Biomedical subjects

William T Heller

Publications and source records attributed to William T Heller.

11 recordsLinked to original sources

PubMed2006

Self-assembled colloidal crystals from ZrO2 nanoparticles.

Ordered three-dimensional (3-D) assemblies of nanocrystalline zirconia were synthesized from aqueous suspensions of ZrO(2) nanoparticles without the need for hydrocarbon surfactants or solvents to control colloidal crystal growth. Nanoparticles were suspended in mild acid and subsequently titrated from low to neutral pH. The solubility was reduced as the surfaces were neutralized, promoting assembly of the nanoparticles into ordered monoliths. TEM measurements indicated the formation of three-dimensional, hexagonal faceted, micrometer-sized colloidal crystals composed of 4 nm diameter ZrO(2) nanoparticles. Lacking organic surfactants, the colloidal crystals were exceptionally robust and were sintered at high temperatures (300-500 degrees C) for further stability. Small-angle X-ray scattering (SAXS) measurements demonstrate that the samples become progressively more amorphous above 350 degrees C, although some ordered domains of nanoparticles persist. Additionally, the heat treatment dramatically increases the surface area of the colloidal crystals as water and residual organics are desorbed, revealing highly controlled interstitial spaces and pores.

Journal Article↗

PubMed2006

Structural basis for the differential regulation of DNA by the methionine repressor MetJ.

The Met regulon in Escherichia coli encodes several proteins responsible for the biosynthesis of methionine. Regulation of the expression of most of these proteins is governed by the methionine repressor protein MetJ and its co-repressor, the methionine derivative S-adenosylmethionine. Genes controlled by MetJ contain from two to five sequential copies of a homologous 8-bp sequence called the metbox. A crystal structure for one of the complexes, the repressor tetramer bound to two metboxes, has been reported (Somers, W. S., and S. E. Phillips (1992) Nature 359, 387-393), but little structural work on the larger assemblies has been done presumably because of the difficulties in crystallization and the variability in the number and sequences of metboxes for the various genes. Small angle neutron scattering was used to study complexes of MetJ and S-adenosylmethionine with double-stranded DNA containing two, three, and five metboxes. Our results demonstrate that the crystal structure of the two-metbox complex is not the native solution conformation of the complex. Instead, the system adopts a less compact conformation in which there is decreased interaction between the adjacent MetJ dimers. Models built of the higher order complexes from the scattering data show that the three-metbox complex is organized much like the two-metbox complex. However, the five-metbox complex differs significantly from the smaller complexes, providing much closer packing of the adjacent MetJ dimers and allowing additional contacts not available in the crystal structure. The results suggest that there is a structural basis for the differences observed in the regulatory effectiveness of MetJ for the various genes of the Met regulon.

Crystallography, X-Ray↗

PubMed2005

Ca2+-induced structural changes in phosphorylase kinase detected by small-angle X-ray scattering.

Phosphorylase kinase (PhK), a 1.3-MDa (alphabetagammadelta)(4) hexadecameric complex, is a Ca(2+)-dependent regulatory enzyme in the cascade activation of glycogenolysis. PhK comprises two arched (alphabetagammadelta)(2) octameric lobes that are oriented back-to-back with overall D(2) symmetry and joined by connecting bridges. From chemical cross-linking and electron microscopy, it is known that the binding of Ca(2+) by PhK perturbs the structure of all its subunits and promotes redistribution of density throughout both its lobes and bridges; however, little is known concerning the interrelationship of these effects. To measure structural changes induced by Ca(2+) in the PhK complex in solution, small-angle X-ray scattering was performed on nonactivated and Ca(2+)-activated PhK. Although the overall dimensions of the complex were not affected by Ca(2+), the cation did promote a shift in the distribution of the scattering density within the hydrated volume occupied by the PhK molecule, indicating a Ca(2+)-induced conformational change. Computer-generated models, based on elements of the known structure of PhK from electron microscopy, were constructed to aid in the interpretation of the scattering data. Models containing two ellipsoids and four cylinders to represent, respectively, the lobes and bridges of the PhK complex provided theoretical scattering profiles that accurately fit the experimental data. Structural differences between the models representing the nonactivated and Ca(2+)-activated conformers of PhK are consistent with Ca(2+)-induced conformational changes in both the lobes and the interlobal bridges.

Animals↗

PubMed2005

A model for the three-dimensional structure of human plasma vitronectin from small-angle scattering measurements.

Small-angle X-ray scattering (SAXS) measurements were used to characterize vitronectin, a circulatory protein found in human plasma that functions in regulating cell adhesion and migration, as well as proteolytic cascades that affect blood coagulation, fibrinolysis, and pericellular proteolysis. SAXS measurements were taken over a 3-fold range of protein concentrations, yielding data that characterize a monodisperse system of particles with an average radius of gyration of 30.3 +/- 0.6 A and a maximum linear dimension of 110 A. Shape restoration was applied to the data to produce two models of the solution structure of the ligand-free protein. A low-resolution model of the protein was generated that indicates the protein to be roughly peanut-shaped. A better understanding of the domain structure of vitronectin resulted from low-resolution models developed from available high-resolution structures of the domains. These domains include the N-terminal domain that was determined experimentally by NMR [Mayasundari, A., Whittemore, N. A., Serpersu, E. H., and Peterson, C. B. (2004) J. Biol. Chem. 279, 29359-29366] and the docked structure of the central and C-terminal domains that were determined by computational threading [Xu, D., Baburaj, K., Peterson, C. B., and Xu, Y. (2001) Proteins: Struct., Funct., Genet. 44, 312-320]. This model provides an indication of the disposition of the central domain and C-terminal heparin-binding domains of vitronectin with respect to the N-terminal somatomedin B (SMB) domain. This model constructed from the available domain structures, which agrees with the low-resolution model produced from the SAXS data, shows the SMB domain well separated from the central and heparin-binding domains by a disordered linker (residues 54-130). Also, binding sites within the SMB domain are predicted to be well exposed to the surrounding solvent for ease of access to its various ligands.

Computational Biology↗

PubMed2004

Influence of multiple well defined conformations on small-angle scattering of proteins in solution.

A common structural motif for many proteins comprises rigid domains connected by a flexible hinge or linker. The flexibility afforded by these domains is important for proper function and such proteins may be able to adopt more than one conformation in solution under equilibrium conditions. Small-angle scattering of proteins in solution samples all conformations that exist in the sampled volume during the time of the measurement, providing an ensemble-averaged intensity. In this paper, the influence of sampling an ensemble of well defined protein structures on the small-angle solution scattering intensity profile is examined through common analysis methods. Two tests were performed using simulated data: one with the extended and collapsed states of the bilobal calcium-binding protein calmodulin and the second with the catalytic subunit of protein kinase A, which has two globular domains connected by a glycine hinge. In addition to analyzing the simulated data for the radii of gyration Rg, distance distribution function P(r) and particle volume, shape restoration was applied to the simulated data. Rg and P(r) of the ensemble profiles could be easily mistaken for a single intermediate state. The particle volumes and models of the ensemble intensity profiles show that some indication of multiple conformations exists in the case of calmodulin, which manifests an enlarged volume and shapes that are clear superpositions of the conformations used. The effect on the structural parameters and models is much more subtle in the case of the catalytic subunit of protein kinase A. Examples of how noise influences the data and analyses are also presented. These examples demonstrate the loss of the indications of multiple conformations in cases where even broad distributions of structures exist. While the tests using calmodulin show that the ensemble states remain discernible from the other ensembles tested or a single partially collapsed state, the tests performed using the simulated catalytic subunit of protein kinase A with noise added demonstrate that it can mask out the ensemble-dependent effects observed for the noiseless profiles.

Amino Acid Motifs↗

PubMed2004

Conformational differences among solution structures of the type Ialpha, IIalpha and IIbeta protein kinase A regulatory subunit homodimers: role of the linker regions.

The regulatory (R) subunits of the cAMP-dependent protein kinase (protein kinase A or PKA) are multi-domain proteins responsible for conferring cAMP-dependence and localizing PKA to specific subcellular locations. There are four isoforms of the R subunit in mammals that are similar in molecular mass and domain organization, but clearly serve different biological functions. Although high-resolution structures are available for the cAMP-binding domains and dimerization/docking domains of two isoforms, there are no high-resolution structures of any of the intact R subunit homodimer isoforms. The results of small-angle X-ray scattering studies presented here indicate that the RIalpha, RIIalpha, and RIIbeta homodimers differ markedly in overall shape, despite extensive sequence homology and similar molecular masses. The RIIalpha and RIIbeta homodimers have very extended, rod-like shapes, whereas the RIalpha homodimer likely has a compact Y-shape. Based on a comparison of the R subunit sequences, we predict that the linker regions are the likely cause of these large differences in shape among the isoforms. In addition, we show that cAMP binding does not cause large conformational changes in type Ialpha or IIalpha R subunit homodimers, suggesting that the activation of PKA by cAMP involves only local conformational changes in the R subunits.

Animals↗

PubMed2004

C subunits binding to the protein kinase A RI alpha dimer induce a large conformational change.

We present structural data on the RI alpha isoform of the cAMP-dependent protein kinase A that reveal, for the first time, a large scale conformational change within the RI alpha homodimer upon catalytic subunit binding. This result infers that the inhibition of catalytic subunit activity is not the result of a simple docking process but rather is a multi-step process involving local conformational changes both in the cAMP-binding domains as well as in the linker region of the regulatory subunit that impact the global structure of the regulatory homodimer. The results were obtained using small-angle neutron scattering with contrast variation and deuterium labeling. From these experiments we derived information on the shapes and dispositions of the catalytic subunits and regulatory homodimer within a holoenzyme reconstituted with a deuterated regulatory subunit. The scattering data also show that, despite extensive sequence homology between the isoforms, the overall structure of the type I alpha holoenzyme is significantly more compact than the type II alpha isoform. We present a model of the type I alpha holoenzyme, built using available high-resolution structures of the component subunits and domains, which best fits the neutron-scattering data. In this model, the type I alpha holoenzyme forms a flattened V shape with the RI alpha dimerization domain at the point of the V and the cAMP-binding domains of the RI alpha subunits with their bound catalytic subunits at the ends.

Animals↗

PubMed2003

Further insights into calmodulin-myosin light chain kinase interaction from solution scattering and shape restoration.

We have gained new insight into the interactions between the second-messenger protein calmodulin (CaM) and myosin light chain kinase from skeletal muscle (skMLCK) using small-angle solution scattering and shape restoration. Specifically, we explored the nature of a 2Ca(2+)-CaM-skMLCK complex and compared it to a 4Ca(2+)-CaM-skMLCK complex under the same conditions. The 2Ca(2+) complex has been proposed to be physiologically relevant. To aid in the interpretation of the data, we developed a shape restoration approach, implemented in GA_STRUCT, that combines many of the best features of other available methods into a single, automated package. Importantly, GA_STRUCT explicitly addresses the problem of the existence of multiple solutions to the inverse scattering problem and produces a consensus envelope from a set of shapes that fit the input intensity. Small-angle scattering intensity profiles measured or calculated from known structures were used to test GA_STRUCT, which was then used to generate low-resolution models for three complexes: 2Ca(2+)-CaM-skMLCK, 4Ca(2+)-CaM-skMLCK, and 4Ca(2+)-CaM-skMLCK with a bound substrate. These models were used in conjunction with high-resolution structures of the protein components to better understand the interactions among them. In the case of the 2Ca(2+)-CaM-skMLCK complex, the consensus envelope is consistent with CaM in a fully collapsed state with its two globular lobes in close contact with each other while the catalytic cleft of the kinase is open. The consensus envelope for the 4Ca(2+)-CaM-skMLCK complex indicates that the collapsed CaM has swung further away from the open catalytic cleft of the skMLCK than in the 2Ca(2+) complex, and further that substrate binding to this complex results in closure of the kinase catalytic cleft, in agreement with previous neutron scattering results. These results indicate that activation of MLCK by CaM can only occur once CaM is fully translocated away from the catalytic cleft, which is presumably linked to full release of the pseudo-substrate/inhibitory sequence. Our scattering data indicate that this step is completed only when all four calcium binding sites are loaded.

Amino Acid Sequence↗

PubMed2003

Small-angle neutron scattering with contrast variation reveals spatial relationships between the three subunits in the ternary cardiac troponin complex and the effects of troponin I phosphorylation.

Small-angle neutron scattering with contrast variation has been used to determine the shapes and dispositions of the three subunits of cardiac troponin and to study the influence of phosphorylation on the structure. Three contrast variation series were collected on three different isotopically labeled variants of the cTnC/cTnI/cTnT(198-298) complex, one of which contained deuterated and bisphosphorylated cTnI. Analysis of the scattering data shows cTnT(198-298) interacting with a single lobe of a somewhat compacted cTnC that sits at one end of an elongated rodlike cTnI, covering about one-third of its length. The cTnT(198-298) sits near the center of the long cTnI axis. The components undergo significant conformational changes and reorientations in response to protein kinase A phosphorylation of cTnI. The rodlike cTnI bends sharply at the end interacting with the cTnC/cTnT(198-298) component, which reorients so as to maintain its contacts with cTnI while undergoing only a relatively small change in shape.

Animals↗

PubMed2002

The solution structure of a cardiac troponin C-troponin I-troponin T complex shows a somewhat compact troponin C interacting with an extended troponin I-troponin T component.

We have investigated the structure of the cTnC-cTnI-cTnT(198-298) calcium-saturated, ternary cardiac troponin complex by small-angle scattering with contrast variation. Shape restoration was also applied to the scattering information resulting from the deuterated cTnC subunit, the unlabeled cTnI-cTnT(198-298) subunits, and the entire complex. The experimental results and modeling indicate that cTnC adopts a partially collapsed conformation, while the cTnI-cTnT(198-298) components have an extended, rod-like structure. Shape restoration applied to the X-ray scattering data and the entire contrast variation series suggest that cTnC and the cTnI-cTnT(198-298) component lie with their long axes roughly parallel to one another with a relatively small surface area for interaction. Our findings indicate that the nature of the interactions between TnC and the TnI-TnT component differs significantly between the cardiac and skeletal isoforms as evidenced by the different degrees of compactness between the cardiac TnC and skeletal TnC in their respective ternary complexes and the fact that the cTnC subunit is not highly intertwined with the other subunits, as observed in the binary complex of the skeletal isoforms [Olah, G. A., and Trewhella, J. (1994) Biochemistry 33, 12800-12806].

Animals↗

PubMed2002

Solution structure of heavy meromyosin by small-angle scattering.

Elucidation of x-ray crystal structures for the S1 subfragment of myosin afforded atomic resolution of the nucleotide and actin binding sites of the enzyme. The structures have led to more detailed hypotheses regarding the mechanisms by which force generation is coupled to ATP hydrolysis. However, the three-dimensional structure of double-headed myosin consisting of two S1 subfragments has not yet been solved. Therefore, to investigate the overall shape and relative orientations of the two heads of myosin, we performed small-angle x-ray and neutron scattering measurements of heavy meromyosin containing all three light chains (LC(1-3)) in solution. The resulting small-angle scattering intensity profiles were best fit by models of the heavy meromyosin head-tail junction in which the angular separation between heads was less than 180 degrees. The S1 heads of the best fit models are not related by an axis of symmetry, and one of the two S1 heads is bent back along the rod. These results provide new information on the structure of the head-tail junction of myosin and indicate that combining scattering measurements with high resolution structural modeling is a feasible approach for investigating myosin head-head interactions in solution.

Animals↗