Protein Interactions In Solution Research Articles

The accurate measurement of second virial coefficients using self-interaction chromatography: Experimental considerations

Measurement of B22, the second virial coefficient, is an important technique for describing the solution behaviour of proteins, especially as it relates to precipitation, aggregation and crystallisation phenomena. This paper describes the best practise for calculating B22 values from self-interaction chromatograms (SIC) for aqueous protein solutions. Detailed analysis of SIC peak shapes for lysozyme shows that non-Gaussian peaks are commonly encountered for SIC, with typical peak asymmetries of 10%. This asymmetry reflects a non-linear chromatographic retention process, in this case heterogeneity of the protein–protein interactions. Therefore, it is important to use the centre of mass calculations for determining accurate retention volumes and thus B22 values. Empirical peak maximum chromatogram analysis, often reported in the literature, can result in errors of up to 50% in B22 values. A methodology is reported here for determining both the mean and the variance in B22 from SIC experiments, includes a correction for normal longitudinal peak broadening. The variance in B22 due to chemical effects is quantified statistically and is a measure of the heterogeneity of protein–protein interactions in solution. In the case of lysozyme, a wide range of B22 values are measured which can vary significantly from the average B22 values.

In-Cell NMR Spectroscopy–In vivo Monitoring of the Structure, Dynamics, Folding, and Interactions of Proteins at Atomic Resolution

Thallapuranam K Suresh Kumar, Ryan Thurman and Srinivas Jayanthi-In-Cell NMR Spectroscopy–<em>In vivo</em> Monitoring of the Structure, Dynamics, Folding, and Interactions of Proteins at Atomic Resolution

An Application of Ultraviolet Spectroscopy to Study Interactions in Proteins Solutions at High Concentrations

Studies of protein-protein interactions (PPIs), especially in high-concentration solutions, have become increasingly important from a pharmaceutical perspective. Analytical methods used to study protein interactions, however, rely primarily on the detection of nonideality in relatively dilute (<50 mg/mL) solutions. We present here an application of variable-pathlength ultraviolet (UV)-visible absorption spectroscopy to examine and better understand such interactions over a wide concentration range (5-240 mg/mL) using several representative proteins. In this study, the change in UV absorption (or extinction coefficient) was monitored by determining delta absorbance (ΔAbs), the difference between the measured absorbance and the corresponding theoretical absorbance (calculated from gravimetric dilution), over a wide range of protein concentrations. The ΔAbs, corrected for light scattering, was found to increase with protein concentration for three model proteins (bovine serum albumin, lysozyme, and monoclonal antibody). Because PPIs influence solution viscosity, we studied the correlation between ΔAbs measurements and viscosity as a function of protein concentration. The magnitude of ΔAbs and solution viscosity followed similar trends with increasing protein concentration, albeit to different extents for different proteins. These data support the use of such ΔAbs measurements as an alternative approach to monitor and evaluate interactions in protein solutions at high concentration.

Ultrasensitive analysis of binding affinity of HIV receptor and neutralizing antibodies using solution-phase electrochemiluminescence assay

Binding of a few ligand molecules with its receptors on cell surface can initiate cellular signaling transduction pathways, and trigger viral infection of host cells. HIV-1 infects host T-cells by binding its viral envelope protein (gp120) with its receptor (a glycoprotein, CD4) on T cells. Primary strategies to prevent and treat HIV infection is to develop therapies (e.g., neutralizing antibodies) that can block specific binding of CD4 with gp120. The infection often leads to the lower counts of CD4 cells, which makes it an effective biomarker to monitor the AIDS progression and treatment. Despite research over decades, quantitative assays for effective measurements of binding affinities of protein–protein (ligand–receptor, antigen–antibody) interactions remains highly sought. Solid-phase electrochemiluminescence (ECL) immunoassay has been commonly used to capture analytes from the solution for analysis, which involves immobilization of antibody on solid surfaces (micron-sized beads), but it cannot quantitatively measure binding affinities of molecular interactions. In this study, we have developed solution-phase ECL assay with a wide dynamic range (0–2nM) and high sensitivity and specificity for quantitative analysis of CD4 at femtomolar level and their binding affinity with gp120 and monoclonal antibodies (MABs). We found that binding affinities of CD4 with gp120 and MAB (Q4120) are 9.5×108 and 1.2×109M−1, respectively. The results also show that MAB (Q4120) of CD4 can completely block the binding of gp120 with CD4, while MAB (17b) of gp120 can only partially block their interaction. This study demonstrates that the solution-phase ECL assay can be used for ultrasensitive and quantitative analysis of binding affinities of protein–protein interactions in solution for better understating of protein functions and identification of effective therapies to block their interactions.

Selective Domain Stabilization as a Strategy to Reduce Fusion Protein Aggregation

A human serum albumin-human growth hormone (HSA-hGH) fusion protein was used as a model to understand the contributions of individual domains to the aggregation behavior of the overall fusion protein. Aggregation of HSA-hGH was studied at two different pH conditions, pH 5 and pH 7. Conformational stability of the HSA domain was modulated by addition of octanoic acid, a binding ligand. Conformational stability of the fusion protein and the HSA domain were determined from experimentally measured values for free energies of unfolding (ΔG(unf) ) with midpoint of apparent unfolding temperatures (T(m) ) used as surrogate in some cases. Apparent T(m) s of both HSA and HSA-hGH were increased by octanoic acid binding. Osmotic second virial coefficients were measured to monitor protein-protein interactions in solution. Reductions in rates of aggregation were observed under solution conditions that increased protein-protein repulsive interactions even when no changes in conformational stability were detected. The results indicate that colloidal instabilities are responsible for HSA-hGH aggregation and that conformational stability of the HSA domain does not play a dominant role in the aggregation of HSA-hGH.

Rapid‐Throughput Competitive Colorimetric Assay Based on Monosaccharide‐Capped Gold Nanoparticles for Detecting Lectin–Protein Interactions

AbstractIdentification of protein binding partners is one of key challenges in proteomics. A rapid‐throughput competitive colorimetric assay is presented that uses gold nanoparticles capped by sugars such as mannopyranoside (Man–GNPs), N‐acetylglucosamine (GlcNAc–GNPs), glucose (Glc–GNPs), or N‐acetylgalactosamine (GalNAc–GNPs). The assay expediently detects protein–protein interactions in solution, particularly protein–lectin interactions. The competitive assays were conducted in microtiter plates; ten proteins (two glycoproteins and eight lectins) and three sugar‐binding lectins (concanavalin A (ConA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA120)) were combinatorially arranged in a 30‐well plate, and constant concentrations of the monosaccharide‐capped GNPs (sugar–GNPs) were added to each well. If interactions occurred between the proteins, the sugar–GNPs retained their burgundy color. If no interactions occurred between the proteins, the sugar–GNPs were agglomerated by the corresponding binding lectin, thus producing a blue color. Several new binding pairs were identified for the first time by using this assay, and the binding constants and stoichiometric ratios were determined on the basis of the wavelength shifts. The results were further verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and fluorescence resonance energy transfer (FRET) spectroscopy. The assay is very sensitive, requiring only nanomolar protein concentrations.

Protein–Protein Interactions in Solution and Their Interplay with Protein Specific Functions

We summarize recent developments of our small angle scattering studies on protein–protein interactions in solution. We have been focusing especially on representative proteins that function at exceptionally high concentration in human body, whose collective nature, rather than a specialized function of an isolated single molecule, plays an important role for their specific biological functions; for instance, human serum albumin (HSA), actin, and human hemoglobin (Hb). We use a static structure factor analysis, which offers information about spatial distributions of the proteins, as well as a Fourier inversion technique for the form factor, giving a real-space picture of the protein assemblies.

Polymeric vesicles mimicking glycocalyx (PV-Gx) for studying carbohydrate–protein interactions in solution

Glycocalyx, the carbohydrate coat on cell surfaces, has been proved to be particularly important in a variety of biological events. In this work, polymeric vesicle mimicking of glycocalyx (PV-Gx), as a simplified model system, is achieved via our NCCM (non-covalently connected micelles) strategy. Briefly, a thermal responsive poly(N-isopropylacrylamide) with phenylboronic acid (BA) end (BA-PNIPAM) and two novel hydrophilic glycopolymers, PGal and PGlc are prepared by RAFT polymerization, where the latter are prepared from N-linked β-pyranoside monomers containing respective units of galactoside (Gal) and glucoside (Glc). Upon heating PNIPAM-BA and PGal (PGlc) self-assemble into vesicles V-PGal (V-PGlc), driven by the dynamic covalent bond between sugars and BA. Both V-PGal and V-PGlc could serve as our target artificial glycocalyx (PV-Gx) because they have sugar-coated surfaces and disperse stably in water. Dynamic light scattering (DLS) has been employed to monitor the binding process between the sugars on PV-Gx and three lectins i.e. Arachis hypogaea (PNA), Erythrina cristagalli (ECA) and Concanavalin A (Con A), concluding that the PV-Gx shows clear specificities in the sugar-protein interactions: V-PGal interacts well with PNA and ECA to form aggregates but not with ConA, while V-PGlc interacts with none of the lectins. The results clearly prove that the PV-Gx constructed from the polymers with well-defined sugar units is a new and promising platform for the study of carbohydrate-protein interactions in solution.

Interaction of C70 fullerene with the Kv1.2 potassium channel

Fullerene C(70) is known to partition into lipid membranes and change their physical properties. Together with gallic acid (GA), C(70) induces cell contraction and cell death. How C(70) and GA-induced perturbations of lipid membranes affect cellular function and membrane protein activity is not understood, though. Meanwhile, fullerene is also known to interfere with the activity of potassium channel proteins, but the mechanisms of protein inhibition are not known. Here we consider the possibility that membrane protein function would be inhibited by C(70) and/or GA through direct contact or through lipid-mediated interactions. To this end, we use microsecond time scale atomistic simulations to explore (a) modifications of membrane properties in the presence of C(70) and/or GA, and (b) the possible conformational changes in Kv1.2, a voltage-gated potassium channel, upon exposure to C(70), or GA, or both. C(70) is found to have an observable effect on structural and elastic properties of protein-free membranes, while the effects of GA on the membrane are less evident. Fullerene–GA interaction is strong and affects significantly the partitioning of C(70) in the membrane, stabilizing C(70) in the aqueous phase. When Kv1.2 is exposed to the solutes, only small conformational changes are observed on the microsecond time scale – comparable to the fluctuations observed in the absence of any solute. Blocking of the channel entrance is not observed, as fullerene binds mainly to hydrophobic residues, both in the water-exposed loops and in the transmembrane helices. The tilt angle of transmembrane helices in the voltage-sensing domain appears to be affected by direct contact with fullerene, but a generic effect due to the small increase in membrane thickness might also play a role. A small rotation of the S3 and S4 helices in the voltage-sensing domain is noticed when C(70) is embedded in the membrane. The interpretation of the observed conformational changes is not straightforward due to the associated time scales, which are difficult to sample with state-of-the-art computing resources. We cannot exclude that both membrane-mediated interactions and specific protein–solute interactions affect the conformation of the protein.

Hydration and interactions in protein solutions containing concentrated electrolytes studied by small-angle scattering

During protein crystallization and purification, proteins are commonly found in concentrated salt solutions. The exact interplay of the hydration shell, the salt ions, and protein-protein interactions under these conditions is far from being understood on a fundamental level, despite the obvious practical relevance. We have studied a model globular protein (bovine serum albumin, BSA) in concentrated salt solutions by small-angle neutron scattering (SANS). The data are also compared to previous studies using SAXS. The SANS results for dilute protein solutions give an averaged volume of BSA of 91,700 Å(3), which is about 37% smaller than that determined by SAXS. The difference in volume corresponds to the contribution of a hydration shell with a hydration level of 0.30 g g(-1) protein. The forward intensity I(0) determined from Guinier analysis is used to determine the second virial coefficient, A(2), which describes the overall protein interactions in solution. It is found that A(2) follows the reverse order of the Hofmeister series, i.e. (NH(4))(2)SO(4) < Na(2)SO(4) < NaOAc < NaCl < NaNO(3) < NaSCN. The dimensionless second virial coefficient B(2), corrected for the particle volume and molecular weight, has been calculated using different approaches, and shows that B(2) with corrections for hydration and the non-spherical shape of the protein describes the interactions better than those determined from the bare protein. SANS data are further analyzed in the full q-range using liquid theoretical approaches, which gives results consistent with the A(2) analysis and the experimental structure factor.

Assembly states of the nucleosome assembly protein 1 (NAP-1) revealed by sedimentation velocity and non-denaturing MS

Proteins often exist as ensembles of interconverting states in solution which are often difficult to quantify. In the present manuscript we show that the combination of MS under nondenaturing conditions and AUC-SV (analytical ultracentrifugation sedimentation velocity) unambiguously clarifies a distribution of states and hydrodynamic shapes of assembled oligomers for the NAP-1 (nucleosome assembly protein 1). MS established the number of associated units, which was utilized as input for the numerical analysis of AUC-SV profiles. The AUC-SV analysis revealed that less than 1% of NAP-1 monomer exists at the micromolar concentration range and that the basic assembly unit consists of dimers of yeast or human NAP-1. These dimers interact non-covalently to form even-numbered higher-assembly states, such as tetramers, hexamers, octamers and decamers. MS and AUC-SV consistently showed that the formation of the higher oligomers was suppressed with increasing ionic strength, implicating electrostatic interactions in the formation of higher oligomers. The hydrodynamic shapes of the NAP-1 tetramer estimated from AUC-SV agreed with the previously proposed assembly models built using the known three-dimensional structure of yeast NAP-1. Those of the hexamer and octamer could be represented by new models shown in the present study. Additionally, MS was used to measure the stoichiometry of the interaction between the human NAP-1 dimer and the histone H2A-H2B dimer or H3-H4 tetramer. The present study illustrates a rigorous procedure for the analysis of protein assembly and protein-protein interactions in solution.

Self-interaction chromatography of proteins on a microfluidic monolith

A novel miniaturized system has been developed for measuring protein–protein interactions in solution with high efficiency and speed, and minimal use of protein. A chromatographic monolith synthesized in a capillary is used in the method to make interaction measurements by self-interaction chromatography (SIC) in a manner that, compared to column methods, is more efficient as well as more readily practicable even if only small amounts of protein are available. The microfluidic monolith requires much less protein for both column preparation and the chromatographic measurements than a conventional SIC system, and in addition offers improved mass transfer and hence higher chromatographic efficiency than for previous SIC miniaturization systems. Protein self-interactions for catalase as a model protein, quantified by measurement of second virial coefficients, B22, were determined by SIC and follow trends that are consistent with previously reported values. Different column derivatization conditions were studied in order to optimize the chromatographic behavior of the microfluidic system for SIC measurements. Chromatographic sensitivity can be further increased by using different column synthesis conditions.

Fluorescence Lifetime Cross Correlation Spectroscopy Resolves EGFR and Antagonist Interaction in Live Cells

Fluorescence correlation or cross-correlation spectroscopy (FCS or FCCS), a single molecule technique, has the ability to provide highly sensitive information on interaction and dynamics of biomolecules both in vitro and in vivo. However, the inherent drawback of FCS is that species with similar molecular weight could not be differentiated. Although FCCS could resolve this through cross-correlation, it suffers from nonideal confocal volume overlap and spectral cross-talk which limits its application. In this work, we demonstrate for the first time the applicability of fluorescence lifetime correlation spectroscopy (FLCS) to monitor the interaction of an antagonist antibody with the epidermal growth factor receptor (EGFR) in live cells. As a proof of concept, we demonstrate the interaction of Cy5 labeled IgG and Alexa633 labeled anti-IgG using a single laser source (636 nm excitation) in vitro. The autocorrelation functions were separated based on their respective lifetime with a single detector and their K(d) value was determined to be 11 +/- 3 nM. An in vivo application constituting the interaction of EGFR neutralizing antibody labeled with Alexa488 and EGFR-GFP in live HEK293 cells was successfully demonstrated. The binding specificity of EGFR neutralizing antibody was confirmed by fluorescence lifetime cross-correlation measurements and fluorescence lifetime imaging (FLIM). The dissociation constant of this complex was found to be 9.2 +/- 2.7 nM. A quantitative assessment of receptor density calculations show that the density of EGFR significantly decreased, from 540 +/- 64 receptors/microm(2) to 38 +/- 7 receptors/microm(2) upon addition of the neutralizing EGFR antibody, indicating that the antagonist could induce receptor internalization. The demonstrated work not only opens up new opportunities in studying protein-protein interactions in solutions and in live cells but also provide new insights in biology to understand how the antagonists influence EGFR through live cell quantification and imaging.

Molecular Mechanism for the Denaturation of Proteins by Urea

Understanding protein-solute interactions is one of the sizable challenges of protein chemistry; therefore, numerous experimental studies have attempted to explain the mechanism by which proteins unfold in aqueous urea solutions. On the basis of kinetic evidence at low urea concentrations, (1)H NMR spectroscopic analysis, and molecular orbital calculations, we propose a mechanistic model for the denaturation of RNase A in urea. Our results support a direct interaction between urea and protonated histidine as the initial step for protein inactivation followed by hydrogen bond formation with polar residues, and the breaking of hydrophobic collapse as the final steps for protein denaturation. With the proposed model, we can rationalize apparently conflicting results in the literature about the mechanism of protein denaturation with urea.

High-throughput inference of protein-protein interfaces from unassigned NMR data

We cast the problem of identifying protein-protein interfaces, using only unassigned NMR spectra, into a geometric clustering problem. Identifying protein-protein interfaces is critical to understanding inter- and intra-cellular communication, and NMR allows the study of protein interaction in solution. However it is often the case that NMR studies of a protein complex are very time-consuming, mainly due to the bottleneck in assigning the chemical shifts, even if the apo structures of the constituent proteins are known. We study whether it is possible, in a high-throughput manner, to identify the interface region of a protein complex using only unassigned chemical shifts and residual dipolar coupling (RDC) data. We introduce a geometric optimization problem where we must cluster the cells in an arrangement on the boundary of a 3-manifold, where the arrangement is induced by a spherical quadratic form [corrected] The arrangement is induced by a spherical quadratic form, which in turn is parameterized by a SO(3)xR2. We show that this formalism derives directly from the physics of RDCs. We present an optimal algorithm for this problem that runs in O(n3 log n) time for an n-residue protein. We then use this clustering algorithm as a subroutine in a practical algorithm for identifying the interface region of a protein complex from unassigned NMR data. We present the results of our algorithm on NMR data for seven proteins from five protein complexes, and show that our approach is useful for high-throughput applications in which we seek to rapidly identify the interface region of a protein complex. Contact authors for source code.

Molecular Effects of Anionic Surfactants on Lysozyme Precipitation and Crystallization

Surfactants are used as additives in some protein separations and crystallization procedures, but the mechanism of their action is still poorly understood. By measuring the osmotic second virial coefficients of lysozyme solutions by static light scattering, we show that small amounts of anionic surfactants of varying molecular weight always make the protein−protein interactions in solution more attractive and that both the charge of the headgroup and the length of the hydrophobic tail mediate the interactions. The same surfactants also modify lysozyme crystallization and promote formation of twinned phases with gross morphologies different from those seen in the absence of surfactant, some of which display remarkably structured patterns on a micrometer scale. The surfactant effects are, however, often only kinetic, as the phases obtained initially recrystallize slowly into large stable crystals. These crystals are of excellent X-ray diffraction quality and resolution (up to 1.4 Å). Their symmetry, unit cell dimensions, crystal contacts, and protein backbone conformation are the same as those commonly observed for lysozyme, but sometimes occur at atypical pH values. The data suggest new techniques for modification of protein crystallization.

Perturbation of folding and reassociation of lactate dehydrogenase by proline and trimethylamine oxide.

Investigations of protein-solute interactions typically show that osmolytes favor native conformations. In this study, the effects of representative compatible and counteracting osmolytes on the reactivation of lactate dehydrogenase from two different conformational states were explored. Contrary to expectations, proline and trimethylamine oxide inhibited both the initial time course and the extent of reactivation of lactate dehydrogenase from bovine heart following denaturation in guanidine hydrochloride, as well as following inactivation at pH 2.3. Reactivation of acid-dissociated porcine heart lactate dehydrogenase was inhibited by both proline and trimethylamine oxide (2 M). In all instances, trimethylamine oxide was the more effective inhibitor of reactivation. Analysis of the catalytic properties of the reactivating enzyme provided evidence that the molecular species that was enzymatically active during the initial stages of reactivation of acid-inactivated porcine heart lactate dehydrogenase reflects a non-native conformation. Proline and trimethylamine oxide stabilize polypeptides through exclusion from the polypeptide backbone; the inhibition of renaturation/reassociation described here is probably due to attenuation of this stabilizing influence through favorable interactions of the osmolytes with sidechains of residues that lie at the interfaces of the monomers and dimers that associate to form the active tetramer. In addition, these osmolytes may stabilize non-native intermediates in the folding pathway. The high viscosity of solutions containing more than 3 m proline was a major factor in the inhibition of reassociation of acid-dissociated porcine heart lactate dehydrogenase as well as other viscosity-dependent transformations that may occur during reactivation following unfolding in guanidine hydrochloride.

The effect of colloid osmotic pressure in human spermatozoa exposed to hypoosmotic conditions.

The use of a protein source such as serum and albumin had been extensively employed as supplements of culture media for handling and culture of gametes and embryos. Protein molecules behave as colloids in solution and contribute to the osmotic pressure of fluids. The interaction of proteins in solution and spermatozoa needs to be assessed in order to determine their possible role in osmoregulation. The aim of this study was to assess possible osmoregulatory mechanisms of protein supplementation against exposure to hypoosmotic conditions by assessing the sperm's response to those environments. A stock hypoosmotic solution (HOS) was prepared by using a mixture of fructose and sodium citrate and adjusted to an osmotic pressure of 150 mOsm l-1. Another stock solution was prepared by diluting a preparation of synthetic serum supplement [SSS; 6% (v/v) total protein] with distilled water to obtain an osmotic pressure of 150 mOsm l-1 (hypoosmotic SSS or H-SSS). Three additional solutions were prepared by mixing the stock HOS and H-SSS solutions in the following proportions (v/v): (i) 75% H-SSS/25% HOS, (ii) 50% H-SSS/50% HOS and (iii) 25% H-SSS/75% HOS. Aliquots of washed spermatozoa from 18 men were diluted 1 : 10 (v/v) with each of the testing solutions and incubated for 60 min. Specimens were assessed on wet mounts for total and specific swelling patterns. Swelling patterns were classified as maximal (>50% tail length swollen) and minimal (<50% tail length swollen) swelling with or without associated sperm motility. The major finding of this study was that increasing the concentration of protein supplementation resulted in a decrease in the proportion of maximal sperm tail swelling patterns and an increase in the proportion of minimal tail swelling patterns. A proportion of spermatozoa which exhibited minimal swelling patterns were still motile in all solutions tested, and the percentage of those spermatozoa increased as the protein supplementation was also increased in the testing solutions. Incorporation of protein supplementation as described in this study delays the effect of sperm swelling in hypoosmotic conditions.

Interaction of Proteins in Solution from Small-Angle Scattering: A Perturbative Approach

In this work an improved methodology for studying interactions of proteins in solution by small-angle scattering is presented. Unlike the most common approach, where the protein-protein correlation functions g ij( r) are approximated by their zero-density limit (i.e., the Boltzmann factor), we propose a more accurate representation of g ij( r) that takes into account terms up to the first order in the density expansion of the mean-force potential. This improvement is expected to be particulary effective in the case of strong protein-protein interactions at intermediate concentrations. The method is applied to analyze small-angle x-ray scattering data obtained as a function of the ionic strength (from 7 to 507 mM) from acidic solutions of β-lactoglobulin at the fixed concentration of 10 gl −1. The results are compared with those obtained using the zero-density approximation and show significant improvement, particularly in the more demanding case of low ionic strength.

Mapping protein-protein interactions in solution by NMR spectroscopy.

NMR is very well suited to the study of especially weak protein-protein interactions, as no crystallization is required. The available NMR methods to this end are reviewed and illustrated with applications from the recent biochemical literature: intermolecular NOEs, cross-saturation, chemical shift perturbation, dynamics and exchange perturbation, paramagnetic methods, and dipolar orientation. Most of these methods are now routinely applied for complexes with total molecular mass of 60 kDa and can likely be applied to systems up to 1000 kDa. A substantial fraction of complexes studied show distinct effects of induced fit affecting structural and dynamical properties beyond the contact interface.