Dynamic Light Scattering Application to Study Protein Interactions in Electrolyte Solutions

The concentration dependence of the diffusion coefficient of particles suspended in solution depends primarily on the occupied volume fraction and on repulsive and attractive forces. This dependency is expressed by the interaction parameter, which can be assessed experimentally by light scattering measurements and have been determined for the diffusion coefficient of BSA under different salt concentration conditions in the present work. The result shows that the diffusion coefficient of protein grows up with increasing protein concentration, and when the ionic strength turns up gradually the diffusion coefficient decreases with protein concentrations increasing. The concentration dependence of BSA diffusion coefficients is interpreted in the context of a two-body potential of mean force, which includes repulsive hard-sphere and Coulombic interactions and attractive dispersion. With the increase of ionic strength, Debye screening decreases, protein interaction changes from repulsion to attraction, and protein begins to aggregate. By means of the concentration dependence of BSA diffusion coefficients, one can obtain the parameters of protein interactions and can find that protein bears a net effective charge of –9.0 e and has a Hamaker constant of 2.8k_BT. This work demonstrates that DLS is an effective technique of studying protein interactions.     

Prion Protein-Detergent Micelle Interactions Studied by NMR in Solution

Cellular prion proteins, PrP^C, carrying the amino acid substitutions P102L, P105L, or A117V, which confer increased susceptibility to human transmissible spongiform encephalopathies, are known to form structures that include transmembrane polypeptide segments. Herein, we investigated the interactions between dodecylphosphocholine micelles and the polypeptide fragments 90–231 of the recombinant mouse PrP variants carrying the amino acid replacements P102L, P105L, A117V, A113V/A115V/A118V, K110I/H111I, M129V, P105L/M129V, and A117V/M129V. Wild-type mPrP-(90–231) and mPrP[M129V]-(91–231) showed only weak interactions with dodecylphosphocholine micelles in aqueous solution at pH 7.0, whereas discrete interaction sites within the polypeptide segment 102–127 were identified for all other aforementioned mPrP variants by NMR chemical shift mapping. These model studies thus provide evidence that amino acid substitutions within the polypeptide segment 102–127 affect the interactions of PrP^C with membranous structures, which might in turn modulate the physiological function of the protein in health and disease.Transmissible spongiform encephalopathies (TSEs),² such as Creutzfeldt-Jakob disease and the Gerstmann-Sträussler-Scheinker syndrome in humans, are accompanied by the appearance in the brain of an aggregated “scrapie” isoform of the host-encoded prion protein, PrP^Sc (1–3). The cellular form, PrP^C, consists of an unstructured N-terminal “tail” of residues 23–125 and a globular domain of residues 126–231, and is attached by a C-terminal glycosylphosphatidylinositol (GPI) anchor to the outer plasma membrane. This structure ensures a role of membrane interactions in the physiological function of PrP^C and probably also in the disease-related events leading to TSEs. For example, transgenic mice expressing a prion protein variant lacking the GPI membrane anchor did not develop the typical clinical signs of TSE after inoculation with infectious brain homogenate, although significant amounts of PrP^Sc accumulated in the brain (4). This finding led to the conclusion that membrane-association of PrP^C is necessary for the development of a TSE. Independent evidence for the importance of membrane interactions for the onset of prion diseases was derived from cell-free conversion assays and cell culture experiments (5, 6).Data have also been presented that indicate that in addition to the normal form with the C terminus linked to a GPI anchor and the C-terminal domain located on the cell surface, PrP^C can adopt two different transmembrane topologies, ^CtmPrP and ^NtmPrP, which have the C-terminal polypeptide segment located in the lumen of the endoplasmic reticulum (^CtmPrP) or in the cytoplasm (^NtmPrP) (7–9). The population of the ^CtmPrP variant is <10% of the total wild-type prion protein present during cellular biosynthesis but is increased to 20–30% for the pathogenic mutations P102L, P105L, and A117V of human PrP and the designed variant mouse PrPs obtained with the amino acid exchanges A113V/A115V/A118V and K110I/H111I (10–13). The population of ^CtmPrP was further increased when an additional mutation, L9R, was present in the N-terminal signal sequence (14), so that ∼50% of the PrP was synthesized as the ^CtmPrP variant in granule neurons obtained from transgenic mice expressing a prion protein construct carrying the four amino acid replacements L9R, A113V, A115V, and A118V (15). Quite generally, an increase in the population of ^CtmPrP was also shown to be associated with severe neurodegeneration in transgenic mice, and it has been suggested that ^CtmPrP may be the proximate cause of neuronal death in certain prion disorders (10, 11, 15).In vitro studies on interactions of full-length and N-terminally truncated forms of recombinant PrP showed that acidic membranes caused the N-terminal part of the protein to become more structured, whereas the C-terminal domain was destabilized (16–19). Furthermore, zwitterionic gel-phase dipalmitoylphosphatidylcholine or raft-like membranes were shown to induce increased α-helical structure in recombinant Syrian hamster PrP-(90–231) at pH 7.0 (18, 19). Membrane interactions of polypeptides representing sequence motifs found in the prion protein have also been studied (20–23).In this report we describe investigations of PrP interactions with a membrane mimetic and focus on the mutations P102L, P105L, and A117V, which have been linked with familial Gerstmann-Sträussler-Scheinker syndrome in humans (2, 24, 25). Our interest in these variant proteins is related to open questions about the mechanisms by which pathogenic mutations predispose humans for prion diseases. We studied the interactions of a recombinant wild-type mouse prion protein fragment, mPrP-(90–231), and the variants mPrP[P102L]-(91–231), mPrP[P105L]-(91–231), mPrP[A117V]-(90–231), mPrP[A113V,A115V,A118V]-(90–231), and mPrP[K110I,H111I]-(90–231). For these studies, we used the N-terminally truncated protein composed of residues 90–231. This region contains the transmembrane segment, all known disease-associated point mutations, the entire polypeptide fragment with proteinase K-resistance in PrP^Sc, which is also sufficient to transmit disease (1, 25, 26). The amino acid substitutions in these variant PrPs are located either within a hydrophobic stretch of residues 112–127, which is highly conserved in mammalian PrPs (27, 28), or in the positively charged segment of residues 95–111 (Fig. 1, B and C). We also included the M129V polymorphism into this study, which was reported to have a significant influence on the susceptibility of humans to prion diseases and on the disease phenotype. For example, the mutations P105L and A117V are only pathogenic in the presence of valine at position 129 (2, 24). The zwitterionic detergent dodecylphosphocholine (DPC, Fig. 1A) was used as a biomembrane mimetic model system, and NMR spectroscopy was employed to screen for protein-detergent micelle interactions, and for the structural characterization of the various prion protein constructs interacting with the detergent micelles.Open in a separate windowFIGURE 1.Detergent and proteins used in this study. A, zwitterionic form of DPC. B, schematic diagram of the mPrP-(90–231) polypeptide indicating the locations of the regular secondary structures, i.e. three α-helices and two strands of an antiparallel β-sheet, a “positively charged cluster” (CC) of amino acid residues in positions 95–111, and a “hydrophobic polypeptide segment” (HPS) comprising residues 112–127. C, amino acid sequence alignment of residues 90–135 for wild-type mPrP-(90–231) and the protein variants studied in this paper, where for each variant mPrP the amino acid replacements are given and identical residues are indicated by dots; the numbering is according to Schätzl et al. (27).     

Free-Solution, Label-Free Protein-Protein Interactions Characterized by Dynamic Light Scattering

We report a free-solution, label-free method for quantitative characterization of macromolecular interactions using dynamic light scattering, a temperature controlled plate reader, and a multiwell concentration gradient. This nondestructive technique enabled determination of stoichiometry of binding, equilibrium dissociation constant, and thermodynamic parameters, as well as the impact of temperature, buffer salinity, and a small-molecule inhibitor. The low volume capability of dynamic light scattering reduced the required sample to 426 pmol/experiment, with detection limits for 150-kDa proteins anticipated to be in the low femtomole range.     

Proliferating Cell Nuclear Antigen (PCNA) Interactions in Solution Studied by NMR

PCNA is an essential factor for DNA replication and repair. It forms a ring shaped structure of 86 kDa by the symmetric association of three identical protomers. The ring encircles the DNA and acts as a docking platform for other proteins, most of them containing the PCNA Interaction Protein sequence (PIP-box). We have used NMR to characterize the interactions of PCNA with several other proteins and fragments in solution. The binding of the PIP-box peptide of the cell cycle inhibitor p21 to PCNA is consistent with the crystal structure of the complex. A shorter p21 peptide binds with reduced affinity but retains most of the molecular recognition determinants. However the binding of the corresponding peptide of the tumor suppressor ING1 is extremely weak, indicating that slight deviations from the consensus PIP-box sequence dramatically reduce the affinity for PCNA, in contrast with a proposed less stringent PIP-box sequence requirement. We could not detect any binding between PCNA and the MCL-1 or the CDK2 protein, reported to interact with PCNA in biochemical assays. This suggests that they do not bind directly to PCNA, or they do but very weakly, with additional unidentified factors stabilizing the interactions in the cell. Backbone dynamics measurements show three PCNA regions with high relative flexibility, including the interdomain connector loop (IDCL) and the C-terminus, both of them involved in the interaction with the PIP-box. Our work provides the basis for high resolution studies of direct ligand binding to PCNA in solution.     

Dynamic Light Scattering Of Biopolymers And Biocolloid

Abstract

Of the physical techniques available for the study of biopolymers and other macromolecular and colloidal systems, dynamic light scattering is, perhaps, the most fascinating. The very idea of extracting useful dynamical and structural information from random fluctuations in the intensity of light scattered from otherwise unperturbed equilibrium solutions^1-3 seems, at first glance, almost too good to be true. Indeed, for fragile preparations that are prone to suffer undesirable fates at the slightest change in solution conditions, dynamic light scattering may be the perfect technique. It is nondestructive, relatively rapid, and typically requires only modest amounts of material. In routine applications, even on unfamiliar samples, the dust-removal procedure (either gravity flow Millipore ® filtration or low-speed centrifugation) requires 10 to 20 min, and determination of the translational diffusion coefficient to an accuracy of a few percent, or less, requires 5 min. Verification that the dynamical motions, in fact, obey the diffusion equation requires an additional 5 to 20 min. The amounts of solution required are typically a few milliliters, although similar experiments have been run on volumes as small as 15 μl.⁴ The concentrations needed, which depend on salt concentration and refractive index increment, and inversely on molecular weight, generally lie in the range 0.5 to 2.0 mg/ml for polypeptides with M_r ～ 100, 000.     

Cell Aggregation in Cultures of <Emphasis Type="Italic">Micrococcus luteus</Emphasis>, Studied by Dynamic Light Scattering

Cell aggregation was studied using the method of dynamic light scattering in the course of growth of Micrococcus luteus cultures in a liquid medium. The method detects particles ranging in size from 0.5 to 1000 μm in samples containing no more than 10⁵ cells/ml. When grown in liquid media, M. luteus forms aggregates; during the lag phase, 80% of the cells are found in aggregates of 10–1000 μm, only minor amounts being represented by single cells. With the onset of exponential growth, the aggregates were decomposed and single cells became prevalent in the culture liquid. This observation confirms that the aggregation of the cells during the lag phase is prerequisite to the initiation of bacterial growth. The method may be used in biotechnology for monitoring the state of bacterial cultures. __________ Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 6, 2005, pp. 647–651. Original Russian Text Copyright ? 2005 by Voloshin, Kaprelyants.     

Monitoring the Stability of Perfluorocarbon Nanoemulsions by Cryo-TEM Image Analysis and Dynamic Light Scattering

Perfluorocarbon nanoemulsions (PFC-NE) are disperse systems consisting of nanoscale liquid perfluorocarbon droplets stabilized by an emulsifier, usually phospholipids. Perfluorocarbons are chemically inert and non-toxic substances that are exhaled after in vivo administration. The manufacture of PFC-NE can be done in large scales by means of high pressure homogenization or microfluidization. Originally investigated as oxygen carriers for cases of severe blood loss, their application nowadays is more focused on using them as marker agents in ¹⁹F Magnetic Resonance Imaging (¹⁹F MRI). ¹⁹F is scarce in organisms and thus PFC-NE are a promising tool for highly specific and non-invasive imaging of inflammation via ¹⁹F MRI. Neutrophils, monocytes and macrophages phagocytize PFC-NE and subsequently migrate to inflamed tissues. This technique has proven feasibility in numerous disease models in mice, rabbits and mini pigs. The translation to clinical trials in human needs the development of a stable nanoemulsion whose droplet size is well characterized over a long storage time. Usually dynamic light scattering (DLS) is applied as the standard method for determining particle sizes in the nanometer range. Our study uses a second method, analysis of transmission electron microscopy images of cryo-fixed samples (Cryo-TEM), to evaluate stability of PFC-NE in comparison to DLS. Four nanoemulsions of different composition are observed for one year. The results indicate that DLS alone cannot reveal the changes in particle size, but can even mislead to a positive estimation of stability. The combination with Cryo-TEM images gives more insight in the particulate evolution, both techniques supporting one another. The study is one further step in the development of analytical tools for the evaluation of a clinically applicable perfluorooctylbromide nanoemulsion.     

Dynamic Light Scattering Techniques and Their Applications in Food Science

This paper reviews recent developments in dynamic light scattering and their application to the study of particle sizes, structures and interactions in food materials. Results obtained in concentrated and highly turbid suspensions via the recently developed technique of diffusing wave spectroscopy (DWS) are described. Problems in the detailed analysis of the information contained in DWS are described, and the possible future uses of the techniques are discussed.     

Dynamic Light Scattering Studies on Hydrodynamic Properties of Fibrinogen-Fibronectin Complex

Abstract

A high molecular weight ‘cryogel’ was obtained as insoluble complexes by cold incubation at near-freezing temperatures from heparinized plasma of patients with rheumatoid arthritis. After the cryogel was solubilized at 37°C, 1:1 complex of fibrinogen and fibronectin was purified at room temperature by affinity chromatography on a gelatin-Sepharose 4B. Hydrodynamic properties of the complex were investigated as a function of temperature and NaCl concentration using a dynamic light scattering. The diffusion coefficients of the complex at 20°C decreased with increasing of NaCl concentration as free fibronectin. The complex appears to be a more compact form at low ionic concentration, which is associated with conformational changes of fibronectin. The diffusion coefficient of the complex at 20°C in 0.05 M Tris- HCl(pH7.4) containing 0.5 M NaCl was estimated as 8.5× 10^?8 cm²s^?1. The complex did not dissociate over the temperature range from 20 to 37°C. The diffusion coefficients of the complex decreased significantly at 12°C and 40°C. The thermal denaturation of fibrinogen molecule in the complex was observed at 40°C. The CONTIN analysis of the light scattering data showed that the complex associated to form higher aggregates at 15°C, but not at near- freezing temperature. The equilibrium between the complex and higher aggregates appeared reversible.     

Vesicle Adhesion and Fusion Studied by Small-Angle X-Ray Scattering

We have studied the adhesion state (also denoted by docking state) of lipid vesicles as induced by the divalent ions Ca²⁺ or Mg²⁺ at well-controlled ion concentration, lipid composition, and charge density. The bilayer structure and the interbilayer distance in the docking state were analyzed by small-angle x-ray scattering. A strong adhesion state was observed for DOPC:DOPS vesicles, indicating like-charge attraction resulting from ion correlations. The observed interbilayer separations of ～1.6 nm agree quantitatively with the predictions of electrostatics in the strong coupling regime. Although this phenomenon was observed when mixing anionic and zwitterionic (or neutral) lipids, pure anionic membranes (DOPS) with highest charge density σ resulted in a direct phase transition to a multilamellar state, which must be accompanied by rupture and fusion of vesicles. To extend the structural assay toward protein-controlled docking and fusion, we have characterized reconstituted N-ethylmaleimide-sensitive factor attachment protein receptors in controlled proteoliposome suspensions by small-angle x-ray scattering.     

Self Crowding of Globular Proteins Studied by Small-Angle X-Ray Scattering

Small-angle x-ray scattering (SAXS) was used to study the behavior of equine metmyoglobin (Mb) and bovine pancreatic trypsin inhibitor (BPTI) at concentrations up to 0.4 and 0.15 g/mL, respectively, in solutions also containing 50% D₂O and 1 M urea. For both proteins, significant effects because of interference between x-rays scattered by different molecules (interparticle interference) were observed, indicating nonideal behavior at high concentrations. The experimental data were analyzed by comparison of the observed scattering profiles with those predicted by crystal structures of the proteins and a hard-sphere fluid model used to represent steric exclusion effects. The Mb scattering data were well fit by the hard-sphere model using a sphere radius of 18 Å, only slightly smaller than that estimated from the three-dimensional structure (20 Å). In contrast, the scattering profiles for BPTI in phosphate buffer displayed substantially less pronounced interparticle interference than predicted by the hard-sphere model and the radius estimated from the known structure of the protein (15 Å). Replacing the phosphate buffer with 3-(N-morpolino)propane sulfonic acid (MOPS) led to increased interparticle interference, consistent with a larger effective radius and suggesting that phosphate ions may mediate attractive intermolecular interactions, as observed in some BPTI crystal structures, without the formation of stable oligomers. The scattering data were also used to estimate second virial coefficients for the two proteins: 2.0 ×10^-4 cm³mol/g² for Mb in phosphate buffer, 1.6 ×10^-4 cm³mol/g² for BPTI in phosphate buffer and 9.2 ×10^-4 cm³mol/g² for BPTI in MOPS. The results indicate that the behavior of Mb, which is nearly isoelectric under the conditions used, is well described by the hard-sphere model, but that of BPTI is considerably more complex and is likely influenced by both repulsive and attractive electrostatic interactions. The hard-sphere model may be a generally useful tool for the analysis of small-angle scattering data from concentrated macromolecular solutions.     

Self Crowding of Globular Proteins Studied by Small-Angle X-Ray Scattering

Small-angle x-ray scattering (SAXS) was used to study the behavior of equine metmyoglobin (Mb) and bovine pancreatic trypsin inhibitor (BPTI) at concentrations up to 0.4 and 0.15 g/mL, respectively, in solutions also containing 50% D₂O and 1 M urea. For both proteins, significant effects because of interference between x-rays scattered by different molecules (interparticle interference) were observed, indicating nonideal behavior at high concentrations. The experimental data were analyzed by comparison of the observed scattering profiles with those predicted by crystal structures of the proteins and a hard-sphere fluid model used to represent steric exclusion effects. The Mb scattering data were well fit by the hard-sphere model using a sphere radius of 18 Å, only slightly smaller than that estimated from the three-dimensional structure (20 Å). In contrast, the scattering profiles for BPTI in phosphate buffer displayed substantially less pronounced interparticle interference than predicted by the hard-sphere model and the radius estimated from the known structure of the protein (15 Å). Replacing the phosphate buffer with 3-(N-morpolino)propane sulfonic acid (MOPS) led to increased interparticle interference, consistent with a larger effective radius and suggesting that phosphate ions may mediate attractive intermolecular interactions, as observed in some BPTI crystal structures, without the formation of stable oligomers. The scattering data were also used to estimate second virial coefficients for the two proteins: 2.0 ×10^-4 cm³mol/g² for Mb in phosphate buffer, 1.6 ×10^-4 cm³mol/g² for BPTI in phosphate buffer and 9.2 ×10^-4 cm³mol/g² for BPTI in MOPS. The results indicate that the behavior of Mb, which is nearly isoelectric under the conditions used, is well described by the hard-sphere model, but that of BPTI is considerably more complex and is likely influenced by both repulsive and attractive electrostatic interactions. The hard-sphere model may be a generally useful tool for the analysis of small-angle scattering data from concentrated macromolecular solutions.     

Dynamic Light Scattering Study of Inhibition of Nucleation and Growth of Hydroxyapatite Crystals by Osteopontin

We study the effect of isoforms of osteopontin (OPN) on the nucleation and growth of crystals from a supersaturated solution of calcium and phosphate ions. Dynamic light scattering is used to monitor the size of the precipitating particles and to provide information about their concentration. At the ion concentrations studied, immediate precipitation was observed in control experiments with no osteopontin in the solution, and the size of the precipitating particles increased steadily with time. The precipitate was identified as hydroxyapatite by X-ray diffraction. Addition of native osteopontin (nOPN) extracted from rat bone caused a delay in the onset of precipitation and reduced the number of particles that formed, but the few particles that did form grew to a larger size than in the absence of the protein. Recombinant osteopontin (rOPN), which lacks phosphorylation, caused no delay in initial calcium phosphate precipitation but severely slowed crystal growth, suggesting that rOPN inhibits growth but not nucleation. rOPN treated with protein kinase CK2 to phosphorylate the molecule (p-rOPN) produced an effect similar to that of nOPN, but at higher protein concentrations and to a lesser extent. These results suggest that phosphorylations are critical to OPN’s ability to inhibit nucleation, whereas the growth of the hydroxyapatite crystals is effectively controlled by the highly acidic OPN polypeptide. This work also demonstrates that dynamic light scattering can be a powerful tool for delineating the mechanism of protein modulation of mineral formation.     

多分散性动态光散射数据反演研究

动态光散射数据反演属于第一类Fredholm积分方程的求解,存在着极大的困难.利用最小二乘法和奇异值分解法对不同分布的多分散性动态光散射模拟数据进行了反演计算.数值测试的结果表明,非负最小二乘法对分布形式较为敏感,奇异值分解法能反演双峰的分布,对窄峰的反演不及非负最小二乘法.     

Inelastic Light Scattering by Large Structured Particles

Autocorrelation functions are computed for nonspherical particles whose dimensions are comparable to or greater than the wavelength of scattered light. Particular attention is given to models of motile microorganisms. Results for Gaussian ellipsoids, finite thin rods, ellipsoids with internal structures, and dumbbell-shaped scatterers are derived and compared.     

Simultaneous Measurement of Amyloid Fibril Formation by Dynamic Light Scattering and Fluorescence Reveals Complex Aggregation Kinetics

An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington''s disease, in vitro. Huntington''s disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer''s and Parkinson''s disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation.