Anastral meiotic spindle morphogenesis: role of the non-claret disjunctional kinesin-like protein

We have used time-lapse laser scanning confocal microscopy to directly examine microtubule reorganization during meiotic spindle assembly in living Drosophila oocytes. These studies indicate that the bipolarity of the meiosis I spindle is not the result of a duplication and separation of centrosomal microtubule organizing centers (MTOCs). Instead, microtubules first associate with a tight chromatin mass, and then bundle to form a bipolar spindle that lacks asters. Analysis of mutant oocytes indicates that the Non-Claret Disjunctional (NCD) kinesin-like protein is required for normal spindle assembly kinetics and stabilization of the spindle during metaphase arrest. Immunolocalization analyses demonstrate that NCD is associated with spindle microtubules, and that the centrosomal components gamma- tubulin, CP-190, and CP-60 are not concentrated at the meiotic spindle poles. Based on these observations, we propose that microtubule bundling by the NCD kinesin-like protein promotes assembly of a stable bipolar spindle in the absence of typical MTOCs.     

Conformation of full-length Bruton tyrosine kinase (Btk) from synchrotron X-ray solution scattering

Brutons's tyrosine kinase (Btk) is a non-receptor protein tyrosine kinase (nrPTK) essential for the development of B lymphocytes in humans and mice. Like Src and Abl PTKs, Btk contains a conserved cassette formed by SH3, SH2 and protein kinase domains, but differs from them by the presence of an N-terminal PH domain and the Tec homology region. The domain structure of Btk was analysed using X-ray synchrotron radiation scattering in solution. Low resolution shapes of the full-length protein and several deletion mutants determined ab initio from the scattering data indicated a linear arrangement of domains. This arrangement was further confirmed by rigid body modelling using known high resolution structures of individual domains. The final model of Btk displays an extended conformation with no, or little, inter-domain interactions. In agreement with these results, deletion of non-catalytic domains failed to enhance the activity of Btk. Taken together, our results indicate that, contrary to Src and Abl, Btk might not require an assembled conformation for the regulation of its activity.     

Conformation of human IgG subclasses in solution. Small-angle X-ray scattering and hydrodynamic studies

The structure of six human myeloma proteins: IgG1(Bal), IgG2(Klu), IgG3(Bak), IgG3(Het), IgG4(Kov) and IgG4(Pol), was studied in solution using small-angle X-ray scattering and hydrodynamic methods. For IgG1(Bal) and IgG3(Het) the experimental data, including radius of gyration (Rg degree), radii of gyration of the cross-section (Rq1, Rq2), intrinsic viscosity [eta], sedimentation coefficient (S degree 20,w) and molecular mass, were interpreted in terms of structural models based on the Fab and Fc conformations, observed in crystal, by varying the relative positions of the Fab and Fc parts, i.e. their relative angles and distances. The values Rg degree = (6.00 +/- 0.05) nm, S degree 20,w = (6.81 +/- 0.10) S and [eta] = 0.0062 +/- 0.0005 cm3/mg obtained for IgG1(Bal) are compatible with a planar model in which the angle between the Fab arms is about 120 degrees. For IgG3(Het) the following data were obtained: Rg degree = (4.90 +/- 0.05) nm, S degree 20,w = (6.32 +/- 0.01) S and [eta] = (0.0065 +/- 0.0005) cm3/mg. The apparent contradiction between the higher molecular mass and lower Rg degree and S degree 20,w values for IgG3(Het) in comparison to IgG1(Bal) can be resolved by proposing a 'non-planar' (tetrahedral) molecular shape, in which the long hinge peptide is in a folded conformation and the two Fab and Fc parts are in a closely packed arrangement. In this model the angle between the two Fab arms is about 90 degrees, in the average position. The X-ray scattering and hydrodynamic behaviour of the IgG2 and IgG4 types of antibodies appeared to be similar to IgG1(Bal). The parameters of the two IgG3 proteins are similar while they are different to the others.     

Analysis of X-ray and neutron scattering from biomacromolecular solutions

New developments in small-angle X-ray and neutron scattering studies of biological macromolecules in solution are presented. Small-angle scattering is rapidly becoming a streamline tool in structural molecular biology providing unique information about overall structure and conformational changes of native individual proteins, functional complexes, flexible macromolecules and assembly processes.     

Conformation of poly(sodium ethacrylate) in solution studied by small-angle X-ray scattering

The pH-induced conformational transition of poly(sodium ethacrylate) PNaEA in aqueous solution, which occurs between a compact form at low charge-density and an extended coil at high charge-density, was studied by small-angle X-ray scattering and the structure at an each conformational state was analyzed and compared with the corresponding one of poly(sodium methacrylate) PNaMA. The conformational transition for PNaEA induced a remarkable change in the scattering data plotted in the form of the Kratky plot. By comparing the scattering data with theoretical scattering functions, it was clarified that the structures of the compact form and the extended coil are well mimicked by a swollen gel having a network structure and by a wormlike chain, respectively. Although such a structure of the extended coil of PNaEA is similar to the corresponding one of PNaMA, the structure of the compact form of PNaEA is different from the corresponding one of PNaMA, which is still represented by a wormlike chain in a Theta medium.     

Joint use of small-angle X-ray and neutron scattering to study biological macromolecules in solution

Novel techniques for simultaneous analysis of X-ray and neutron scattering patterns from macromolecular complexes in solution are presented. They include ab initio shape and internal structure determination of multicomponent particles and more detailed rigid body modeling of complexes using high resolution structures of subunits. The methods fit simultaneously X-ray and neutron scattering curves including contrast variation data sets from selectively deuterated complexes. Biochemically sound interconnected models without steric clashes between the components displaying a pre-defined symmetry are generated. For rigid body modeling, distance restraints between specified residues/nucleotides or their ranges are taken into account. The efficiency of the methods is demonstrated in model examples, and potential sources of ambiguity are discussed.     

Characterization of protein fold by wide-angle X-ray solution scattering

Wide-angle X-ray solution scattering (WAXS) patterns contain substantial information about the three-dimensional structure of a protein. Although WAXS data have far less information than is required for determination of a full three-dimensional structure, the actual amount of information contained in a WAXS pattern has not been carefully quantified. Here we carry out an analysis of the amount of information that can be extracted from a WAXS pattern and demonstrate that it is adequate to estimate the secondary-structure content of a protein and to strongly limit its possible tertiary structures. WAXS patterns computed from the atomic coordinates of a set of 498 protein domains representing all of known fold space were used as the basis for constructing a multidimensional space of all corresponding WAXS patterns (‘WAXS space’). Within WAXS space, each scattering pattern is represented by a single vector. A principal components analysis was carried out to identify those directions in WAXS space that provide the greatest discrimination among patterns. The number of dimensions that provide significant discrimination among protein folds agrees well with the number of independent parameters estimated from a naïve Shannon sampling theorem approach. Estimates of the relative abundances of secondary structures were made using training/test sets derived from this data set. The average error in the estimate of α-helical content was 11%, and of β-sheet content was 9%. The distribution of proteins that are members of the four structure classes, α, β, α/β and α+β, are well separated in WAXS space when data extending to a spacing of 2.2 Å are used. Quantification of the information embedded within a WAXS pattern indicates that these data can be used as a powerful constraint in homology modeling of protein structures.     

Self-assembly of brome mosaic virus capsids: Kinetic study using neutron and X-ray solution scattering

The self-assembly of brome mosaic virus capsid has been studied kinetically by means of X-ray and neutron scattering. It appears to be a very fast process: for the concentrations used (5 to 8 mg/ml) the forward scattering reaches 50% of its maximal value in less than one second. Further, the assembly seems to proceed through intermediate states whose nature is still speculative.     

Oligomeric domain structure of human complement factor H by X-ray and neutron solution scattering.

Factor H is a regulatory component of the complement system. It has a monomer Mr of 150,000. Primary structure analysis shows that the polypeptide is divided into 20 homologous regions, each 60 amino acid residues long. These are independently folding domains and are termed "short consensus repeats" (SCRs) or "complement control protein" (CCP) repeats. High-flux synchrotron X-ray and neutron scattering studies were performed in order to define its solution structure in conditions close to physiological. The Mr of factor H was determined as 250,000-320,000 to show that factor H is dimeric. This structure is maintained at concentrations between 1 and 11 mg/mL in the pH range 5-9. Zn2+ ions are an inhibitor of C3b cleavage by factor I, a reaction in which factor H acts as a cofactor. Additions of Zn2+ to factor H caused it to form oligomers containing 4-10 monomers. The radius of gyration RG of native factor H by X-rays or by neutrons in 0% or 100% 2H2O buffers is not measurable but is greater than 12.5 nm. Two cross-sectional radii of gyration RXS-1 and RXS-2 were determined as 3.0-3.1 and 1.8 nm, respectively. Analyses of the cross-sectional intensities show that factor H is composed of two distinct subunits. The RXS-1 corresponds to the cross-sectional properties of both subunits and exhibits an unusual radiation dependence on the X-ray flux. Since RXS-2 is close to the corresponding RXS of C4b binding protein (91% of which is formed from SCR/CCP domains), it is inferred that the SCR/CCP domains of factor H and C4b binding protein have similar solution structures. The use of hydrodynamic spheres to reproduce literature sedimentation coefficients of 5.5-5.6 S showed that these were compatible with a V-shaped arrangement of two rods (36 spheres each, length 87 +/- 5 nm) joined at an angle of 5 degrees. The use of a similar arrangement of 244 spheres arranged in two rods (length 77 nm) to fit the experimental X-ray and neutron scattering curves showed that the two rods are joined at an angle of 5 degrees. This model corresponds to an actual RG of 21-23 nm. The separation between each SCR/CCP in factor H is close to 4 nm. In the solution structure of factor H, the SCR/CCP domains are in a highly extended conformation.     

X-ray solution scattering studies of the structural diversity intrinsic to protein ensembles

It is becoming increasingly clear that characterization of the protein ensemble-the collection of all conformations of which the protein is capable-will be a critical step in developing a full understanding of the linkage between structure, dynamics, and function. X-ray solution scattering in the small angle (SAXS) and wide-angle (WAXS) regimes represents an important new window to exploring the behavior of ensembles. The characteristics of the ensemble express themselves in X-ray solution scattering data in predictable ways. Here we present an overview of the effect that structural diversity intrinsic to protein ensembles has on scattering data. We then demonstrate the observation of these effects in scattering from four molecular systems; myoglobin; ubiquitin; alcohol dehydrogenase; and HIV protease; and demonstrate the modulation of these ensembles by ligand binding, mutation, and environmental factors. The observations are analyzed quantitatively in terms of the average spatial extent of structural fluctuations occurring within these proteins under different experimental conditions. The insights which these analyses support are discussed in terms of the function of the various proteins.     

Conformation of poly(methacrylic acid) in acidic aqueous solution studied by small angle X-ray scattering

The nature of the contracted form of poly(methacrylic acid) PMA chain in salt-free acidic aqueous solution was studied by analyzing scattering curves registered by small-angle X-ray scattering, comparing it with those of PMA in methanol at 26 degrees C and of partially neutralized PMA in aqueous solution containing added salt (the concentration of added salt, Cs=0.1 M NaF). It is shown that the distribution of segments in the contracted form as well as that of PMA in methanol is that of a random-coil in a theta medium and that this distribution of segments is stable over a fair range of degrees of ionization alpha for Cs below 0.1 M. Moreover, the persistence length of PMA at Cs=0.1 M (4+/-0.5 A) is substantially constant throughout the entire range of alpha, indicating that the contracted form of PMA changes to an expanded random-coil in a higher pH region without a significant change in the chain flexibility.     

Molecular dynamics decomposition of temperature-dependent elastic neutron scattering by a protein solution

Molecular dynamics simulations are performed of bovine pancreatic trypsin inhibitor in a cryosolution over a range of temperatures from 80 to 300 K and the origins identified of elastic dynamic neutron scattering from the solution. The elastic scattering and mean-square displacement calculated from the molecular dynamics trajectories are in reasonable agreement with experiments on a larger protein in the same solvent. The solvent and protein contributions to the scattering from the simulation model are determined. At lower temperatures (< approximately 200 K) or on shorter timescales ( approximately 10 ps) the scattering contributions are proportional to the isotopic nuclear scattering cross-sections of each component. However, for T > 200 K marked deviations from these cross-sections are seen due to differences in the dynamics of the components of the solution. Rapid activation of solvent diffusion leads to the variation with temperature of the total elastic intensity being determined largely by that of the solvent. At higher temperatures (>240 K) and longer times ( approximately 100 ps) the protein makes the only significant contribution to the scattering, the solvent scattering having moved out of the accessible time-space window. Decomposition of the protein mean-square displacement shows that the observed dynamical transition in the solution at 200-220 K involves activation of both internal motions and external whole-molecule rotational and translational diffusion. The proportion that the external dynamics contributes to the protein mean-square displacement increases to approximately 30 and 60% at 300 K on the 10- and 100-ps timescales, respectively.     

Reconstruction of protein form with X-ray solution scattering and a genetic algorithm

We have reconstructed, from experimental approximately 2 nm resolution X-ray solution scattering profiles, the corresponding shapes and sizes of myoglobin, troponin C, spermadhesin PSP-I/PSP-II, chymotrypsinogen A, superoxide dismutase, ovalbumin, tubulin, nitrite reductase, catalase, the structural change of troponin C upon dissociation of the two high affinity Ca(2+), and the solution model structure of a tandem pair of fibronectin type III cytoplasmic domains of integrin alpha6beta4 before determination of its crystal structure. To this purpose we have designed a new genetic algorithm which gradually explores a discrete search space and evolves convergent models made of several hundred beads (down to 0.3 nm radius) best fitting the scattering profile upon Debye calculation, without geometrical constraints or penalty for loose beads. This is a procedure of effective numerical transformation of the one-dimensional scattering profiles into three-dimensional model structures. The number of beads in models is correlated with the protein molecular mass (with one exception). The shape and approximate dimensions of each protein have been retrieved by a set of ten solution models, essentially superimposable with the available crystal structures.     

Domain conformation of tau protein studied by solution small-angle X-ray scattering

Tau is one of the two main proteins involved in the pathology of Alzheimer's disease via formation of beta-sheet rich intracellular aggregates named paired helical filaments (PHFs). Given that tau is a natively unfolded protein with no folded core (even upon binding to physiological partners such as microtubules), its structural analysis by high-resolution techniques has been difficult. In this study, employing solution small-angle X-ray scattering from the full length isoforms and from a variety of deletion and point mutants the conformation of tau in solution is structurally characterized. A recently developed ensemble optimization method was employed to generate pools of random models and to select ensembles of coexisting conformations, which fitted simultaneously the scattering data from the full length protein and deletion mutants. The analysis of the structural properties of these selected ensembles allowed us to extract information about residual structure in different domains of the native protein. The short deletion mutants containing the repeat domain (considered the core constituent of the PHFs) are significantly more extended than random coils, suggesting an extended conformation of the repeat domain. The longer tau constructs are comparable in size with the random coils, pointing to long-range contacts between the N- and C-termini compensating for the extension of the repeat domain. Moreover, most of the aggregation-promoting mutants did not show major differences in structure from their wild-type counterparts, indicating that their increased pathological effect is triggered only after an aggregation core has been formed.     

Small angle neutron and X-ray scattering in structural biology: recent examples from the literature

Small angle scattering can provide unique structural information on the shape, domain organisation, and interactions of biomacromolecules in solution. Small angle neutron scattering (SANS) combined with deuterium labelling makes it possible to define the positions of specific components within a complex while small angle X-ray scattering (SAXS) provides more precise data on the overall shape. Here I review four recent publications, three of which were presented at the Neutrons in Biology meeting at the STFC Rutherford Appleton Laboratory in July 2007, that utilise SANS, SAXS, and complementary techniques to define the solution structure of large multidomain proteins and macromolecular complexes. These four papers emphasise the critical importance of sample quality and characterisation as well as the important role played by complementary techniques in building structural models based on small angle scattering data. They show the ability of SANS and SAXS in determining solution structures provides an important complementary structural technique for large, flexible, and glycosylated proteins where high resolution structural techniques, such as crystallography and NMR, cannot be applied.     

Conformation of the HIV-1 Gag protein in solution

A single multi-domain viral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. We have purified the human immunodeficiency virus type 1 (HIV-1) Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) from bacteria. This protein is capable of assembly into virus-like particles in a defined in vitro system. We have reported that it is in monomer-dimer equilibrium in solution, and have described a mutant Gag protein that remains monomeric at high concentrations in solution. We report that the mutant protein retains several properties of wild-type Gag. This mutant enabled us to analyze solutions of monomeric protein. Hydrodynamic studies on the mutant protein showed that it is highly asymmetric, with a frictional ratio of 1.66. Small-angle neutron scattering (SANS) experiments confirmed its asymmetry and yielded an R(g) value of 34 A. Atomic-level structures of individual domains within Gag have previously been determined, but these domains are connected in Gag by flexible linkers. We constructed a series of models of the mutant Gag protein based on these domain structures, and tested each model computationally for its agreement with the experimental hydrodynamic and SANS data. The only models consistent with the data were those in which Gag was folded over, with its N-terminal matrix domain near its C-terminal nucleocapsid domain in three-dimensional space. Since Gag is a rod-shaped molecule in the assembled immature virion, these findings imply that Gag undergoes a major conformational change upon virus assembly.     

Complexes of RecA protein in solution. A study by small angle neutron scattering

RecA complexes on DNA and self-polymers were analysed by small-angle neutron scattering in solution. By Guinier analysis at small angles and by model analysis of a subsidiary peak at wider angles, we find that the filaments fall into two groups: the DNA complex in the presence of ATP gamma S, an open helix with pitch 95 A, a cross-sectional radius of gyration of 33 A and a mass per length of about six RecA units per turn, which corresponds to the state of active enzyme; and the compact form (bound to single-stranded DNA in the absence of ATP, or binding ATP gamma S in the absence of DNA, or just the protein on its own), a helical structure with pitch 70 A, cross-sectional radius of gyration 40 A and mass per length about five RecA units per turn, which corresponds to the conditions of inactive enzyme. The results are discussed in the perspective of unifying previous conflicting structural results obtained by electron microscopy.     

Molecular modeling of human complement component C4 and its fragments by X-ray and neutron solution scattering

The solution structures of human complement component C4 and five derived fragments, C4u, C4(a + b), C4b, C4c, and C4d, were analyzed by synchrotron X-ray and neutron scattering. The X-ray radii of gyration RG for C4, C4u, and C4(a + b) in H2O buffers are similar at 5.23-5.28 nm, and likewise the cross-sectional radii of gyration RXS are similar at 2.48-2.52 nm. Molecular mass calculations using X-rays and neutrons show unexpectedly that C4c is dimeric; however, all the other forms are monomeric. C4c2 has an X-ray RG of 5.18 nm and an RXS of 2.89 nm. Neutron contrast variation gives RG values at infinite contrast of 4.87-4.93 nm for C4 and C4u, 4.79 nm for C4b, 4.94 nm for C4c2, and 2.69 nm for C4d. The RXS values at infinite contrast are 2.23-2.25 nm for C4 and C4u, 1.89 nm for C4b, and 2.62 nm for C4c2. These data show that a large conformational change occurs on going from C4 to C4b, but not on going from C4 to C4u, and this is attributed to the presence of the C4a moiety in C4u. Comparisons of the C4 and C4u scattering curves show that these are very similar out to a nominal resolution of 4 nm. Scattering-curve models were developed to account for the neutron scattering curves of C4, C4c2, and C4d in 2H2O buffers. The C4c monomer could be represented by a lamellar ellipsoid of size 8 nm x 2 nm x 18 nm. C4d was found to be 4 nm x 2 nm x 9 nm. The combination of these structures gave good accounts of the neutron data for C4, C4b, and C4c2 to resolutions of 5-6 nm. The C4 model was obtained by placing the long axis of C4d parallel to that of C4c such that the cross section is extended. C4b was best modeled by repositioning C4d relative to C4c such that this cross section becomes more compact. The C4 and C4b models are compared with possible structures for the C1 component of complement to show the importance of the surface accessibility of the protease domains and short consensus repeat domains in C1 for C4 activation.     

Determination of domain structure of proteins from X-ray solution scattering

An ab initio method for building structural models of proteins from x-ray solution scattering data is presented. Simulated annealing is employed to find a chain-compatible spatial distribution of dummy residues which fits the experimental scattering pattern up to a resolution of 0.5 nm. The efficiency of the method is illustrated by the ab initio reconstruction of models of several proteins, with known and unknown crystal structure, from experimental scattering data. The new method substantially improves the resolution and reliability of models derived from scattering data and makes solution scattering a useful technique in large-scale structural characterization of proteins.