Small-angle X-ray Scattering Model Research Articles

Structural and Biophysical Properties of Supercharged and Circularized Nanodiscs.

Nanodiscs based on membrane scaffold proteins (MSPs) and phospholipids are used as membrane mimics to stabilize membrane proteins in solution for structural and functional studies. Combining small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and time-resolved small-angle neutron scattering (TR-SANS), we characterized the structure and lipid bilayer properties of five different nanodiscs made with dimyristoylphosphatidylcholine and different MSPs varying in size, charge, and circularization. Our SAXS modeling showed that the structural parameters of the embedded lipids are all similar, irrespective of the MSP properties. DSC showed that the lipid packing is not homogeneous in the nanodiscs and that a 20 Å wide boundary layer of lipids with perturbed packing is located close to the MSP, while the packing of central lipids is tighter than in large unilamellar vesicles. Finally, TR-SANS showed that lipid exchange rates in nanodiscs decrease with increasing nanodisc size and are lower for the nanodiscs made with supercharged MSPs compared to conventional nanodiscs. Altogether, the results provide a thorough biophysical understanding of the nanodisc as a model membrane system, which is important in order to carry out and interpret experiments on membrane proteins embedded in such systems.

A multimethod approach for analyzing FapC fibrillation and determining mass per length

Amyloid proteins are found in a wide range of organisms owing to the high stability of the β-sheet core of the amyloid fibrils. There are both pathological amyloids involved in various diseases and functional amyloids that play a beneficial role for the organism. The aggregation process is complex and often involves many different species. Full understanding of this process requires parallel acquisition of data by complementary techniques monitoring the time course of aggregation. This is not an easy task, given the often-stochastic nature of aggregation, which can lead to significant variations in lag time. Here, we investigate the aggregation process of the functional amyloid FapC by simultaneous use of four different techniques, namely dynamic light scattering, small-angle x-ray scattering (SAXS), circular dichroism, and Thioflavin T fluorescence. All these approaches are applied to the same FapC sample just after desalting. Our data allow us to construct a master time-course graph showing the same time-course of aggregation by all techniques. This allows us to integrate insights from approaches that report on different structural and length scales. During the lag phase, loosely aggregated oligomers with random-coil structure are formed, which subsequently transform to fibrils without accumulation of additional significant species. Subsequently, the loosely associated protofilaments/subfilaments, which form side by side, mature to more compact fibrils. Furthermore, we determine the mass per length of the mature fibrils, obtaining very similar results by SAXS (33 kDa/nm) and tilted-beam transmission electron microscopy (31 kDa/nm). Transmission electron microscopy showed that the fibrils consist of primarily two protofilaments and similar dimensions of the cross section of the fibrils as revealed by SAXS modeling when the number of protofilaments per fibril was taken into account. Mass per length information underscores the general usefulness of SAXS in fibrillation analysis and provides an important constraint for further modeling the fibril structures.

Quantitative characterization and comparsion of bentonite microstructure by small angle X-ray scattering and nitrogen adsorption

Quantitative characterization of the pore structure of typical bentonite in China is beneficial to nuclear waste treatment and sludge solidification modeling. A new small angle X-ray scattering (SAXS) method and conventional method low-temperature nitrogen adsorption and desorption (LTNAD) were combined to investigate the pore structure of four Chinese typical bentonites. The pore size and pore size distribution measured by the novel SAXS and conventional LTNAD methods were compared. The results suggested that Gaomiaozi bentonite exhibited Porod positive deviation effect. The GMZ01 had better sortability than GMZ07, and the pore size was concentrated. The average pore size of GMZ01 and GMZ07 calculated by the maximum entropy method were close. LTNAD experiment indicated that the specific surface area (SSA) of bentonite depended on the pore volume of mesopores and micropores and the types of exchangeable cations between layers. The SSA was positively correlated with the adsorption capacity. SAXS revealed that pore size estimated by maximum entropy method was larger than that evaluated by Guinier method. The log-normal distribution method in SAXS model was more suitable for characterizing small pores than maximum entropy method. Compared with maximum entropy method, The LTNAD method was more compatible with the SAXS lognormal distribution method. Compared with LTNAD, the SAXS has a wider detection range. These findings fill a gap in SAXS research on Gaomiaozi bentonite (GMZ01, GMZ07, CaGMZ).

SAXS signature of the lamellar ordering of ionic domains of perfluorinated sulfonic-acid ionomers by electric and magnetic field-assisted casting.

At present, small angle X-ray scattering (SAXS) studies of perfluorinated sulfonic-acid ionomers (PFSAs) are unable to fully determine the true shape of their building blocks, as recent SAXS modelling predicts disk- and rod-like nanoionic domains as being equally possible. This scenario requires evidence-based findings to unravel the real shape of PFSA building blocks. Herein, a SAXS pattern signature for a lamellar nanophase separation of the ionic domains of Nafion is presented, backed by mid and far infrared spectroscopy (MIR and FIR) and wide angle X-ray scattering (WAXS) data of Nafion in different ionic forms, a broad range of ionic phase contents (EW ∼ 859-42 252 g eq-1) and temperatures. The study indicates that the lamellar arrangement of the ionic domains is the most representative morphology that accounts for the physical properties of this ionomer. The lamellar SAXS reflections of Nafion are enhanced in electric and magnetic field-aligned membranes, as confirmed by atomic force microscopy (AFM). Electric and magnetic field-assisted casting of Nafion allowed producing nanostructured and anisotropic films with the lamellas stacked perpendicularly to the field vector, which is the direction of interest for several applications. Such nanostructured Nafion membranes are bestowed with advanced optical and proton transport properties, making them promising materials for solar and fuel cells.

Four steps for revealing and adjusting the 3D structure of aptamers in solution by small-angle X-ray scattering and computer simulation

Nucleic acid (NA) aptamers bind to their targets with high affinity and selectivity. The three-dimensional (3D) structures of aptamers play a major role in these non-covalent interactions. Here, we use a four-step approach to determine a true 3D structure of aptamers in solution using small-angle X-ray scattering (SAXS) and molecular structure restoration (MSR). The approach consists of (i) acquiring SAXS experimental data of an aptamer in solution, (ii) building a spatial distribution of the molecule's electron density using SAXS results, (iii) constructing a 3D model of the aptamer from its nucleotide primary sequence and secondary structure, and (iv) comparing and refining the modeled 3D structures with the experimental SAXS model. In the proof-of-principle we analyzed the 3D structure of RE31 aptamer to thrombin in a native free state at different temperatures and validated it by circular dichroism (CD). The resulting 3D structure of RE31 has the most energetically favorable conformation and the same elements such as a B-form duplex, non-complementary region, and two G-quartets which were previously reported by X-ray diffraction (XRD) from a single crystal. More broadly, this study demonstrates the complementary approach for constructing and adjusting the 3D structures of aptamers, DNAzymes, and ribozymes in solution, and could supply new opportunities for developing functional nucleic acids. Graphical abstract.

Correlation of precipitate evolution with Vickers hardness in Haynes® 282® superalloy: In-situ high-energy SAXS/WAXS investigation

The aim of this work is to characterize the precipitation kinetics in Haynes® 282® superalloys using in-situ high-energy Small Angle X-ray Scattering (SAXS) together with Wide Angle X-ray Scattering (WAXS). The phases identified by WAXS include γ (matrix), γ′ (hardening precipitates), MC (metallic carbides), and M23C6/M6C (secondary metallic carbides). The γ’-precipitates are spheroids with a diameter of several nanometres, depending on the temperature and ageing time. From the SAXS data, quantitative parameters such as volume fraction, number density and inter-particle distance were determined and correlated with ex-situ Vickers microhardness measurements. The strengthening components associated with precipitates and solid solutions are differentiated using the measured Vickers microhardness and SAXS model parameters. A square root dependence between strengthening attributable to the precipitates and the product of volume fraction and mean precipitate radius is found. The solid solution strengthening component correlates with the total volume fraction of precipitates.

Determining Atomistic SAXS Models of Tri-Ubiquitin Chains from Bayesian Analysis of Accelerated Molecular Dynamics Simulations.

Small-angle X-ray scattering (SAXS) has become an increasingly popular technique for characterizing the solution ensemble of flexible biomolecules. However, data resulting from SAXS is typically low-dimensional and is therefore difficult to interpret without additional structural knowledge. In theory, molecular dynamics (MD) trajectories can provide this information, but conventional simulations rarely sample the complete ensemble. Here, we demonstrate that accelerated MD simulations can be used to produce higher quality models in shorter time scales than standard simulations, and we present an iterative Bayesian Monte Carlo method that is able to identify multistate ensembles without overfitting. This methodology is applied to several ubiquitin trimers to demonstrate the effect of linkage type on the solution states of the signaling protein. We observe that the linkage site directly affects the solution flexibility of the trimer and theorize that this difference in plasticity contributes to their disparate roles in vivo.

Heterogeneity of Collagen VI Microfibrils: STRUCTURAL ANALYSIS OF NON-COLLAGENOUS REGIONS

Collagen VI, a collagen with uncharacteristically large N- and C-terminal non-collagenous regions, forms a distinct microfibrillar network in most connective tissues. It was long considered to consist of three genetically distinct α chains (α1, α2, and α3). Intracellularly, heterotrimeric molecules associate to form dimers and tetramers, which are then secreted and assembled to microfibrils. The identification of three novel long collagen VI α chains, α4, α5, and α6, led to the question if and how these may substitute for the long α3 chain in collagen VI assembly. Here, we studied structural features of the novel long chains and analyzed the assembly of these into tetramers and microfibrils. N- and C-terminal globular regions of collagen VI were recombinantly expressed and studied by small angle x-ray scattering (SAXS). Ab initio models of the N-terminal globular regions of the α4, α5, and α6 chains showed a C-shaped structure similar to that found for the α3 chain. Single particle EM nanostructure of the N-terminal globular region of the α4 chain confirmed the C-shaped structure revealed by SAXS. Immuno-EM of collagen VI extracted from tissue revealed that like the α3 chain the novel long chains assemble to homotetramers that are incorporated into mixed microfibrils. Moreover, SAXS models of the C-terminal globular regions of the α1, α2, α4, and α6 chains were generated. Interestingly, the α1, α2, and α4 C-terminal globular regions dimerize. These self-interactions may play a role in tetramer formation.

The PKA regulatory subunit from yeast forms a homotetramer: Low-resolution structure of the N-terminal oligomerization domain.

The cAMP dependent protein kinase (PKA) is a key enzyme involved in many cellular processes in eukaryotes. In mammals, the regulatory (R) subunit localises the catalytic (C) subunit to specific subcellular sites through the interaction of its N-terminal homodimeric docking and dimerization (D/D) domain with specific scaffold proteins. The structure of the D/D domain has been extensively studied in mammals, but there is little information from non-mammalian species. In this work, we present the structural analysis of the D/D domain of Bcy1, the R subunit of PKA from Saccharomyces cerevisiae. Using chemical crosslinking experiments and static light scattering measurements we found that this R subunit forms a tetramer in solution, unlike its dimeric mammalian counterparts. We determined that the D/D domain is responsible for this unusual oligomeric state. Using biophysical techniques including size-exclusion chromatography, sucrose gradient sedimentation, small angle X-ray scattering (SAXS), and circular dichroism, we performed a detailed structural characterization of the tetrameric D/D domain of Bcy1. We used homology modelling in combination with computer-aided docking methods and ab initio SAXS modelling methods to develop structural models for the D/D domain tetramer. The models consist of two homodimers with a canonical D/D domain fold that generate a dimer of dimers with novel putative interaction surfaces. These findings indicate that the oligomerization states of PKA R subunits is more diverse than previously thought, and suggest that this might allow some forms of PKA to interact with a wide range of intracellular partners.

Physico-chemical stability and structural characterization of thickened multilamellar beta-carotene-loaded liposome dispersions produced using a proliposome method

The objective of this study was to investigate in more detail the structure of multilamellar beta-carotene-loaded liposome dispersions produced by proliposome hydration. Such dispersions were stabilized using xanthan gum as a thickener in different concentrations, and their stabilities were monitored for 90 days. The vesicles exhibited an average diameter in the range of 700 to 3000 nm, and the liposomes were capable of protecting β-carotene from degradation for a period of 90 days. The dispersions were also characterized by transmission electron microscopy, differential scanning calorimetry, rheology, and small-angle X-ray scattering (SAXS). The thermal analyses showed that neither the β-carotene nor the xanthan gum affected the liposome bilayer structure. The presence of the xanthan gum, which affects the scattering intensity, was not an obstacle to obtain the structural parameters by SAXS modeling, as a modified modeling strategy (Gaussian deconvolution) was applied. This modeling resulted in 40 symmetric layers, and the results obtained with the variation of temperature were in agreement with the gel-liquid crystalline transition temperature obtained by calorimetric measurements. Additionally, the rheological data showed that xanthan gum was not as effective as a mixture of xanthan gum and guar gum at stabilizing the liposomes, most likely due to the higher stiffness of the gum alone compared with that of its combination with guar gum.

Determination of the three-dimensional structure of ferrihydrite nanoparticle aggregates.

Aggregation impacts the reactivity, colloidal stability, and transport behavior of nanomaterials, yet methods to characterize basic structural features of aggregates are limited. Here, cryo-transmission electron microscope (cryo-TEM) based tomography is utilized as a method for directly imaging fragile aggregates of nanoparticles in aqueous suspension and an approach for extracting quantitative fractal dimensions from the resulting three-dimensional structural models is introduced. The structural quantification approach is based upon the mass autocorrelation function, and is directly comparable with small-angle X-ray scattering (SAXS) models. This enables accurate characterization of aggregate structure, even in suspensions where the aggregate cluster size is highly polydisperse and traditional SAXS modeling is not reliable. This technique is applied to study real suspensions of ferrihydrite nanoparticles. By comparing tomographic measurements with SAXS-based measurements, we infer that certain suspensions contain polydisperse aggregate size distributions. In other suspensions, fractal-type structures are identified with low intrinsic fractal dimensions. The fractal dimensions are lower than would be predicted by simple models of particle aggregation, and this low dimensionality enables large, low-density aggregates to exist in stable colloidal suspension.

Analysis of macromolecular flexibility in solution by combining SAXS with MX

Proteins containing significant disordered regions are difficult for crystallization. Consequently, individual structured domains or subunits are often crystallized to determine their high resolution structures. These structures, not containing the disordered parts, are available from the protein databases. Small-Angle X-ray Scattering (SAXS) in solution is a complementary structural technique offering the possibility to characterize full length proteins including inter-domain disordered regions. Modern approaches for the analysis of the SAXS data from flexible systems utilize the representation of the system as ensembles of co-existing structures (e.g. Ensemble Optimization Method (EOM), Bernado et al. 2007). Here, the high resolution models of the domains are employed as rigid bodies together with the flexible parts represented by chains of dummy residues in order to construct models covering the configurational space of the full length proteins. Until now, the use of the ensemble approach was mostly limited to relatively simple cases like single chain proteins. We present improvements for the SAXS modeling including the possibilities to characterize flexible particles for complicated scenarios, in particular, for the cases when disorder comes in combination with point group symmetry. A new strategy for missing sections generation allows a rapid and accurate construction of the full length protein. Further, two metrics, Rflex and Rrat, are introduced for a quantitative assessment of the EOM results. Rflex is used as a measure of flexibility – based on the concept of entropy (information communication) – whereas Rrat is employed as a control parameter to detect potential artifacts. These developments implemented in the new version EOM 2.0 further promote the hybrid approach synergistically employing SAXS and MX in the analysis of complicated flexible systems. The capacity of the enhanced method will be illustrated by practical examples.

Agonist ligands mediate the transcriptional response of nuclear receptor heterodimers through distinct stoichiometric assemblies with coactivators.

The constitutive androstane (CAR) and retinoid X receptors (RXR) are ligand-mediated transcription factors of the nuclear receptor protein superfamily. Functional CAR:RXR heterodimers recruit coactivator proteins, such as the steroid receptor coactivator-1 (SRC1). Here, we show that agonist ligands can potentiate transactivation through both coactivator binding sites on CAR:RXR, which distinctly bind two SRC1 molecules. We also observe that SRC1 transitions from a structurally plastic to a compact form upon binding CAR:RXR. Using small angle x-ray scattering (SAXS) we show that the CAR(tcp):RXR(9c)·SRC1 complex can encompass two SRC1 molecules compared with the CAR(tcp):RXR·SRC1, which binds only a single SRC1. Moreover, sedimentation coefficients and molecular weights determined by analytical ultracentrifugation confirm the SAXS model. Cell-based transcription assays show that disrupting the SRC1 binding site on RXR alters the transactivation by CAR:RXR. These data suggest a broader role for RXR within heterodimers, whereas offering multiple strategies for the assembly of the transcription complex.

Structural Characterization of Heparin-induced Glyceraldehyde-3-phosphate Dehydrogenase Protofibrils Preventing α-Synuclein Oligomeric Species Toxicity

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme that has been associated with neurodegenerative diseases. GAPDH colocalizes with α-synuclein in amyloid aggregates in post-mortem tissue of patients with sporadic Parkinson disease and promotes the formation of Lewy body-like inclusions in cell culture. In a previous work, we showed that glycosaminoglycan-induced GAPDH prefibrillar species accelerate the conversion of α-synuclein to fibrils. However, it remains to be determined whether the interplay among glycosaminoglycans, GAPDH, and α-synuclein has a role in pathological states. Here, we demonstrate that the toxic effect exerted by α-synuclein oligomers in dopaminergic cell culture is abolished in the presence of GAPDH prefibrillar species. Structural analysis of prefibrillar GAPDH performed by small angle x-ray scattering showed a particle compatible with a protofibril. This protofibril is shaped as a cylinder 22 nm long and a cross-section diameter of 12 nm. Using biocomputational techniques, we obtained the first all-atom model of the GAPDH protofibril, which was validated by cross-linking coupled to mass spectrometry experiments. Because GAPDH can be secreted outside the cell where glycosaminoglycans are present, it seems plausible that GAPDH protofibrils could be assembled in the extracellular space kidnapping α-synuclein toxic oligomers. Thus, the role of GAPDH protofibrils in neuronal proteostasis must be considered. The data reported here could open alternative ways in the development of therapeutic strategies against synucleinopathies like Parkinson disease.

Structure of the Streptococcus pneumoniae Surface Protein and Adhesin PfbA

PfbA (plasmin- and fibronectin-binding protein A) is an extracellular Streptococcus pneumoniae cell-wall attached surface protein that binds to fibronectin, plasmin, and plasminogen. Here we present a structural analysis of the surface exposed domains of PfbA using a combined approach of X-ray crystallography and small-angle X-ray scattering (SAXS). The crystal structure of the PfbA core domain, here called PfbAβ, determined to 2.28 Å resolution revealed an elongated 12-stranded parallel β-helix fold, which structure-based comparisons reveal is most similar to proteins with carbohydrate modifying activity. A notable feature of the PfbAβ is an extensive cleft on one face of the protein with electrochemical and spatial features that are analogous to structurally similar carbohydrate-active enzymes utilizing this feature for substrate accommodation. Though this cleft displays a combination of basic amino acid residues and solvent exposed aromatic amino acids that are distinct features for recognition of carbohydrates, no obvious arrangement of amino acid side chains that would constitute catalytic machinery is evident. The pseudo-atomic SAXS model of a larger fragment of PfbA suggests that it has a relatively well-ordered structure with the N-terminal and core domains of PfbA adopting an extend organization and reveals a novel structural class of surface exposed pneumococcal matrix molecule adhesins.

The structure of soy soluble polysaccharide in aqueous solution

Soybean soluble polysaccharide (SSPS) extracted from soybean cotyledons is an acidic polysaccharide containing a protein fraction. Small angle X-ray scattering (SAXS) and static light scattering (SLS) were used to reveal the structural conformation and nano-structural arrangement of SSPS in aqueous solutions aiming to gain more information on the functional properties of SSPS. Three samples, differentiated by their extraction processes, namely SSPS-L; -M; and -H, were used. NMR results show that SSPS has a similar structure regardless of the differences in the extraction conditions. The Mark–Houwink constants were determined to be 7.52 × 10−4 and 0.473 for K and α respectively indicating that SSPS is a flexible random coil in solution. SAXS profiles revealed power law functions (I(q) ∼ q−α) with an exponent of −1 signifying the presence of rigid rods. Due to the overall large size of SSPS, it is thought that SAXS focused on single branches of SSPS. SAXS models confirmed that the nano-structural conformation of SSPS remains unaltered due to the presence of sodium nitrate salt, neither is the conformation affected by changes in pH nor concentration.

Solution structure of the tandem acyl carrier protein domains from a polyunsaturated fatty acid synthase reveals beads-on-a-string configuration.

The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP domains for increasing the yield of fatty acids in bacterial cultures.

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)(4) complex that regulates glycogenolysis in skeletal muscle. Activity of the catalytic γ subunit is regulated by allosteric activators targeting the regulatory α, β, and δ subunits. Three-dimensional EM reconstructions of PhK show it to be two large (αβγδ)(2) lobes joined with D(2) symmetry through interconnecting bridges. The subunit composition of these bridges was unknown, although indirect evidence suggested the β subunits may be involved in their formation. We have used biochemical, biophysical, and computational approaches to not only address the quaternary structure of the β subunits within the PhK complex, i.e. whether they compose the bridges, but also their secondary and tertiary structures. The secondary structure of β was determined to be predominantly helical by comparing the CD spectrum of an αγδ subcomplex with that of the native (αβγδ)(4) complex. An atomic model displaying tertiary structure for the entire β subunit was constructed using chemical cross-linking, MS, threading, and ab initio approaches. Nearly all this model is covered by two templates corresponding to glycosyl hydrolase 15 family members and the A subunit of protein phosphatase 2A. Regarding the quaternary structure of the β subunits, they were directly determined to compose the four interconnecting bridges in the (αβγδ)(4) kinase core, because a β(4) subcomplex was observed through both chemical cross-linking and top-down MS of PhK. The predicted model of the β subunit was docked within the bridges of a cryoelectron microscopic density envelope of PhK utilizing known surface features of the subunit.

Activation of p115-RhoGEF Requires Direct Association of Gα13 and the Dbl Homology Domain

RGS-containing RhoGEFs (RGS-RhoGEFs) represent a direct link between the G(12) class of heterotrimeric G proteins and the monomeric GTPases. In addition to the canonical Dbl homology (DH) and pleckstrin homology domains that carry out the guanine nucleotide exchange factor (GEF) activity toward RhoA, these RhoGEFs also possess RGS homology (RH) domains that interact with activated α subunits of G(12) and G(13). Although the GEF activity of p115-RhoGEF (p115), an RGS-RhoGEF, can be stimulated by Gα(13), the exact mechanism of the stimulation has remained unclear. Using combined studies with small angle x-ray scattering, biochemistry, and mutagenesis, we identify an additional binding site for activated Gα(13) in the DH domain of p115. Small angle x-ray scattering reveals that the helical domain of Gα(13) docks onto the DH domain, opposite to the surface of DH that binds RhoA. Mutation of a single tryptophan residue in the α3b helix of DH reduces binding to activated Gα(13) and ablates the stimulation of p115 by Gα(13). Complementary mutations at the predicted DH-binding site in the αB-αC loop of the helical domain of Gα(13) also affect stimulation of p115 by Gα(13). Although the GAP activity of p115 is not required for stimulation by Gα(13), two hydrophobic motifs in RH outside of the consensus RGS box are critical for this process. Therefore, the binding of Gα(13) to the RH domain facilitates direct association of Gα(13) to the DH domain to regulate its exchange activity. This study provides new insight into the mechanism of regulation of the RGS-RhoGEF and broadens our understanding of G protein signaling.

Solution Conformation of Extracellular Matrix Proteins

The extracellular matrix (ECM) is composed of large, multi-domain proteins with varying degree of shapes and post-translational modifications. Therefore, it is challenging to study such proteins with a single technique to gain reliable information about their size, shape and conformation. We have employed a multidisciplinary approach using a variety of techniques to investigate the solution conformation of laminin gamma-1 short arm, netrin-4 and G3 domain of agrin fused with human IgG-Fc (G3Fc). Briefly, the hydrodynamic properties of entire multi-domain proteins were studied using analytical ultracentrifugation, dynamic light scattering, and small angle X-ray scattering (SAXS). Further, the information from SAXS for entire multi-domain protein was then either used to determine ab initio structure or combined with high-resolution data for individual domains to obtain a detailed solution structure of desired multi-domain protein. Additionally, the SAXS models were verified by comparing the experimentally determined hydrodynamic parameters with the parameters calculated from solution structures. The ab initio structures for laminin gamma-1 short arm revealed an extended and curved assembly. We could also combine small portion of high-resolution structure from X-ray Crystallography data to obtain rigid-body models. Further, we found that the netrin4 has an elongated shape in solution using ab initio methods. Remarkably, we found that the G3Fc chimera is T-shaped and not Y-shaped like an antibody in solution by rigid body modelling. Thus, we conclude that by combining various methods, it is possible to explore large muldi-domain protein and protein-protein assemblies that will help enable us to understand structure-function relationship of these proteins with various forms of diseases.