Small-angle X-ray scattering studies of calmodulin mutants with deletions in the linker region of the central helix indicate that the linker region retains a predominantly alpha-helical conformation

Two mutant forms of calmodulin were examined by small-angle X-ray scattering in solution and compared with the wild-type protein. Each mutant has deletions in the linker region of the central helix: one lacks residues Glu-83 and Glu-84 (Des2) and the other lacks residues Ser-81 through Glu-84 (Des4). The deletions change both the radii of gyration and the maximum dimensions of the molecules. In the presence of Ca2+, the observed radii of gyration are 22.4 A for wild-type bacterially expressed calmodulin, 19.5 A for Des2 calmodulin, and 20.3 A for Des4 calmodulin. A reduction in the radius of gyration by 1-2 A on removal of calcium, previously observed in the native protein, was also found in the wild type and the Des4 mutant; however, no significant size change was observed in the Des2 mutant. The large calcium-dependent conformational change in calmodulin induced by the binding of melittin [Kataoka, M., Head, J.F., Seaton, B.A., & Engelman, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6944-6948] was observed in all the bacterially expressed proteins. Each protein appears to undergo a transition from a dumbbell shape to a more globular conformation on binding melittin in the presence of calcium, although quantitatively the changes in the wild-type and Des4 proteins greatly exceed those in Des2. Modeling shows the central linker region of the molecule. Thus, the structure of the linker region is stable enough to maintain the average orientation and separation of the lobes yet flexible enough to permit the lobes to approach each other upon binding a peptide.     

Solution structure of phosphorylase kinase studied using small-angle X-ray and neutron scattering.

Small-angle X-ray and neutron scattering have been used to characterize the solution structure of rabbit skeletal phosphorylase kinase. The radius of gyration of the unactivated holoenzyme determined from neutron scattering is 94 A, and its maximum dimension is approximately 275-295 A. A planar model has been constructed that is in general agreement with the dimensions of the transmission electron microscope images of negatively stained phosphorylase kinase and that gives values for the radius of gyration, maximum linear dimension, and a pair distribution function for the structure that are consistent with the scattering data.     

The linker of calmodulin lacking Glu84 is elongated in solution,although it is bent in the crystal

The solution structure of a mutant calmodulin (des84) lacking Glu84 in the central helix linking the two calmodulin lobes is substantially different from its crystal structure. As determined by small-angle X-ray scattering, the radius of gyration and the maximum linear dimension of des84 in the presence of 0.1 mM calcium are 20.8 Å and 62.5 Å, respectively. These respective dimensions are larger than those expected from the crystal structure of des84, 18.5 Å and 55.0 Å, and smaller than those expected from the crystal structure of wild type, 22.8 Å and 67.5 Å. The distance distribution function of des84 indicates that it assumes an elongated, dumbbell shape in solution. The solution scattering profile of des84 is indistinguishable from that of wild-type calmodulin. The calcium-dependent binding of melittin to des84 causes a change in its shape from elongated to spherical, as seen with other calmodulins. In comparison with calcium-saturated des84, calcium-free des84 is slightly elongated; a slight compaction is observed with native calmodulin. The observed differences between the averaged solution structure and the crystal structure of des84 suggests that an ensemble of structures is available to calmodulin in solution and that its target need not induce a change in its conformation. These results support the theory that the linker region of the central helix of calmodulin functions as a flexible tether. © 1996 Wiley-Liss, Inc.     

Formation of actin dimers as studied by small angle neutron scattering

Small angle neutron scattering has been used to study the dimensions of G-actin and the formation of low molecular weight actin oligomers under conditions where rapid polymerization does not take place. In the presence of 200 microM Ca2+, actin in solution consists of a single component with a radius of gyration (Rg) of 19.9 +/- 0.4 A, consistent with the known molecular dimensions of the G-actin molecule. In the presence of 50 microM Mg2+, however, formation of an actin species with a larger Rg occurs over a 4-h period. Multicomponent fits were tried and the data were best fit assuming two components, the monomer and a species with an Rg of 29 +/- 1 A. This latter value is consistent with the dimensions expected for certain actin dimers. The apparent dissociation constant for dimer formation is approximately 150 microM with forward and reverse rate constants of 6.0 X 10(-7) microM-1 s-1 and 8.8 X 10(-5) s-1, respectively. Kinetic fluorescence experiments show that the dimer formed in the presence of low levels of Mg2+ is a nonproductive complex which does not participate in the polymerization process. However, the addition of cytochalasin D to actin in the presence of 50 microM Mg2+ rapidly induces the formation of dimers, presumably related to cytochalasin's ability to nucleate actin polymerization.     

Conformational changes in arginine kinase upon ligand binding seen by small-angle X-ray scattering

Small-angle X-ray scattering is used to study the effects of substrate binding to lobster arginine kinase in solution. We measure the radius of gyration of the enzyme in the absence and in the presence of ligands. We find that the radius of gyration decreases by 1.20 ± 0.25 Å upon binding ADP-Mg and L-arginine to form the ternary complex. The same decrease is also observed upon binding ADP-Mg alone or ATP-Mg. These results indicate a large conformational change consistent with the hinge motion of domains observed in other phosphokinases.     

A neutron solution scattering study of the structure of annexin-V and its binding to lipid vesicles.

Low-angle neutron solution scattering has been used to study the structure of annexin-V and its interaction with small single-bilayer vesicles consisting of phosphatidylserine and phosphatidylcholine at a 33:66 (mol:mol) ratio. There was no evidence for a change in the state of aggregation of annexin-V, which remained as a monomer in the presence of 3 mM-free calcium. The only difference between presence and absence of free calcium was the increase of the radius of gyration, from 19(+/- 0.4) A to 22(+/- 0.4) A in 2H2O buffer and from 19.7(+/- 1.2) A to 22.2(+/- 1.2) A in H2O buffer. The relative molecular weight, outer radius and average surface area per lipid of vesicles alone were respectively 2.5(+/- 0.5) x 10(6), 127 A and 90(+/- 19) A2. These parameters were not modified in the presence of free calcium, which testified to the absence of vesicle coalescence. The calcium-dependent binding of annexin-V was essentially interfacial and therefore did not alter significantly the structural characteristics of the vesicles. At saturation, 80(+/- 10) annexin-V molecules were bound per vesicle, the available area per molecule being 2500(+/- 300) A2 thus covering approximately 28 lipid head groups. The protein shell was approximately 35 A thick. The apparent dissociation constant was probably less than 1 nM. These data contribute to a more accurate definition of annexin-V as a possible probe of those cytodynamic events involving exposure of sequestered membrane aminophospholipids.     

Changes in the structure of calmodulin induced by a peptide based on the calmodulin-binding domain of myosin light chain kinase

Small-angle X-ray and neutron scattering data were used to study the solution structure of calmodulin complexed with a synthetic peptide corresponding to residues 577-603 of rabbit skeletal muscle myosin light chain kinase. The X-ray data indicate that, in the presence of Ca2+, the calmodulin-peptide complex has a structure that is considerably more compact than uncomplexed calmodulin. The radius of gyration, Rg, for the complex is approximately 20% smaller than that of uncomplexed Ca2+.calmodulin (16 vs 21 A), and the maximum dimension, dmax, for the complex is also about 20% smaller (49 vs 67 A). The peptide-induced conformational rearrangement of calmodulin is [Ca2+] dependent. The length distribution function for the complex is more symmetric than that for uncomplexed Ca2+.calmodulin, indicating that more of the mass is distributed toward the center of mass for the complex, compared with the dumbell-shaped Ca2+.calmodulin. The solvent contrast dependence of Rg for neutron scattering indicates that the peptide is located more toward the center of the complex, while the calmodulin is located more peripherally, and that the centers of mass of the calmodulin and the peptide are not coincident. The scattering data support the hypothesis that the interconnecting helix region observed in the crystal structure for calmodulin is quite flexible in solution, allowing the two lobes of calmodulin to form close contacts on binding the peptide. This flexibility of the central helix may play a critical role in activating target enzymes such as myosin light chain kinase.     

Size of a human serum albumin molecule in solution]

The size of a human serum albumin molecule in aqueous solution containing 150 mM NaCl was studied using small-angle neutron scattering. The molecular radius of gyration was estimated to be 27.4 +/- 0.35 A. The compact sphere should have a smaller radius of gyration, whereas the popular human serum albumin model, a "cigar" 136 A long, should correspond to a greater radius of gyration. Possible shapes of the human serum albumin molecule which are in accordance with the results obtained, are the following: an extended ellipsoid less than 110 A of length or a nonsymmetrical oblate ellipsoid with a diameter of 85 A. The oblate ellipsoid might be close to the heart"-shaped structure of the crystalline human serum albumin molecule. The size of the albumin molecule does not change significantly as pH increases to 8.9. The possibility of the dynamic coexistence of various human serum albumin conformers in solution is discussed.     

Small angle x-ray scattering (SAXS) study of the extracellular Hemoglobin of Glossoscolex paulistus: effect of pH, iron oxidation state, and interaction with anionic SDS surfactant

pH effects on the oligomeric structure of giant Glossoscolex paulistus extracellular hemoglobin in the oxyand met-forms have been studied as well as effects of the addition of anionic sodium dodecyl sulfate surfactant. A radius of gyration of 110 A is observed for a macromolecule. At 2 mm surfactant, the radius of gyration diminishes slightly for the oxy-form. However, the extrapolated initial scattering intensity (I0) decreases a factor of 2.5, indicating protein dissociation. At 20 mm surfactant, further I0 decrease is observed, with a reduction of radius of gyration to approximately 30 A consistent with dissociation into smaller subunits. At pH 9.0, the scattering curves are similar to that obtained for the protein in the presence of 20 mm surfactant at pH 7.0. A radius of gyration of approximately 35 A shows that the giant hemoglobin dissociation into small subunits also occurs at alkaline pH. From the I0 value, one can suggest that the tetramer is the main scatter at pH 9.0. At pH 7.0, the met-form dissociates to a larger extent at 2 mm surfactant as compared with the oxy-form, and the main scatters seem to be the 1/12 subunit. At pH 9.0, for the oxy-form, the addition of surfactant does not modify the scattering curve and a radius of gyration approximately 30 A is obtained, while for the met-form some kind of aggregation is observed. Our results give support to conclude that the iron oxidation state is an important factor modulating the oligomeric dissociation.     

Calmodulin-peptide interactions: apocalmodulin binding to the myosin light chain kinase target-site

Noncovalent binding of the synthetic peptide RS20 to calmodulin in the presence of calcium was confirmed by electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry to form a complex with a 1:1:4 calmodulin/RS20/calcium stoichiometry. There was no evidence for formation of a calmodulin-RS20-Ca(2) species. The absence of calmodulin-RS20-Ca(2) would be consistent with models in which the two globular domains are coupled functionally. There was evidence that calmodulin, RS20-calmodulin without associated calcium, and calmodulin-RS20-Ca(4) existed together in solution, whereas calmodulin-calcium complexes were absent. It is proposed that calcium binding to form the calmodulin-RS20-Ca(4) complex occurs after an initial RS20-calmodulin binding event, and serves to secure the target within the calmodulin structure. The binding of more than one RS20 molecule to calmodulin was observed to induce unfolding of calmodulin.     

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.     

The structural basis for the regulation of tissue transglutaminase by calcium ions.

The role of calcium ions in the regulation of tissue transglutaminase is investigated by experimental approaches and computer modeling. A three-dimensional model of the transglutaminase is computed by homology building on crystallized human factor XIII and is used to interpret structural and functional results. The molecule is a prolate ellipsoid (6.2 x 4.2 x 11 nm) and comprises four domains, assembled pairwise into N-terminal and C-terminal regions. The active site is hidden in a cleft between these regions and is inaccessible to macromolecular substrates in the calcium-free form. Protein dynamics simulation indicates that these regions move apart upon addition of calcium ions, revealing the active site for catalysis. The protein dimensions are consistent with results obtained with small-angle neutron and X-ray scattering. The gyration radius of the protein (3 nm) increases in the presence of calcium ions (3.9 nm), but it is virtually unaffected in the presence of GTP, suggesting that only calcium ions can promote major structural changes in the native protein. Proteolysis of an exposed loop connecting the N-terminal and C-terminal regions is linearly correlated with enzyme inactivation and prevents the calcium-induced conformational changes.     

Formation of cyclic imide-like structures upon the treatment of calmodulin and a calmodulin peptide with heat

Protein cyclic imide is the putative intermediate in the formation of sites of carboxyl-methylation in eukaryotic proteins. Conditions known to induce the formation of a cyclic imide in model peptides have been applied to a protein, calmodulin. Heating of calmodulin in the dry state at 100 degrees C for 24 h after lyophilization from a pH 2.0 or pH 6.0 solution produces derivatives with altered chromatographic properties in anion-exchange HPLC. At pH 6.0, complete activity of calmodulin was retained. Analysis with Fourier transform infrared (FTIR)-photoacoustic spectroscopy demonstrated the presence of a new structure in the calmodulin molecule consistent with modification of carboxylic acid groups. The conversion of calmodulin is dependent upon the absence of Ca2+ (the presence of 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid). A peptide analogous to the calcium binding regions of calmodulin, Asp-Lys-Asp-Gly-Asn-Gly-Thr-Ile-Thr-Thr-Lys-Glu, is also converted, upon heating, to chromatographically different forms in reversed-phase chromatography. This process is also dependent upon the absence of calcium. Sequence analysis of the peptide derivatives reveals a second amino terminus, implicating peptide bond hydrolysis in the product. A dipeptide, Asp-Gly, known to form a cyclic imide structure under similar conditions is also hydrolyzed during sequence analysis consistent with cleavage occurring at the position of the cyclic imide structure. Asp3 is suggested to be the site of cyclic imide formation in the calmodulin peptide. The presence of a cyclic imide structure is also confirmed by the application of FTIR-photoacoustic spectroscopy. These data suggest that cyclic imide formation in calmodulin has been induced, possibly at one, or more, of the calcium binding loops of the protein. These modification reactions may provide a basis for future investigations of cyclic imide formation in proteins.     

Binding of both Ca2+ and mastoparan to calmodulin induces a large change in the tertiary structure

The technique of small-angle X-ray scattering has been employed to examine the solution conformation of calmodulin and its complexes with Ca2+ alone, and with both Ca2+ and mastoparan. The radius of gyration decreased by 3.1 +/- 0.3 A upon binding of both 4 mol Ca2+/mol of protein and 1 mol mastoparan/mol of protein to form the ternary complex. A smaller increase was found for the separate binding of 4 mol Ca2+/mol of protein in the absence of mastoparan (0.6 +/- 0.3 A). The analyses of pair distance distribution function showed that the maximal pair distance in calmodulin complex with both Ca2+ and mastoparan decreased by 20-30% in comparison with calmodulin or its complex with Ca2+, and a shoulder near 40 A, which characterizes the dumbbell-shaped molecule of calmodulin, disappeared. These results indicate that the two globular domains of the calmodulin complex with Ca2+ and mastoparan come close together by 8.0-9.5 A on average, if the size and the overall shape of the globular domains are the same in Ca2+-calmodulin-mastoparan complex as in calmodulin or Ca2+-calmodulin complex.     

Detection of ligand- and solvent-induced shape alterations of cell-growth-regulatory human lectin galectin-1 in solution by small angle neutron and x-ray scattering

The bioactivity of galectin-1 in cell growth regulation and adhesion prompted us to answer the questions whether ligand presence and a shift to an aprotic solvent typical for bioaffinity chromatography might alter the shape of the homodimeric human lectin in solution. We used small angle neutron and synchrotron x-ray scattering studies for this purpose. Upon ligand accommodation, the radius of gyration of human galectin-1 decreased from 19.1 +/- 0.1 A in the absence of ligand to 18.2 +/- 0.1 A. In the aprotic solvent dimethyl sulfoxide, which did not impair binding capacity, galectin-1 formed dimers of a dimer, yielding tetramers with a cylindrical shape. Intriguingly, no dissociation into subunits occurred. In parallel, NMR monitoring was performed. The spectral resolution was in accord with these data. In contrast to the properties of the human protein, a nonhomologous agglutinin from mistletoe sharing galactose specificity was subject to a reduction in the radius of gyration from approximately 62 A in water to 48.7 A in dimethyl sulfoxide. Evidently, the solvent caused opposite responses in the two tested galactoside-binding lectins with different folding patterns. We have hereby proven that ligand presence and an aprotic solvent significantly affect the shape of galectin-1 in solution.     

Time-resolved fluorescence study of VU-9 calmodulin, an engineered calmodulin possessing a single tryptophan residue

An engineered calmodulin (VU-9 calmodulin), which possesses a single tryptophan residue at position 99 in calcium binding domain III, was studied by time-resolved fluorescence. At least two exponential terms are needed to describe the tryptophan fluorescence decays, either in the presence or in the absence of calcium. The characteristics of the fluorescence decays are strongly dependent upon the number of calcium ions bound per molecule of VU-9 calmodulin until half of the calcium sites are occupied, i.e., three in the absence of magnesium and two in the presence of 5 mM magnesium. A clear time-dependent spectral shift is observed in the presence of calcium. The existence of an isosbestic point in the time-resolved spectra is in agreement with a two-state model. The biexponential analysis of the 340-nm fluorescence decay during calcium titration gives parameters consistent with a two-state model in which tryptophan 99 interconverts between two different conformations, characterized by a different lifetime value, with rates altered by calcium binding. This model explains the decrease in the protein quantum yield induced by calcium binding [Kilhoffer, M. C., Roberts, D. M. Adibi, A. O., Watterson, D. M., & Haiech, J. (1989) Biochemistry (preceding paper in this issue)].     

Calcium-induced shape change of calmodulin with mastoparan studied by solution X-ray scattering

Solution x-ray scattering using synchrotron radiation as an x-ray source was used to analyze the Ca2+-dependent shape change of pig brain calmodulin in detail. The radius of gyration of calmodulin at 10 mg/ml was increased by 0.9 A. The increase was nearly completed when 2.5 mol of Ca2+/mol of calmodulin was added, whereas the radius of gyration of calmodulin with mastoparan decreased by about 3 A with an increasing Ca2+ concentration up to 4 mol of Ca2+/mol of calmodulin. At a moderate angle of region, both scattering profiles from calmodulin with or without Ca2+ displayed clear humps at s = 0.03 A-1 which are characteristic of a dumbbell structure. However, in the presence of mastoparan, the hump in the scattering profile became obscure and later disappeared with the third and fourth Ca2+ binding to calmodulin. These findings are attributable to the Ca2+-induced shape change of calmodulin with mastoparan from a dumbbell structure to a non-dumbbell structure in which the distance between the two lobes of calmodulin become closer by a bend in the central helix.     

Study of the structure of immunoglobins by small-angle x-ray diffraction. I. The structure of IgMCep in solution

The data of small-angle X-ray scattering from monoclonal immunoglobulin MCep (IgM) enabled the shape and geometrical parameters of the molecule in solution at 23 degrees C to be established. The molecule is a flat, strongly anisometric particle with radius of gyration 115 A, volume 1,8 X 10(6) A3, maximum size 380 A, thickness 35-40 A. The most probable molecular model in the approximation of homogeneous electron density in the molecule was suggested, its geometry fitting the experimental parameters. The five IgM subunits are located in the equatorial plane, low-electronic-density regions are located in the centre and at the periphery of the macromolecule. In addition, the absence of fixed angle values between Fab-regions in each subunit is indicative of rather high structural mobility at the periphery of the IgM molecule.     

The conformation of polyriboadenylic acid at low temperature and neutral pH. A single-stranded rodlike structure.

The conformation of single-stranded polyrA in aqueous solution has been measured at temperatures down to ?12°C. The radius of gyration of low-molecular-weight polyrA varies very little with temperature in this range. By studying the dependence of radius of gyration on temperature for several polyrA fractions, we show that the dependence of the radius upon chain length is consistent with formation of a single-stranded rodlike structure at low temperature. The structure has an approximate length of 3.2 Å/nucleotide.     

Evidence for major structural changes in the Manduca sexta midgut V1 ATPase due to redox modulation. A small angle X-ray scattering study

The shape and overall dimensions of the oxidized and reduced form of the V(1) ATPase from Manduca sexta were investigated by synchrotron radiation x-ray solution scattering. The radius of gyration of the oxidized and reduced complex differ noticeably, with dimensions of 6. 20 +/- 0.06 and 5.84 +/- 0.06 nm, respectively, whereas the maximum dimensions remain constant at 22.0 +/- 0.1 nm. Comparison of the low resolution shapes of both forms, determined ab initio, indicates that the main structural alteration occurs in the head piece, where the major subunits A and B are located, and at the bottom of the stalk. In conjunction with the solution scattering data, decreased susceptibility to tryptic digestion and tryptophan fluorescence of the reduced V(1) molecule provide the first strong evidence for major structural changes in the V(1) ATPase because of redox modulation.