Structure of clathrin-coated vesicles from small-angle scattering experiments

Previously published small-angle neutron and X-ray scattering data from coated vesicles, reassembled coats, and stripped vesicles have been analyzed in terms of one common model. The neutron data sets include contrast variation measurements at three different D20 solvent concentrations. The model used for interpreting the data has spherical symmetry and explicitly takes into account polydispersity, which is described by a Gaussian distribution. Å constant thickness of the clathrin coats is assumed. The fitting of the model shows that the coated vesicles consist of a low-density outer protein shell (clathrin) and a central protein shell (accessory polypeptides and receptors) of approximately six times higher density. For the X-ray scattering and neutron contrast variation data, the polydispersity of the samples is of the order of 90 Å (full-width-at-half-maximum value) and the average outer radius is approximately 400 Å. The inner high-density shell has inner and outer radii of 115 and 190 Å, respectively. Å simultaneous fit to the three neutron contrast variation data sets identifies the lipid membrane with a thickness of 40 Å and an outer radius of 196 Å. Thus, the membrane and the high-density protein shell overlap in space, which shows that the lipid membrane contains protein. The molecular mass of the average particle is 27 × 106 Da. The coated vesicles consist, on average, of approximately 85 % protein and 15 lipids. About 40% of the protein mass is situated in the central high-density shell, which gives a large amount of protein in the lipid membrane. The densities of the central shell and the lipid membrane show that the hydration is small in the central region. Å comparison of the total mass, the mass distribution, and the structure of the average-size particles with the barrel structure shows that the accessory polypeptides are incorporated in the lipid membrane. The results from the neutron data for the reassembled coats show that the structure of these particles is very similar to the structure of the native coats. The main difference is a higher density of the central protein shell, which shows that the membrane is replaced by protein in the reassembled coats.     

Structural themes and variations in protein kinase A as seen by small-angle scattering and neutron contrast variation

Using small-angle solution scattering and neutron contrast variation, we have studied the structure of the multi-subunit protein kinase A. We have gained insights into how nature can take a set of common structural domains (or themes) and modulate their interactions via sequence variations and second messenger mediated signaling to affect enzyme activity and receptor binding important for targeting this multi-function enzyme to specific sub-cellular locations. These studies demonstrate the power of neutron contrast variation to expand our knowledge of the dynamic supra-molecular structures that carry out biological function.     

Structure of casein micelles studied by small-angle neutron scattering

The structure of casein micelles has been studied by small-angle neutron scattering and static light scattering. Alterations in structure upon variation of pH and scattering contrast, as well as after addition of chymosin, were investigated. The experimental data were analyzed by a model in which the casein micelle consists of spherical submicelles. This model gave good agreement with the data and gave an average micellar radius of about 100–120 nm and a submicellar radius of about 7 nm both with a polydispersity of about 40–50%. The contrast variation indicated that the scattering length density of the submicelles was largest at the center of the submicelles. The submicelles were found to be closely packed, the volume fraction varying slightly with pH. Upon addition of chymosin the submicellar structure remained unchanged within the experimental accuracy.Correspondence to: S. Hansen     

The aggregation behavior of zinc-free insulin studied by small-angle neutron scattering

The aggregation behavior of zinc-free insulin has been studied by small-angle neutron scattering as a function of pH and ionic strength of the solution. The pair distance distribution functions for the 12 samples have been obtained by indirect Fourier transformation. The results show that the diameter of the aggregates is 40 Å at pH 11 and 10 mM NaCl, independent of the protein concentration. The largest diameter of about 120 Å is found for pH 8, 100 mM NaCl, and a protein concentration of 10 mg/ml. Estimates of the pair distance distribution functions, free of inter-particle correlation effects, were obtained by an indirect Fourier transformation, omitting the data at small scattering vectors, which are influenced by these effects. By this procedure the weight-averaged molecular mass and the average radius of gyration were determined. These parameters vary from 1.3 times the monomer mass and 14 Å, to 6.8 times the monomer mass and 31 Å, respectively. The mass distribution between the oligomers was determined by a model based on the crystal structure of zinc-free insulin. The results from this model and the Fourier transformations have been compared to an equilibrium model recently introduced by Kadima et al. (1993). The neutron scattering results agree well with the predictions of this model except that broader mass distributions are suggested by neutron scattering.Correspondence to: J. Skov Pedersen     

Protein kinase A targeting and activation as seen by small-angle solution scattering

We have studied the solution structures of the multi-functional protein kinase A using small-angle X-ray and neutron scattering and have found a remarkable structural diversity in the different isoforms of this multi-subunit enzyme, in spite of its having high sequence homology and a common domain organization within its sequences. The available high-resolution crystal and NMR structural data for the protein kinase A components have aided in the interpretation of the solution scattering data and enabled us to develop models that bring insights into protein kinase A activation and targeting mechanisms, such as the opening and closing of the catalytic cleft to facilitate substrate binding or inhibition, respectively, and the role of sequence segments that join functional domains in the R subunit in providing a structurally flexible scaffold for interactions with the C subunit and A kinase-anchoring proteins (AKAPs).     

Interaction of GroEL and GroEL/GroES complexes with a nonnative subtilisin variant: a small-angle neutron scattering study

Small-angle neutron scattering and contrast variation were used to study the solution structure of GroEL and GroEL/GroES chaperonins complexed with a nonnative variant of the polypeptide substrate, subtilisin (PJ9). The subtilisin was 86% deuterated (dPJ9) so that it contrasted sufficiently with the chaperonin, allowing the contrast variation technique to be used to separate the scattering from the two components bound in the complex. Both the native double-ring GroEL and a single-ring mutant were used with dPJ9 bound in a 1:1 stoichiometry per GroEL toroid. This allowed both the position and the shape of dPJ9 in the GroEL/dPJ9 complexes to be determined. A single-ring GroEL/GroES variant complexed with one dPJ9 molecule was used to study the structural changes of dPJ9 in GroEL/GroES/dPJ9 complexes formed with ADP and with ATP. It was found that both the shape and the position of the bound dPJ9 in the GroEL/GroES/dPJ9 complex with ADP were the same as those in the GroEL/dPJ9 complex. However, dPJ9 assumed a more symmetric shape when bound in the GroEL/GroES/dPJ9 complex with ATP. This important observation reflects the relative ability of ATP to promote refolding of protein substrates relative to that of ADP.     

Vibrational mode analysis of guanine by neutron inelastic scattering

The low-temperature neutron inelastic spectrum of guanine has been measured. In order to assign the intense peaks observed in this spectrum, a normal mode analysis has been performed, using the Wilson GF-method. The theoretical treatment is based on a non-redundant set of internal coordinates, and a simplified valence force-field approximation. Only the fundamentals have been considered for simulating the internal vibrational mode spectrum. The calculations account for the spectral shape as well as the main observed peaks.Offprint requests to: M. Ghomi     

X-ray and neutron scattering studies of the hydration structure of alkali ions in concentrated aqueous solutions

The presence of ions in water provides a rich and varied environment in which many natural processes occur with important consequences in biology, geology and chemistry. This article will focus on the structural properties of ions in water and it will be shown how the 'difference' methods of neutron diffraction with isotopic substitution (NDIS) and anomalous X-ray diffraction (AXD) can be used to obtain direct information regarding the radial pair distribution functions of many cations and anions in solution. This information can subsequently be used to calculate coordination numbers and to determine ion-water conformation in great detail. As well as enabling comparisons to be made amongst ions in particular groups in the periodic table, such information can also be contrasted with results provided by molecular dynamics (MD) simulation techniques. To illustrate the power of these 'difference' methods, reference will be made to the alkali group of ions, all of which have been successfully investigated by the above methods, with the exception of the radioactive element francium. Additional comments will be made on how NDIS measurements are currently being combined with MD simulations to determine the structure around complex ions and molecules, many of which are common in biological systems.     

The onset of amelogenin nanosphere aggregation studied by small-angle X-ray scattering and dynamic light scattering

Proteins with predominantly hydrophobic character called amelogenins play a key role in the formation of the highly organized enamel tissue by forming nanospheres that interact with hydroxyapatite crystals. In the present investigation, we have studied the temperature and pH-dependent self-assembly of two recombinant mouse amelogenins, rM179 and rM166, the latter being an engineered version of the protein that lacks a 13 amino acid hydrophilic C-terminus. It has been postulated that this hydrophilic domain plays an important role in controlling the self-assembly behavior of rM179. By small-angle X-ray and neutron scattering, as well as by dynamic light scattering, we observed the onset of an aggregation of the rM179 protein nanospheres at pH 8. This behavior of the full-length recombinant protein is best explained by a core-shell model for the nanospheres, where hydrophilic and negatively charged side chains prevent the agglomeration of hydrophobic cores of the protein nanospheres at lower temperatures, while clusters consisting of several nanospheres start to form at elevated temperatures. In contrast, while capable of forming nanospheres, rM166 shows a very different aggregation behavior resulting in the formation of larger precipitates just above room temperature. These results, together with recent observations that rM179, unlike rM166, can regulate mineral organization in vitro, suggest that the aggregation of nanospheres of the full-length amelogenin rM179 is an important step in the self-assembly of the enamel matrix.     

Solution scattering studies of dimeric and tetrameric spectrin

The structure of spectrin dimers and tetramers in solution has been examined by light, low-angle X-ray and neutron scattering. The results show a good correspondence between the solution dimensions of these molecules and their appearance in the electron microscope after shadowing. The scattering profiles are not compatible with an extended rod-like character, but reflect the presence of a considerable degree of bending. The radii of gyration of the dimer and tetramer were determined to be 170 and 375 Å and the cross-section radii of gyration 14 and 12.3 Å. respectively. Both are thus long. thin. rather bent molecules, and the tetramer is twice the length of the dimer.     

Detection of new double-membrane structures in native mitochondria by the method of small-angle neutron scattering

The structure of mitochondrial cristae has been studied for the first time by the method of small-angle neutron scattering. Experiments were performed on intact (functioning) mitochondria from rat liver. Mitochondrial cristae are usually considered to be folds of the inner membrane with arbitrary variable intermembrane distances. Under conditions of low-amplitude swelling, mitochondrial cristae transformed into double-membrane structures with a distance of 190 Å between the central planes of the membranes. The formation of double-membrane structures and their structural parameters did not depend on the method for inducing swelling which was accomplished either by placing the mitochondria into a hypotonic medium or through the opening of nonspecific pores.     

Shape and subunit organisation of the DNA methyltransferase M.AhdI by small-angle neutron scattering

Type I restriction-modification (R-M) systems encode multisubunit/multidomain enzymes. Two genes (M and S) are required to form the methyltransferase (MTase) that methylates a specific base within the recognition sequence and protects DNA from cleavage by the endonuclease. The DNA methyltransferase M.AhdI is a 170 kDa tetramer with the stoichiometry M(2)S(2) and has properties typical of a type I MTase. The M.AhdI enzyme has been prepared with deuterated S subunits, to allow contrast variation using small-angle neutron scattering (SANS) methods. The SANS data were collected in a number of (1)H:(2)H solvent contrasts to allow matching of one or other of the subunits in the multisubunit enzyme. The radius of gyration (R(g)) and maximum dimensions (D(max)) of the M subunits in situ in the multisubunit enzyme (50 A and 190 A, respectively) are close of those of the entire MTase (51 A and 190 A). In contrast, the S subunits in situ have experimentally determined values of R(g)=35 A and D(max)=110 A, indicating their more central location in the enzyme. Ab initio reconstruction methods yield a low-resolution structural model of the shape and subunit organization of M.AhdI, in which the Z-shaped structure of the S subunit dimer can be discerned. In contrast, the M subunits form a much more elongated and extended structure. The core of the MTase comprises the two S subunits and the globular regions of the two M subunits, with the extended portion of the M subunits most probably forming highly mobile regions at the outer extremities, which collapse around the DNA when the MTase binds.     

Dissociation of clathrin from coated vesicles by the uncoating ATPase

The uncoating ATPase has been shown to dissociate clathrin from both clathrin-coated vesicles and synthetic clathrin baskets (Rothman, J. E., and Schmid, S. L. (1986) Cell 46, 5-9). In the present study, we investigated the mechanism of action of the uncoating ATPase using intact coated vesicles isolated from bovine brain. We observed an initial burst of uncoating followed by much slower steady-state uncoating. The initial burst of uncoating was essentially stoichiometric with each molecule of uncoating ATPase apparently binding to one leg of the clathrin triskelion. When the enzyme was preincubated with equimolar ADP, Pi, and ATP, rather than just ATP alone, both the initial burst and the slow steady-state uncoating were markedly inhibited, suggesting that the combination of ADP and Pi is a strong competitive inhibitor of ATP binding. However, kinetic studies suggested that ADP and Pi dissociates from the enzyme relatively rapidly unless clathrin is also bound to the enzyme. These results suggest that, after the uncoating ATPase rapidly removes a stoichiometric amount of clathrin while ATP is hydrolyzed at the active site, slow release of ADP and Pi from the resulting enzyme.clathrin.ADP.Pi complex limits the rate at which further uncoating occurs.     

Characterization of multimetric variants of ubiquitin carboxyl-terminal hydrolase L1 in water by small-angle neutron scattering

Here, we illustrated that the morphological structures of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) variants and Parkinson's disease (PD) exhibit good pathological correlation by a small-angle neutron scattering (SANS). UCH-L1 is a neuro-specific multiple functional enzyme, deubiquitinating, ubiquityl ligase, and also involved in stabilization of mono-ubiquitin. To examine the relationship between multiple functions of UCH-L1 and the configuration of its variants [wild-type, I93M (linked to familial Parkinson's disease), and S18Y (linked to reduced risk of Parkinson's disease)], in this report, we proposed that these were all self-assembled dimers by an application of a rotating ellipsoidal model; the configurations of these dimers were quite different. The wild-type was a rotating ellipsoidal. The globular form of the monomeric component deformed by the I93M mutation. Conversely, the S18Y polymorphism promoted the globularity. Thus, the multiple functional balance is closely linked to the intermolecular interactions between the UCH-L1 monomer and the final dimeric configuration.     

Organization and dynamics of lipids in bovine brain coated and uncoated vesicles

Three characteristics have been demonstrated by the chemical analysis of bovine brain coated vesicles following removal of the coat proteins: a high protein content, a high cholesterol/lipid ratio and a high percentage of phosphatidylethanolamine amongst the phospholipids.The study of lipid bilayer organization and dynamics has been performed using the fluorescent probes pyrene and parinaric acid (cis and trans). This has allowed the study of both lateral mobility and rotational motion in the lipid bilayer of the coated and uncoated vesicles.Lateral mobility in the fluid phase of the lipid is slightly reduced by the presence of the clathrin coat, as indicated by the lower diffusion coefficient of pyrene in coated compared with uncoated vesicles.At all temperatures from 6° to 30°C, solid-phase domains, probed by trans parinaric acid, coexist with fluid-phase domains in the lipid bilayer. The temperature dependence of the parinaric acid lifetimes and of their amplitudes strongly suggests that the solid phase domains decrease in size with temperature, both in coated and uncoated vesicles.However, the difference in the value of the anisotropy at long times (r ), between coated and uncoated vesicles (a difference which is more pronounced for cis than for trans parinaric acid), indicates that the presence of the clathrin coat introduces disorder in the surrounding lipids, thus suggesting a possible role of the clathrin in the formation of the pits on the plasma membrane.Abbreviations CVs coated vesicles - UVs uncoated vesicles - TLC thin layer chromatography - DMSO dimethylsulfoxide - DPPC dipalmitoylphosphatidylcholine - cis Pna cis parinaric acid - (9,11,13,15-cis-trans-trans-cis) octadecatetraenoic acid - Trans Pna Trans parinaric acid - (9,11,13,15-all-trans) octadecatetraenoic acid     

Light scattering studies of tetramethyl ammonium gellan

Tetramethyl ammonium (TMA) gellan does not gel. Light scattering studies suggest that in solutions of TMA gellan, in tetramethyl ammonium chloride (TMACI), the gellan molecules assemble end to end to produce elongated fibrous structures. Such fibrils are envisaged as resulting from double-helix formation between the ends of neighbouring gellan molecules. Fibrils with molecular weights ranging from (1.06 ± 0.06) × 10⁵ to (4.5 ± 0.1) × 10⁶ have been observed. The molecular weights obtained depended upon the pore size of the filters used to clarify the solutions. The formation of strong gels, in the presence of gel promoting cations, is attributed to a localized ordered lateral association, or crystallization of regions of these fibrils. It is suggested that such a model for gelation may be of general applicability to a number of polysaccharide systems.     

Light scattering study of phosphatidylcholine vesicles at the pretransition

Laser light scattering has been used to investigate the thermal pretransition of dipalmitoylglycerophosphocholine vesicles with variable radius as obtained by the mild sonication method. Intensity changes in 90° scattered light are observed at the pretransition for larger vesicles and actually increase with increasing vesicle size, reaching a constant value.This constant value is in good agreement with the value calculated from the refractive index data.The intensity ratio of scattered light at temperatures of 30°C and 40°C (I₄₀/I₃₀) approaches unity at a radius of small single-bilayer vesicle. This result is interpreted as no pretransition for small vesicles in agreement with the calorimetric results. An expression of the particle scattering factor is also presented for multilayered shells composed of anisotropic elements. It is shown numerically, using this expression, that changes in the lipid layer thickness and the tilting angles at the pretransition have no effects on the scattering factor. Therefore it is concluded that the intensity changes in scattered light reflect the changes in the refractive index of the vesicle originating in the polar head groups.     

Determination of bilayer thickness and lipid surface area in unilamellar dimyristoylphosphatidylcholine vesicles from small-angle neutron scattering curves: a comparison of evaluation methods

Small-angle neutron scattering (SANS) experiments were performed on unilamellar 1,2-dimyristoylphosphatidylcholine (DMPC) vesicles prepared in heavy water by extrusion through polycarbonate filters with 500 Å pores. The data obtained at 30±0.1 °C were evaluated using a five-strip function model of the bilayer coherent neutron scattering length density, three different approximate form factors describing scattering from vesicles, and different methods of evaluation of the experimental data. It is shown that the results obtained from the SANS data in the range of scattering vector values 0.0316 Å^–1<q<0.0775 Å^–1 are not sensitive to the vesicle form factor, nor to the evaluation method. Using the hollow sphere model of vesicles convoluted with the Gaussian distribution of their sizes, a constrained bilayer polar region thickness of 9 Å and a DMPC headgroup volume of 325.5 ų, it was possible to obtain from the experimental data the DMPC surface area as 58.9±0.8 Ų, the bilayer thickness as 44.5±0.3 Å and the number of water molecules as 6.8±0.2 per DMPC located in the bilayer polar region.     

Denaturation of a halophilic enzyme monitored by small-angle neutron scattering

Malate dehydrogenase from Halobacterium maris mortui exists in 4 M-NaCl as a stable protein dimer, with which are associated unusually large amounts of salt and water. In 1 M-NaCl at 25 degrees C, it denatures with a time-constant of about 0.5 h-1. Small-angle neutron scattering data from the protein under these conditions were monitored regularly over more than 12 hours during denaturation. They are quantitatively consistent with a model in which the protein dimer loses its exceptional salt and water-binding properties, and dissociates into monomers that partially unfold and have the interactions with solvent expected from their relatively charged amino acid composition. The exceptional salt and water-binding by the native enzyme, therefore, is associated with the native structure of the dimer.     

Analytical ultracentrifugation combined with X-ray and neutron scattering: Experiment and modelling

Analytical ultracentrifugation and solution scattering provide different multi-parameter structural and compositional information on proteins. The joint application of the two methods supplements high resolution structural studies by crystallography and NMR. We summarise the procedures required to obtain equivalent ultracentrifugation and X-ray and neutron scattering data. The constrained modelling of ultracentrifugation and scattering data is important to confirm the experimental data analysis and yields families of best-fit molecular models for comparison with crystallography and NMR structures. This modelling of ultracentrifugation and scattering data is described in terms of starting models, their conformational randomisation in trial-and-error fits, and the identification of the final best-fit models. Seven applications of these methods are described to illustrate the current state-of-the-art. These include the determination of antibody solution structures (the human IgG4 subclass, and oligomeric forms of human IgA and its secretory component), the solution structures of the complement proteins of innate immunity (Factor H and C3/C3u) and their interactions with macromolecular ligands (C-reactive protein), and anionic polysaccharides (heparin). Complementary features of joint ultracentrifugation and scattering experiments facilitate an improved understanding of crystal structures (illustrated for C3/C3u, C-reactive protein and heparin). If a large protein or its complex cannot be crystallised, the joint ultracentrifugation-scattering approach provides a means to obtain an overall macromolecular structure.