated by f at the beginning of the relevant sections.

Protein-Solute Interactions by Density Measurements

Protein-Solute Interactions by Density Measurements