Streptokinase is a flexible multi-domain protein

The structure of streptokinase in solution has been studied by dynamic light scattering, small-angle X-ray scattering and circular dichroism spectroscopy. The Stokes' radius and radius of gyration of the protein monomer are 3.58 nm and 4.03 nm, respectively. The maximum intraparticle distance of the molecule is 14 nm. More than half of the amino acids of the molecule are organized in regular secondary structures. The X-ray scattering curve, the results from dynamic light scattering, and the finding that at least 50% of the amino acid residues are organized in regularly folded secondary structures are consistent with the following structural model. Streptokinase consists of four compact, separately folded, domains linked by mobile segments of the protein chain. The molecule exhibits the conformation of a flexible string-of-beads in solution.     

Size, shape and secondary structure of calponin

The overall size and shape of the chicken gizzard calponin (CaP) h1 molecule was investigated by dynamic light scattering (DLS) measurements. From the DLS experiments, a z-averaged translational diffusion coefficient is derived (5.75 +/- 0.3) x 10(-7) cm(2) s(-1), which corresponds to a hydrodynamic radius of 3.72 nm for calponin. The frictional ratio (1.8 for the unhydrated molecule and 1.5 for the hydrated one) suggests a pronounced anisotropic structure for the molecule. An ellipsoidal model in length 19.4 nm and with a diameter of 2.6 nm used for hydrodynamic calculations was found to reproduce the DLS experimental data. The evaluation of the secondary structure of CaP h1 from the CD spectra by two independent methods has revealed that it contains, on average, 23% helix, 19% beta-strand, 18% beta-turns and loops, and 40% of remainder structures. These values are in good agreement with those predicted from the amino-acid sequence. Predictions used for CaP h1 were applied to other isoforms of known sequences and revealed that all calponins share a common secondary structure. Moreover, the predicted structure of the calponin CH domain is identical to that found by X-ray studies of the spectrin, fimbrin and utrophin CH domains.     

Solution scattering studies of conformation stability of xylanase XYNII from Trichoderma longibrachiatum

Xylanase (endo-1,4-beta-xylanase; EC 3.2.1.8) is an enzyme that catalyzes the hydrolysis reaction of xylan. The structure of the xylanase II (XYNII) molecule from Trichoderma longibrachoatum (formerly Trichoderma reesei) in a solution and at different pH values has been studied by small- and wide-angle scattering of synchrotron radiation (SAXS-WAXS). Analysis of the radius of gyration that characterizes xylanase has confirmed the stability of the above enzyme structure (the radius of gyration varied from 1.65 to 1.74 nm). On the basis of the SAXS data, the low-resolution structure of the xylanase molecule in solution has been reconstructed by using ab initio methods and programs DALAI_GA and DAMMIN. The full SAXS-WAXS data set (0.15 > s > 9.5 nm(-1)) fed to the program GASBOR permitted us to construct a chain-like spatial distribution of a dummy residues model of the xylanase molecule. The shape of the model molecules is similar to that of xylanase molecule in the crystal and shows the characteristic asymmetry that makes the molecule to resemble a right hand.     

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.     

The conformation of a large RNA fragment from the E.coli ribosomal 16S-RNA. An X-ray and neutron small-angle scattering study.

A large 12S RNA fragment which constitutes the 5' two-thirds of 16S-RNA from the E. coli 30S subunit has been investigated by small-angle X-ray and neutron scattering. The results indicate that in reconstitution buffer the 12S-RNA fragment has a molecular weight of 270,000 +/- 20,000 and a radius of gyration of 7.1 nm. The scattering data are compatible with the RNA being folded into two major domains with the shapes of two adjacent, quite similar cylinders.     

Molecular modeling of the domain structure of C9 of human complement by neutron and X-ray solution scattering.

C9 is the most abundant component of the membrane attack complex of the complement system of immune defense. This is a typical mosaic protein with thrombospondin (TSR) and low density lipoprotein receptor (LDLr) domains at its N-terminus and an epidermal growth factor-like (EGF) domain at its C-terminus. Between these lies a perforin-like sequence. In order to define the arrangement in solution of these four moieties in C9, high-flux neutron and synchrotron X-ray solution scattering studies were carried out. The neutron radius of gyration RG at infinite contrast is 3.33 nm, and its cross-sectional RG (RXS) is 1.66 nm. Similar values were obtained by synchrotron X-ray scattering after allowance for radiation effects. Stuhrmann analyses showed that the neutron radial inhomogeneity of scattering density alpha is 35 X 10(-5) from the RG data and 16 X 10(-5) from the RXS data. These values are typical for soluble glycoproteins and show no evidence for the existence of any large hydrophobic surface patches on free C9 that might form contacts with lipids. Indirect transformation of the neutron and X-ray scattering curves into real space showed that C9 had a maximum dimension estimated at 12 +/- 2 nm, and this suggests that the lengths of 7-8 nm deduced from previous electron microscopy studies in vacuo are underestimated. Molecular modeling of the C9 scattering curves utilized small spheres in the Debye equation, in which the analyses were constrained by the known volumes of the four moieties of C9 and the known sizes of the TSR and EGF-like domains. The most likely models for C9 suggest that these four regions of C9 are arranged in a V-shaped structure, with an angle of 10 degrees between the two arms, each of length 11.1 nm. This structure has a more hydrophobic character between the two arms. The scattering model is fully consistent with hydrodynamic sedimentation data on C9. Similar V-shaped hydrodynamic models could be developed for C6, C7, C8, and C9 of complement. Such a compact structure is atypical of other multidomain complement proteins so far studied by solution scattering and is fully compatible with mechanisms in which C9 is postulated, on activation, to undergo a drastic unfolding of its domain structure and to expose a more hydrophobic surface which can be embedded into lipid bilayers.     

Structural changes in cellobiohydrolase I upon binding of a macromolecular ligand as evident by SAXS investigations

Xylan from Rhodymenia palmata binds to the cellobiohydrolase I from Trichoderma reesei (CBH I) or its core protein, inhibiting their activity. Adsorption onto microcrystalline cellulose (Avicel) is reduced approximately 30% for intact CBH I and nearly 50% for the core, whereas the effects with cellobiose are negligible. Structural changes concomitant with this binding are studied in solution by small angle X-ray scattering. In the "tadpole" structure typical for the CBH I [Abuja et al., 1988] the lengthening of the tail part is the most salient observation when xylan is present which accounts for an increase in Dmax (18.0 to 22.0 nm) and radius of gyration (4.74 to 5.18 nm). When xylan binds to the core the radius of gyration remains nearly unchanged. Here a model can be constructed showing a xylan molecule on the surface of the core protein near the tail part.     

Solution structure and conformational changes of the Streptomyces chitin-binding protein (CHB1)

The shape and overall dimensions of the recently discovered Streptomyces alpha-chitin-binding protein, CHB1, were investigated by synchrotron radiation X-ray solution scattering. The radius of gyration and the maximum size of CHB1 were determined to be 1.75 +/- 0.03 nm and 6.0 +/- 0.2 nm, respectively. Using two independent ab initio approaches the low-resolution shape of the protein was found to consist of two domains, an elongated main globule with a length of about 4 nm and a foot-like domain of about 2 nm width. The structural and functional properties of CHB1 depend strongly on the presence of disulfide bonds; upon their reduction, the protein loses its affinity to chitin.     

Small-angle X-ray studies of Lumbricus terrestris haemoglobin

Small-angle X-ray scattering of Lumbricus terrestris haemoglobin was measured in dilute solutions in 0.1 M Tris HCl buffer, pH 7.0. The following molecular parameters were determined: radius of gyration 11.2 nm, volume 7700 nm³, maximum diameter 29 nm, molecular weight 3.95 × 10⁶. The experimental scattering curve was compared with the scattering curves and distance distribution functions calculated for various models. The overall shape of the haemoglobin could be approximated by a hollow cylinder with the following dimensions: outer radius 13.5 nm, inner radius 5.4 nm, height 16.0 nm. The best fit was obtained with a model which consists of 12 large subunits arranged in two superimposed hexagonal rings with a number of smaller subunits between the large subunits and in the centre of the molecule.     

Molecular modeling of the multidomain structures of the proteoglycan binding region and the link protein of cartilage by neutron and synchrotron X-ray scattering

The interaction of proteoglycan monomers with hyaluronate in cartilage is mediated by a globular binding region at the N-terminus of the proteoglycan monomer; this interaction is stabilized by link protein. Sequences show that both the binding region (27% carbohydrate) and the link protein (6% carbohydrate) contain an immunoglobulin (Ig) fold domain and two proteoglycan tandem repeat (PTR) domains. Both proteins were investigated by neutron and synchrotron X-ray solution scattering, in which nonspecific aggregate formation was reduced by the use of citraconylation to modify surface lysine residues. The neutron and X-ray radius of gyration RG of native and citraconylated binding region is 5.1 nm, and the cross-sectional RG (RXS) is 1.9-2.0 nm. No neutron contrast dependence of the RG values was observed; however, a large contrast dependence was seen for the RXS values which is attributed to the high carbohydrate content of the binding region. The neutron RG for citraconylated link protein is 2.9 nm, its RXS is 0.8 nm, and these data are also independent of the neutron contrast. The scattering curves of binding region and link protein were modeled using small spheres. Both protein structures were defined initially by the representation of one domain by a crystal structure for a variable Ig fold and a fixed volume for the two PTR domains calculated from sequence data. The final models showed that the different dimensions and neutron contrast properties of binding region compared to link protein could be attributed to an extended glycosylated C-terminal peptide with extended carbohydrate structures in the binding region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Small-angle x-ray scattering investigation of the solution structure of troponin C

X-ray crystallographic studies of troponin C (Herzberg, O., and James, M.N.G. (1985) Nature 313, 653-659; Sundaralingam, M., Bergstrom, R., Strasburg, G., Rao, S.T., and Roychowdhury, P. (1985a) Science 227, 945-948) have revealed a novel protein structure consisting of two globular domains, each containing two Ca2+-binding sites, connected via a nine-turn alpha-helix, three turns of which are fully exposed to solvent. Since the crystals were grown at pH approximately 5, it is of interest to determine whether this structure is applicable to the protein in solution under physiological conditions. We have used small-angle x-ray scattering to examine the solution structure of troponin C at pH 6.8 and the effect of Ca2+ on the structure. The scattering data are consistent with an elongated structure in solution with a radius of gyration of approximately 23.0 A, which is quite comparable to that computed for the crystal structure. The experimental scattering profile and the scattering profile computed from the crystal structure coordinates do, however, exhibit differences at the 40-A level. A weak Ca2+-facilitated dimerization of troponin C was observed. The data rule out large Ca2+-induced structural changes, indicating rather that the molecule with Ca2+ bound is only slightly more compact than the Ca2+-free molecule.     

Structural insight into the glycosylphosphatidylinositol transamidase subunits PIG-K and PIG-S from yeast

The addition of glycosylphosphatidylinositol (GPI) anchors to eukaryotic proteins in the lumen of the endoplasmic reticulum is catalyzed by the transamidase complex, composed of at least five subunits (PIG-K, PIG-S, PIG-T, PIG-U and GPAA1). Here PIG-K(24-337) and PIG-S(38-467) from yeast, including the residues 24-337 and 38-467 of the entire 411 and 534 residue protein, respectively, was produced in Escherichia coli and purified to homogeneity. Analysis of secondary structure by circular dichroism spectroscopy showed that yPIG-K(24-377) comprises 52% α-helix and 12% β-sheet, whereas yPIG-S(38-467) involves 58% α-helix and 18% β-sheet. The radius of gyration (R(g)) and the maximum size (D(max)) of both proteins have been analyzed by small angle X-ray scattering (SAXS) and determined to be 2.64±0.3 and 10.3±0.1 nm (yPIG-K(24-377)) as well as 3.06±0.02 nm (R(g)) and 16.9±0.4 nm (D(max)) in the case of yPIG-S(38-467), respectively. Using an ab initio approach, the first low-resolution solution structures of both proteins were restored. yPIG-K(24-377) is an elongated particle consisting of an egg-like portion and a small globular segment linked together by an 1.9 nm long stalk. yPIG-S(38-467) forms an elongated molecule in solution with a larger domain of 10.1 nm in length, a diameter of 9.1 nm and a smaller domain of 6.7 nm in length and 3.4 nm in width. The two domains of yPIG-S(38-467) are tilted relative to each other. Finally, the arrangements of PIG-K and PIG-S inside the ensemble of the transamidase complex are discussed.     

Structural change of troponin C molecule and its domains upon Ca2+ binding in the presence of Mg2+ ions measured by a solution X-ray scattering technique

A small-angle X-ray scattering study on troponin C showed that two domains of the molecule move closer to each other and the molecule shrinks along its long axis upon Ca2+ binding in the absence of Mg2+ ions (Fujisawa, T., Ueki, T., & Iida S. (1988) J. Biochem. 105, 377-383). When Mg2+ ions bind to troponin-C, the radius of gyration changes from 27.8 to 24.3 A and the average radius of gyration of the two domains is estimated to be 15.1 A. These radii indicate that the distance between the centers of the two domains is 38.1 A. Such a change is analogous to the previous result for troponin C with two Ca2+ ions bound at the high-affinity sites. Thus, the structural behavior of troponin C molecule is essentially the same when Ca2+/Mg2+ ions bind to its high-affinity sites. On the other hand, the effect of Ca2+ binding to the low-affinity sites in the presence of Mg2+ ions is quite different from the previous result. The binding of Ca2+ ions causes a dimerization of troponin C molecules with an apparent constant of 511 M-1. Such a characteristic behavior, implying the occurrence of a surface property change, may be related to the physiological role of troponin C molecule in the muscle. The scattering experiments on the tryptic fragments of troponin C also had interesting and important results: the C-domain shrinks, with the radius of gyration changing from 17.0 to 14.9 A while the N-domain swells from 13.9 to 15.0 A upon Ca2+ binding. Such an opposite change is consistent with the results of circular dichroism and spectroscopic studies of the domains.     

Small-angle x-ray studies on malate synthase from baker's yeast.

Malate synthase was investigated in solution by the small-angle X-ray scattering technique. The substrate-free enzyme was shown to have a molecular weight of 186000, a radius of gyration of 3.96 nm, a maximum particle diameter of 11.2 nm, a volume of 343 nm³, a radius of gyration of the thickness of 1.04 nm, and an axial ratio of 1:0.33. The enzyme molecule undergoes small changes in overall structure upon binding substrates. Investigation of the enzyme under prolonged exposure to X-rays led to an aggregation of the enzyme and allowed statements concerning the way of aggregation and factors influencing aggregation.     

Models of fibronectin.

The radius of gyration of human plasma fibronectin was determined by light scattering both under conditions in which the molecule is in an extended conformation (ionic strength 1.01 M, pH 8) and close to its native, more compact conformation (ionic strength 0.16 M, pH 8). These values were found to be 17.5 +/- 0.8 nm and 10.7 +/- 0.9 nm respectively, for a constant mol. wt of 533,000 +/- 8000, in excellent agreement with the value of 520,000 deduced from its known composition. A set of models, each made of two identical, end-to-end joined chains of 28 beads, was then constructed, and their calculated physico-chemical parameters were compared with those available for the whole fibronectin molecule and for some of its proteolytic fragments in both conformations. Two possible models for the circulating form are presented here: in both, the fibronectin molecule is in a compact, tangled conformation, with the amino-terminal end of one chain folded over to the carboxy end of itself or of the other chain either in a hairpin or in a circular fashion. With the exception of the carboxy-terminal fibrin(ogen)-binding domains, all the domains appear to be well exposed to the solvent, and thus free to interact with potential ligands.     

Extended conformation of mammalian translation elongation factor 1A in solution

The conformation of mammalian elongation factor eEF1A in solution was examined by the small angle neutron scattering and scanning microcalorimetry. We have found that in contrast to the bacterial analogue the eEF1A molecule has no fixed rigid structure in solution. The radius of gyration of the eEF1A molecule (5.2 nm) is much greater than that of prokaryotic EF1A. The specific heat of denaturation is considerably lower for eEF1A than for EF1A, suggesting that the eEF1A conformation is significantly more disordered. Despite its flexible conformation, eEF1A is found to be highly active in different functional tests. According to the neutron scattering data, eEF1A becomes much more compact in the complex with uncharged tRNA. The absence of a rigid structure and the possibility of large conformational change upon interaction with a partner molecule could be important for eEF1A functioning in channeled protein synthesis and/or for the well-known capability of the protein to interact with different ligands besides the translational components.     

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.     

The tetrameric form of ribosomal protein L7/L12 from Escherichia coli

A tetrameric form of the ribosomal protein L7/L12 has been prepared and its structure studied by using hydrodynamic methods, photon correlation spectroscopy, and small angle x-ray scattering. The tetrameric nature of the protein preparation is confirmed by three independent determinations of its molecular weight, with analysis of accurate sedimentation equilibrium data giving the most reliable estimate. The species has a Stokes radius of 4.0 +/- 0.1 nm and an absolute frictional ratio of 1.7. Taken together, the hydrodynamic measurements suggest the possibility of a flat structure, and this is consistent with the x-ray scattering results. The molecule has a radius of gyration of 3.6 +/- 0.05 nm and a maximum dimension of 11-12 nm. A geometric model consisting of four elongated monomers, arranged in a plane, is proposed.     

X-ray scattering study of hagfish protease inhibitor, a protein structurally related to complement and alpha 2-macroglobulin.

A protease inhibitor from hagfish blood plasma, homologous to human alpha 2-macroglobulin, has been studied in solution using small-angle X-ray scattering; the radius of gyration, R, was found to be 7.0 nm, the molecular weight 340,000 +/- 20,000, and the largest distance within the molecule, Dmax, 22 nm. When the inhibitor reacts with chymotrypsin, its 1:1 chymotrypsin complex is found to be more compact than the native molecule, R = 6.1 nm. A very similar conformational change is observed after the protein is reacted with methylamine. The data are consistent with models consisting of two equal elliptic cylinders with the same size as the one used as a model for the complement proteins C3 and C4 [cf. Osterberg et al. (1989) Eur. J. Biochem. 183, 507-511]. In the model for the native protein, these cylinders are arranged in an extended form, and in the one for the methylamine derivative (or chymotrypsin complex), they are closer together so that the projection of their elliptic surfaces forms an angle of about 70 degrees. These models for the hagfish protease inhibitor were expanded to models for the twice as large human alpha 2-macroglobulin using symmetry operations, and the resulting alpha 2-macroglobulin models were found to agree with those emerged from earlier studies involving electron microscopy and X-ray scattering methods.     

Small-angle X-ray studies of the human immunoglobulin molecule Kol.

The conformation of the human immunoglobulin molecul Kol [IgG I, kappa2 gamma2, Gm(f)+] was studied by small-angle X-ray scattering in 0.15 M NaCl solution. The radius of gyration was found to be 5.84 +/- 0.04 nm, the volume 329 +/- 15 nm3 and the molecular weight 150 000 +/- 10 000. Information on the overall shape was obtained by comparing the experimental scattering curve with the calculated curves for various models. The models were obtained by arranging the models found for the Fab and Fc fragments of the same immunoglobulin molecule in a different manner. A model which fits all the date and the form of the experimental scattering curve is presented.