Hydrodynamic modes of arbitrarily shaped flexible macromolecules: Influence on dynamic light scattering

The diffusional motions of flexible macromolecules are analyzed with an increasingly realistic Rouse–Zimm model, i.e., by modeling the molecule as an arbitrary set of spheres connected by nearly harmonic springs. New features include (1) nearly arbitrary arrangements of spheres, (2) arbitrary arrangements of translational and torsional springs, (3) significant anharmonic corrections to the elastic potential surface, and (4) inclusion of torsional damping and various hydrodynamic cross-coupling effects (including two types of translational-rotational coupling) with no additional fitted parameters. The hydrodynamic interactions [R. F. Goldstein (1985) Journal of Chemical Physics, Vol. 83, pp. 2390–2397] contain no adjustable parameters other than temperature, viscosity, and the radii and positions of the spheres. These hydrodynamic interactions allow accurate calculations of rigid body diffusion as well as flexible motions. Given the positions, radii, and spring constant matrix, one can calculate a full set of three-dimensional diffusional modes. Because one uses an off-diagonal hydrodynamic resistance matrix instead of a diagonal mass matrix, the diffusional modes are different in structure from vacuum normal modes, and give rise to different rms motions in the laboratory frame. These hydrodynamic modes include the effects of vibrational-translational cross-coupling (i.e., motion along a vibrational coordinate may give rise to a translational force, and vice versa). The diffusional modes are used to simulate dynamic light scattering (DLS). I examine various molecules with different shapes, flexibilities, and with different scattering vectors. Radial and angular motions influence DLS decays differently. These effects are dependent upon the molecular shape (straight, bent, or curved) and type of flexibility (stretching or bending). Furthermore, small cubic corrections to the potential surface can be significant for DLS of certain geometries such as straight rods and semicircles. © 1993 John Wiley & Sons, Inc.     

Binding of Janus kinase inhibitor tofacitinib with human serum albumin: multi-technique approach

In this report, we have investigated the binding affinity of tofacitinib with human serum albumin (HSA) under simulated physiological conditions by using UV–visible spectroscopy, fluorescence quenching measurements, dynamic light scattering (DLS), differential scanning calorimetry (DSC) and molecular docking methods. The obtained results demonstrate that fluorescence intensity of HSA gets quenched by tofacitinib and quenching occurs in static manner. Binding parameters calculated from modified Stern–Volmer equation shows that the drug binds to HSA with a binding constant in the order of 10⁵. Synchronous fluorescence data deciphered the change in the microenvironment of tryptophan residue in HSA. UV spectroscopy and DLS measurements deciphered complex formation and reduction in hydrodynamic radii of the protein, respectively. Further DSC results show that tofacitinib increases the thermo stability of HSA. Hydrogen bonding and hydrophobic interaction are the main binding forces between HSA and tofacitinib as revealed by docking results.     

Dynamic light scattering study of peanut agglutinin: Size, shape and urea denaturation

Peanut agglutinin (PNA) is a homotetrameric protein with a unique open quaternary structure. PNA shows non-two state profile in chaotrope induced denaturation. It passes through a monomeric molten globule like state before complete denaturation (Reddyet al 1999). This denaturation profile is associated with the change in hydrodynamic radius of the native protein. Though the molten globule-like state is monomeric in nature it expands in size due to partial denaturation. The size and shape of the native PNA as well as the change in hydrodynamic radius of the protein during denaturation has been studied by dynamic light scattering (DLS). The generation of two species is evident from the profile of hydrodynamic radii. This study also reveals the extent of compactness of the intermediate state.     

Effects of arginine on kinetics of protein aggregation studied by dynamic laser light scattering and tubidimetry techniques

Prevention of undesirable protein aggregation is an extremely important strategy in protein science, medicine, and biotechnology. Arginine is one of the most widely used low molecular weight solution additives effective in suppressing aggregation, assisting refolding of aggregated proteins, and enhancing the solubility of aggregation-prone unfolded molecules in vitro. However, the mechanism of suppression of protein aggregation by arginine is not well understood. To address the mechanism, two model systems have been investigated: protection of alcohol dehydrogenase (ADH) and insulin from heat- and dithiothreitol-induced aggregation, respectively, in the presence of arginine. Using dynamic light scattering (DLS) technique, we have demonstrated the concentration-dependent suppression of light scattering intensity of both ADH and insulin aggregates upon addition of arginine to the incubation medium, a significant effect being revealed in the physiological concentration range of arginine (1-10 mM). DLS studies showed that arginine shifted the populations of nanoparticles with higher hydrodynamic radii to the lower ones, suggesting that the preventive effect of arginine on the protein aggregation process arises because it suppresses intermolecular interactions among aggregation-prone molecules. The results of turbidity measurements were also shown to be consistent with these findings.     

应用动态光散射对一些蛋白质结晶性能的研究

动态光散射是研究生物大分子溶液物化行为和结晶性能的重要工具。以溶菌酶为模型蛋白 ,对其溶液的DLS研究表明 ,一定范围的蛋白质浓度不会影响蛋白质聚合性质 ,而起沉淀剂作用的盐对多色散性的影响较大。考虑这些研究结果 ,应用DLS研究对空间微重力下晶体生长样品的质量做了考察 ,这对于提高空间样品准备水平 ,进而提高空间实验成功率是一重要途径。     

Jack bean urease (EC 3.5.1.5) aggregation monitored by dynamic and static light scattering

Aggregation of jack bean urease (JBU) is involved in many alterations of its biological properties, notably the ureolytic and entomotoxic activities. In order to investigate this phenomenon, protein aggregates were characterized by dynamic (DLS) and static light scattering (SLS) spectroscopies through determination of apparent hydrodynamic radii, the average molecular masses, radii of gyration and second virial coefficients. No effect of disulfide reducing agents on protein association was observed contrasting with previous reports implicating their function in the prevention of JBU aggregation. The influence of freeze-thawing cycles on protein aggregation was also investigated. Our results showed that after freeze-thawing cycles the native form of JBU with apparent hydrodynamic radius of 7 nm and radius of gyration of 12 nm is replaced by high-order oligomers and this aggregation is not reverted neither by dithiothreitol (DTT) treatment nor by high concentration of salts. Altogether the data help to understand the complex behavior of JBU in solution and may correlate with the diversity of biological properties of this enzyme.     

A first-order-like state transition for recombinant protein folding

Normally, proteins will aggregate and precipitate by direct folding processes. In this study, we report that quasi-static processes can restore both the structure and bio-function of two kinds of fish recombinant growth hormones (Plecoglossus altivelis and Epinephelus awoara). The conformational changes and the particle-size-distribution (PSD) of each refolding intermediate can be monitored by circular dichroism spectroscopy (CD) and dynamic light scattering (DLS), respectively. Conformation analysis of the CD spectra of the refolding intermediates indicated that the secondary structures were restored in the initial refolding intermediate. However, the tertiary interactions of the proteins were restored during the last two refolding stages, as elucidated by thermal stability tests. This is consistent with a sequential model. DLS analysis suggested that the average hydrodynamic radii of the refolding intermediates shrank to their native-like sizes after the first refolding stage. This is consistent with a collapse model. After comparison with the data on the direct folding process, it is concluded that the denaturant-containing protein folding reaction is a first-order-like state transition process.     

Hydrodynamic properties of nitrogenase — the MoFe protein from Azotobacter vinelandii studied by dynamic light scattering and hydrodynamic modelling

Experimental results for the nitrogenase MoFe protein from Azotobacter vinelandii obtained by dynamic light scattering (DLS) are presented. The translational diffusion coefficient was determined to D=(4.0±0.2)×10⁻⁷ cm²/s. Complementary, we have performed hydrodynamic model calculations based on the X-ray crystallographic data of the MoFe protein. The calculated transport coefficient suggests that the size and shape of the protein in solution is consistent with that in the crystal structure.     

Dynamic light scattering study of calmodulin-target peptide complexes

Dynamic light scattering (DLS) has been used to assess the influence of eleven different synthetic peptides, comprising the calmodulin (CaM)-binding domains of various CaM-binding proteins, on the structure of apo-CaM (calcium-free) and Ca(2+)-CaM. Peptides that bind CaM in a 1:1 and 2:1 peptide-to-protein ratio were studied, as were solutions of CaM bound simultaneously to two different peptides. DLS was also used to investigate the effect of Ca(2+) on the N- and C-terminal CaM fragments TR1C and TR2C, and to determine whether the two lobes of CaM interact in solution. The results obtained in this study were comparable to similar solution studies performed for some of these peptides using small-angle x-ray scattering. The addition of Ca(2+) to apo-CaM increased the hydrodynamic radius from 2.5 to 3.0 nm. The peptides studied induced a collapse of the elongated Ca(2+)-CaM structure to a more globular form, decreasing its hydrodynamic radius by an average of 25%. None of the peptides had an effect on the conformation of apo-CaM, indicating that either most of the peptides did not interact with apo-CaM, or if bound, they did not cause a large conformational change. The hydrodynamic radii of TR1C and TR2C CaM fragments were not significantly affected by the addition of Ca(2+). The addition of a target peptide and Ca(2+) to the two fragments of CaM, suggest that a globular complex is forming, as has been seen in nuclear magnetic resonance solution studies. This work demonstrates that dynamic light scattering is an inexpensive and efficient technique for assessing large-scale conformational changes that take place in calmodulin and related proteins upon binding of Ca(2+) ions and peptides, and provides a qualitative picture of how this occurs. This work also illustrates that DLS provides a rapid screening method for identifying new CaM targets.     

pH-Dependent Conformational Transitions in Conalbumin (Ovotransferrin), a Metalloproteinase from Hen Egg White

Acid unfolding pathway of conalbumin (CA), a monomeric glycoprotein from hen egg white, has been investigated using far- and near-UV CD spectroscopy, intrinsic fluorescence emission, extrinsic fluorescence probe 1-anilino-8-napthalene sulfonate (ANS) and dynamic light scattering (DLS). We observe pH-dependent changes in secondary and tertiary structure of CA. It has native-like α-helical secondary structure at pH 4.0 but loss structure at pH 3.0. The CA existed exclusively as a pre-molten globule state and molten globule state in solution at pH 4.0 and pH 3.0, respectively. The effect of pH on the conformation and thermostability of CA points toward its heat resistance at neutral pH. DLS results show that MG state existed as compact form in aqueous solutions with hydrodynamic radii of 4.7 nm. Quenching of tryptophan fluorescence by acrylamide further confirmed the accumulation of an intermediate state, partly unfolded, in-between native and unfolded states.     

High-throughput dynamic light scattering method for measuring viscosity of concentrated protein solutions

We propose a new method to measure the viscosity of concentrated protein solutions in a high-throughput format. This method measures the apparent hydrodynamic radius of polystyrene beads with known sizes using a dynamic light scattering (DLS) system with a microplate reader. Glycerol solution viscosities obtained by the DLS method were in good agreement with those reported in the literature. Viscosity of the solutions of two monoclonal antibody molecules was acquired using both DLS and cone-and-plate techniques, and the results were comparable. The DLS method described here has the potential to be used in many aspects of protein characterization.     

Detection of monodisperse aggregates of a recombinant amelogenin by dynamic light scattering

Recombinant murine amelogenins M179 and M166 were expressed in Escherichia coli and purified. The aggregation properties of these amelogenins have been investigated in aqueous solutions as well as acetonitrile-containing solutions using dynamic light scattering. Dynamic light scattering provides direct measurement of the translational diffusion coefficient and hydrodynamic radius, and of an estimate of the molecular weight. Polydispersity and statistical parameters of how to interpret the analysis are also provided. Amelogenin aggregation was examined in solutions of a range of pH, ionic strengths, and protein concentrations. It was shown that at pH 7.8–8 and ionic strength of 0.02–0.05M the M179 molecules form monodispersed aggregates with hydrodynamic radii ranging from 15 to 19 nm. Analysis of hydrodynamic radii and size distribution of M179 aggregates in acetonitrile-containing solvents compared to that in aqueous solutions indicated a primary role for hydrophobic interactions in the association process of amelogenin molecules to form aggregates. Comparison between the aggregates formed by M179 and M166, which lacks the hydrophilic carboxy-terminal 13 residue sequence of M179, suggested that the self-assembly of amelogenin molecules to form stable and monodisperse aggregates requires the presence of the hydrophilic carboxy-terminal sequence of M179. © 1994 John Wiley & Sons, Inc.     

生物科学中一个崭露头角的领域：高静压力研究

通过介绍高静压力作用于生物大分子的机制,提出一些有关高压力生物学的基本概念。此外,还介绍了高静压力在研究蛋白质构象,蛋白质－蛋白质、蛋白质－核酸、蛋白质－配基等相互作用,酶活性的调制以及在生物技术中的应用,表明高压力是一很好的研究手段。     

Neutron and light-scattering studies of DNA gyrase and its complex with DNA

The solution structure of Escherichia coli DNA gyrase, an enzyme that catalyzes the ATP-dependent supercoiling of DNA, has been characterized by small-angle neutron scattering (SANS) and dynamic light-scattering (DLS). The enzyme and its complex with a 172 base-pair fragment of duplex DNA, in H2O or 2H2O solvent, were studied by contrast variation and the measurement of hydrodynamic parameters as a function of scattering angle. The complex was also measured in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP), a non-hydrolyzable ATP analog that is known to support limited supercoiling. The values of the radius of gyration, Rg = 67 A, from SANS and the hydrodynamic radius, Rh = 64 A, from DLS predict a larger than expected volume for the enzyme, supporting the notion of channels or cavities within the molecule. In addition, several classes of models were rejected based on SANS data obtained in 2H2O at larger scattering angles. The best fit to both the SANS and DLS data is obtained for oblate, inhomogeneous particles approximately 175 A wide and 52 A thick. Such particles provide a large surface area for DNA interaction. Both Rg and Rh values change very little upon addition of DNA, suggesting that DNA binds in a manner that does not significantly change the shape of the protein. No appreciable change in structure is found with the addition of ADPNP. However, the higher-angle SANS data indicate a slight rearrangement of the enzyme in the presence of nucleotide.     

Size and shape of detergent-solubilized photochemical reaction centers from two strains of Rhodopseudomonas sphaeroides. A solution X-ray scattering study

Solubilized reaction centers purified from Rhodopseudomonas sphaeroides (wild type and R26 strains) were studied in a nondenaturing detergent, dodecyldimethylamine N-oxide, by solution X-ray scattering. Some thermodynamic parameters were also obtained by coupling the results of this study with sedimentation equilibrium data previously obtained (Rivas, E., Reiss-Husson, F and le Maire, M. (1980) Biochemistry 19, 2943–2950). The particle weight of both types of reaction centers was found to be about 160 000 Da, corresponding to a protein molecular weight close to 90 000. Both hydrodynamic and solution X-ray scattering experiments suggest that the complexes have a globular shape, with a maximal chord of about 90 Å as indicated by the autocorrelation function. This maximal dimension is probably created by the binding of detergent to the solubilized complex. The approach followed in this study to investigate the shape of detergent-protein complexes involved a comparison of the Stokes' radii and the radii of gyration of various proteins.     

Characterization of a fluorophore binding RNA aptamer by fluorescence correlation spectroscopy and small angle X-ray scattering

Using fluorescence correlation spectroscopy (FCS), we have established an in vitro assay to study RNA dynamics by analyzing fluorophore binding RNA aptamers at the single molecule level. The RNA aptamer SRB2m, a minimized variant of the initially selected aptamer SRB-2, has a high affinity to the disulfonated triphenylmethane dye sulforhodamine B. A mobility shift of sulforhodamine B after binding to SRB2m was measured. In contrast, patent blue V (PBV) is visible only if complexed with SRB2m due to increased molecular brightness and minimal background. With small angle X-ray scattering (SAXS), the three-dimensional structure of the RNA aptamer was characterized at low resolution to analyze the effect of fluorophore binding. The aptamer and sulforhodamine B-aptamer complex was found to be predominantly dimeric in solution. Interaction of PBV with SRB2m led to a dissociation of SRB2m dimers into monomers. Radii of gyration and hydrodynamic radii, gained from dynamic light scattering, FCS, and fluorescence cross-correlation experiments, led to comparable conclusions. Our study demonstrates how RNA-aptamer fluorophore complexes can be simultaneously structurally and photophysically characterized by FCS. Furthermore, fluorophore binding RNA aptamers provide a tool for visualizing single RNA molecules.     

Hydration and Hydrodynamic Interactions of Lysozyme: Effects of Chaotropic versus Kosmotropic Ions

Using static and dynamic light scattering we have investigated the effects of either strongly chaotropic, nearly neutral or strongly kosmotropic salt ions on the hydration shell and the mutual hydrodynamic interactions of the protein lysozyme under conditions supportive of protein crystallization. After accounting for the effects of protein interaction and for changes in solution viscosity on protein diffusivity, protein hydrodynamic radii were determined with ±0.25 Å resolution. No changes to the extent of lysozyme hydration were discernible for all salt-types, at any salt concentration and for temperatures between 15-40°C. Combining static with dynamic light scattering, we also investigated salt-induced changes to the hydrodynamic protein interactions. With increased salt concentration, hydrodynamic interactions changed from attractive to repulsive, i.e., in exact opposition to salt-induced changes in direct protein interactions. This anti-correlation was independent of solution temperature or salt identity. Although salt-specific effects on direct protein interactions were prominent, neither protein hydration nor solvent-mediated hydrodynamic interactions displayed any obvious salt-specific effects. We infer that the protein hydration shell is more resistant than bulk water to changes in its local structure by either chaotropic or kosmotropic ions.     

Size-Dependent Diffusion of Dextrans in Excised Porcine Corneal Stroma

Delivery of therapeutic agents to the eye requires efficient transport through cellular and extracellular barriers. We evaluated the rate of diffusive transport in excised porcine corneal stroma using fluorescently labeled dextran molecules with hydrodynamic radii ranging from 1.3 to 34 nm. Fluorescence correlation spectroscopy (FCS) was used to measure diffusion coefficients of dextran molecules in the excised porcine corneal stroma. The preferential sensitivity of FCS to diffusion along two dimensions was used to differentially probe diffusion along the directions parallel to and perpendicular to the collagen lamellae of the corneal stroma. In order to develop an understanding of how size affects diffusion in cornea, diffusion coefficients in cornea were compared to diffusion coefficients measured in a simple buffer solution. Dextran molecules diffuse more slowly in cornea as compared to buffer solution. The reduction in diffusion coefficient is modest however (67% smaller), and is uniform over the range of sizes that we measured. This indicates that, for dextrans in the 1.3 to 34 nm range, the diffusion landscape of corneal stroma can be represented as a simple liquid with a viscosity approximately 1.5 times that of water. Diffusion coefficients measured parallel vs. perpendicular to the collagen lamellae were indistinguishable. This indicates that diffusion in the corneal stroma is not highly anisotropic. Our results support the notion that the corneal stroma is highly permeable and isotropic to transport of hydrophilic molecules and particles with hydrodynamic radii up to at least 34 nm.     

A label-free nanoparticle aggregation assay for protein complex/aggregate detection and study

The detection, analysis, and understanding of protein complexes/aggregates and their formation process are extremely important for biomolecular research, diagnosis, and biopharmaceutical development. Unfortunately, techniques that can be used conveniently for protein complex/aggregate detection and analysis are very limited. Using gold nanoparticle immunoprobes coupled with dynamic light scattering (DLS), we developed a label-free nanoparticle aggregation immunoassay (NanoDLSay) for protein aggregate detection and study. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a protein target used routinely in Western blot as a loading control, is demonstrated here as an example. Through this study, we discovered that GAPDH has a strong tendency to form large aggregates in certain buffer solutions at a concentration range of 10-25 μg/ml. The strong light scattering property of gold nanoparticles immunoprobes greatly enhanced the sensitivity of the dynamic light scattering for protein complex/aggregate detection. In contrast to fluorescence techniques for protein complex and aggregation study, the protein targets do not need to be labeled with fluorescent probe molecules in NanoDLSay. NanoDLSay is a very convenient and sensitive tool for protein complex/aggregate detection and study.