Biomedical subjects

J Trewhella

Publications and source records attributed to J Trewhella.

At least 37 records · Page 2Linked to original sources

PubMed1997

Insights into biomolecular function from small-angle scattering.

Recent advances in neutron and X-ray sources and instrumentation, new and improved scattering techniques, and molecular biology techniques, which have permitted facile preparation of samples, have each led to new opportunities in using small-angle scattering to study the conformations and interactions of biological macromolecules in solution as a function of their properties. For example, new instrumentation on synchrotron sources has facilitated time-resolved studies that yield insights into protein folding. More powerful neutron sources, combined with molecular biology tools that isotopically label samples, have facilitated studies of biomolecular interactions, including those involving active enzymes.

Aspartate Carbamoyltransferase↗

PubMed1996

The relative orientation of Gla and EGF domains in coagulation factor X is altered by Ca2+ binding to the first EGF domain. A combined NMR-small angle X-ray scattering study.

Coagulation factor X is a serine protease containing three noncatalytic domains: an N-terminal gamma-carboxyglutamic acid (Gla)1 domain followed by two epidermal growth factor (EGF)-like domains. The isolated N-terminal EGF domain binds Ca2+ with a Kd of 10(-3) M. When linked to the Gla domain, however, its Ca2+ affinity is increased 10-fold. In this paper, we present the NMR solution structure of the factor X Gla-EGF domain pair with Ca2+ bound to the EGF domain, as well as small angle X-ray scattering (SAXS) data on the Gla-EGF domain pair with and without Ca2+. Our results show that Ca2+ binding to the EGF domain makes the Gla and EGF domains fold toward each other using the Ca2+ site as a hinge. Presumably, a similar mechanism may be responsible for alterations in the relative orientation of protein domains in many other extracellular proteins containing EGF domains with the consensus for Ca2+ binding. The results of the NMR and SAXS measurements reported in this paper confirm our previous result that the Gla domain is folded also in its apo state when linked to the EGF domain [Sunnerhagen, M., et al. (1995) Nat. Struct. Biol. 2, 504-509]. Finally, our study clearly demonstrates the powerful combination of NMR and SAXS in the study of modular proteins, since this enables reliable evaluation of both short-range (NMR) and long-range interactions (SAXS).

1-Carboxyglutamic Acid↗

PubMed1996

The structure of the muscle protein complex 4Ca2+.troponin C.troponin I. Monte Carlo modeling analysis of small-angle X-ray data.

Analysis of scattering data based on a Monte Carlo integration method was used to obtain a low resolution model of the 4Ca2+.troponin C.troponin I complex. This modeling method allows rapid testing of plausible structures where the best fit model can be ascertained by a comparison between model structure scattering profiles and measured scattering data. In the best fit model, troponin I appears as a spiral structure that wraps around 4Ca2+.troponin C which adopts an extended dumbbell conformation similar to that observed in the crystal structures of troponin C. The Monte Carlo modeling method can be applied to other biological systems in which detailed structural information is lacking.

Animals↗

PubMed1995

Structures of fd gene 5 protein.nucleic acid complexes: a combined solution scattering and electron microscopy study.

Small-angle scattering and electron microscopy studies of fd gene 5 protein (g5p) and reconstituted g5p.nucleic acid complexes have been used to test models for the complexes and evaluate their uniqueness. In addition, we have obtained new information on the dependence of nucleotide type and protein/nucleotide (P/N) ratio on the structure of the complexes. Reconstituted complexes were made with single-stranded fd viral DNA (fd ssDNA), poly[d(A)] and poly[r(A)]. All complexes form similar left-handed, flexible superhelices having approximately the same diameter, but the pitch differs among these complexes. The g5p protein is a dimer in solution and the dimers associate to form a superhelical framework to which the polynucleotide is attached. The combined X-ray and neutron scattering data confirm the nucleic acid is inside the protein superhelix. A Monte Carlo integration modeling procedure applied to the scattering data was used to systematically test large numbers of possible models for each complex, and previously proposed models based on parameters obtained from electron microscopy were found to be essentially correct and unique. The data on the complexes with different P/N ratios showed that mass per unit length values decreased while the rise per dimer and pitch of the superhelix increased for g5p.fd-ssDNA complexes with decreasing P/N ratios.

DNA, Single-Stranded↗

PubMed1995

Conformationally constrained analogs of protein kinase inhibitor (6-22)amide: effect of turn structures in the center of the peptide on inhibition of cAMP-dependent protein kinase.

The high-affinity interaction between protein kinase inhibitor (PKI)(6-22)amide(Thr6-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly- Arg-Arg-Asn- Ala-Ile22-NH2) and the catalytic subunit of cAMP-dependent protein kinase requires both the N-terminal Thr6 to Ile11 sequence of the inhibitor peptide and its C-terminal pseudosubstrate site comprised of Arg15 to Ile22. Small angle X-ray scattering data indicate that PKI(6-22)amide has a compact, rather than extended, structure in solution (Reed J et al., 1989, Biochem J 264:371-380). CD spectroscopic analysis of the PKI peptide led to the suggestion that a beta-turn structure might be located in the -Ala12-Ser-Gly-Arg15-connecting sequence in the middle of the molecule (Reed J, Kinzel V, Cheng HC, Walsh DA, 1987, Biochemistry 26:7641-7647). To investigate this possibility further, conformationally constrained and flexible analogs of PKI(6-22)amide were synthesized and used to study the structure-function relationships of this central portion of the inhibitor. (Des12-14)PKI(6-22) amide exhibited over a 200-fold loss in inhibitory activity. Replacement of the omitted -Ala12-Ser-Gly14-sequence with aminocaprylic acid yielded an analog that regained more than 90% of the lost binding energy. The D-alanine14 PKI analog was as potent as the parent peptide, whereas the beta-alanine14 and the sarcosine14 analogs were only 10-fold less active. Several peptides that promoted a beta-turn structure at residues 12-15 showed about 200-fold decreases in inhibitory activity. Two constrained analogs that could not assume a beta-turn conformation were only 30-fold less potent than PKI(6-22)amide. Thus, the structure of the central connecting portion of the PKI peptide, encompassing residues 12-15, greatly influences its ability to effectively bind to and inhibit the catalytic subunit. We conclude, however, that a formal beta-turn at this position is not required and is actually detrimental for a high-affinity interaction of PKI(6-22)amide with the enzyme. These results are interpreted in light of the Fourier-transform infrared spectra of the peptide analogs and the crystal structure of the peptide bound at the active site of the protein kinase (Knighton DR et al., 1991b, Science 253:414-420).

Amino Acid Sequence↗

PubMed1994

A model structure of the muscle protein complex 4Ca2+.troponin C.troponin I derived from small-angle scattering data: implications for regulation.

We report here a model structure for 4Ca2+.troponin C.troponin I derived from small-angle X-ray and neutron scattering data using a Monte Carlo modeling method. In this model, troponin I appears as a spiral structure that wraps around 4Ca2+.troponin C which adopts an extended dumbbell conformation similar to that observed in the crystal structures of troponin C. The troponin I spiral has the approximate dimensions of an alpha-helix and winds through the hydrophobic "cups" in each globular domain of troponin C. The model is consistent with a body of previously published biochemical data on the interactions between troponin C and troponin I, and suggests the molecular mechanism for the Ca(2+)-sensitive switch that regulates the muscle contraction/relaxation cycle involves a signal transmitted via the central spiral region of troponin I.

Amino Acid Sequence↗

PubMed1994

Troponin I encompasses an extended troponin C in the Ca(2+)-bound complex: a small-angle X-ray and neutron scattering study.

We have studied the solution structure of skeletal muscle troponin C complexed with troponin I in the presence of calcium using small-angle X-ray and neutron scattering. 4Ca2+.troponin C in the complex has an extended dumbbell shape with a radius of gyration of 23.9 +/- 0.5 A and a maximum linear dimension of approximately 72 A, similar to the values obtained from the crystal structure coordinates of troponin C (Herzberg & James, 1985). Troponin I is even more extended than troponin C with a radius of gyration of 41 +/- 2 A and a maximum linear dimension of approximately 118 A. The centers-of-mass for each component of the complex are approximately coincident (< 10-A separation) as are their long axes, and the troponin I component encompasses the 4Ca2+.troponin C. These data provide new insights into the nature of the conformational arrangement of this important Ca(2+)-sensitive molecular switch.

Calcium↗

PubMed1993

Leucine/isoleucine/valine-binding protein contracts upon binding of ligand.

Small-angle x-ray scattering and computer modeling have been used to study the effects of ligand binding to the leucine/isoleucine/valine-binding protein, an initial component of the high-affinity active transport system for branched-chain aliphatic amino acids in Escherichia coli. Measurements were made with no ligand present and with either L-leucine or L-valine present. Upon binding of either leucine or valine, there is a decrease in the radius of gyration, from 23.2 +/- 0.2 to 22.2 +/- 0.2 A, and in the maximum particle dimension, from 82 +/- 3 to 73 +/- 3 A. The x-ray structure of the unbound form has been determined and gives a radius of gyration and a maximum dimension consistent with the values found for the solution structure in this study (Sack, J. S., Saper, M. A., and Quiocho, F. A. (1989) J. Mol. Biol. 206, 171-191). The reduction in the radius of gyration and maximum dimension upon ligand binding can be accounted for by a substrate-induced cleft closure in a combined "hinge-twist" motion. Modeling of the substrate-bound state was done by comparison of this protein with another periplasmic binding protein (L-arabinose-binding protein), which possesses a similar two-lobe structure and for which the x-ray structure is known in its ligand-bound form.

Bacterial Proteins↗

PubMed1993

Solution structure of the cAMP-dependent protein kinase catalytic subunit and its contraction upon binding the protein kinase inhibitor peptide.

Small-angle X-ray scattering and Fourier transform infrared (FTIR) spectroscopy experiments have been completed on the catalytic subunit of the cAMP-dependent protein kinase. Measurements were made both with and without the protein kinase inhibitor peptide, PKI alpha(5-22)amide. Binding of the peptide results in an overall contraction of the structure that is characterized by a decrease of 9% in radius of gyration and about 16% in the maximum linear dimension. Both the secondary structure content of the protein/peptide complex, as determined by FTIR, and the solution structure of this binary complex, as determined by X-ray scattering, agree well with the structural characteristics of this complex as elucidated by the crystal structure [Knighton, D.R., Zheng, J., Ten Eyck, L. F., Ashford, V.A., Xuong, N.H., Taylor, S.S., & Sowadsi, J. M. (1991a) Science 253, 407-414]. Further, the contraction of the structure observed by X-ray scattering upon inhibitor peptide binding is not accompanied by any detectable change in secondary structure content of the kinase. We have modeled the contraction of the kinase upon inhibitor peptide binding as a simple rotation of the large and small lobes seen in the crystal structure such that the cleft between them is closed. For a substrate these changes would then allow catalysis to ensue. The hinge for this movement occurs around a glycine that is one of the protein kinase family consensus amino acids.

Amino Acid Sequence↗

PubMed1992

4Ca2+.troponin C forms dimers in solution at neutral pH that dissociate upon binding various peptides: small-angle X-ray scattering studies of peptide-induced structural changes.

Small-angle X-ray scattering data have been measured for rabbit skeletal muscle troponin C and its complexes with the venom peptides melittin and mastoparan as well as synthetic peptides based on regions of the troponin I sequence implicated in troponin C binding. At the neutral pH used in this study (pH 6.8), troponin C shows a tendency to form dimers in the presence of 4 mol equiv of Ca2+, but is monomeric in solution when 2 or less mol equiv of Ca2+ is present. The 4Ca2+.troponin C dimers dissociate upon binding melittin, mastoparan, and peptides based on residues 96-115, 1-30, and 1-40 in the troponin I sequence. This result suggests that the peptide-binding sites overlap with the regions of contact between troponin C molecules forming a dimer. Like the structurally homologous calcium-binding protein calmodulin, troponin C shows conformational flexibility upon binding different peptides. Upon binding melittin, troponin C contracts in a similar manner to calmodulin when it binds peptides known to form amphiphilic helices (e.g., melittin, mastoparan, or MLCK-I). In contrast, mastoparan binding to troponin C does not result in a contracted structure. The scattering data indicate troponin C also remains in an extended structure upon binding the inhibitory peptides having the same sequence as residues 96-115 in troponin I.

Amino Acid Sequence↗

PubMed1992

Denatured states of ribonuclease A have compact dimensions and residual secondary structure.

Using small-angle X-ray scattering and Fourier transform infrared spectroscopy, we have determined that the thermally denatured state of native ribonuclease A is on average a compact structure having residual secondary structure. Under strongly reducing conditions, the protein further unfolds into a looser structure with larger dimensions but still retains a comparable amount of secondary structure. The dimensions of the thermally and chemically denatured states of the reduced protein are different but both are more compact than is predicted for a random coil of the same length. These results demonstrate that thermal denaturation in ribonuclease A is not a simple two-state transition from a native to a completely disordered random coil state.

Animals↗

PubMed1992

Distances between the antigen-binding sites of three murine antibody subclasses measured using neutron and X-ray scattering.

For three different murine immunoglobulins (IgG subclasses 1, 2a, and 2b), the distances between their antigen-binding sites have been measured using neutron scattering from deuterated antigens complexed with proteated IgG. Neutron-scattering data were measured for each antibody-antigen complex in a 41% D2O solvent. Unlike the proteated antibody molecule, the deuterated antigens are strongly contrasted against the 41% D2O solvent and give rise to a scattering profile that contains an interference term related to the distance between the deuterated antigens. For all three subclasses, the damping of this interference term, which gives information on the relative flexibility of the antigen-binding sites, indicates that a single distance is inadequate to describe the observed scattering and a distribution of distances is needed. The scattering profile has been modeled for each subclass to give the mean distance between the antigens and the variance of this distance. For all three IgG subclasses, the mean distance is between 117 and 134 A, and the variance is large (approximately 40 A), indicating a high degree of flexibility of the Fab arms. Small-angle X-ray scattering measurements on the same samples are consistent with the neutron-scattering results.

Animals↗

PubMed1992

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.

Animals↗

PubMed1991

Expression in Escherichia coli and characterization of the heat-stable inhibitor of the cAMP-dependent protein kinase.

Pure heat-stable inhibitor of the cAMP-dependent protein kinase (PKI) has been isolated in high yield by using a bacterial expression vector constructed to synthesize the complete sequence of the rabbit muscle protein kinase inhibitor, plus an amino-terminal initiator methionine and glycine. Bacterially expressed PKI has an inhibitory activity identical to that of the protein isolated from rabbit skeletal muscle and, by gel filtration and gel electrophoresis, has the same physicochemical characteristics as the native physiological form of PKI. Fourier transformed infrared spectroscopy and CD establish that PKI has unusually large amounts of random coil and turn structures, with significantly smaller amounts of alpha-helix and beta structures.

Animals↗

PubMed1990

Small-angle scattering studies show distinct conformations of calmodulin in its complexes with two peptides based on the regulatory domain of the catalytic subunit of phosphorylase kinase.

Small-angle X-ray and neutron scattering have been used to study the solution structures of calmodulin complexed with synthetic peptides corresponding to residues 342-366 and 301-326, designated PhK5 and PhK13, respectively, in the regulatory domain of the catalytic subunit of skeletal muscle phosphorylase kinase. The scattering data show that binding of PhK5 to calmodulin induces a dramatic contraction of calmodulin, similar to that previously observed when calmodulin is complexed with the calmodulin-binding domain peptide from rabbit skeletal muscle myosin light chain kinase. In contrast, calmodulin remains extended upon binding PhK13. In the presence of both peptides, calmodulin also remains extended. Apparently, the presence of PhK13 inhibits calmodulin from undergoing the PhK5-induced contraction. These data indicate that there is a fundamentally different type of calmodulin-target enzyme interaction in the case of the catalytic subunit of phosphorylase kinase compared with that for myosin light chain kinase.

Amino Acid Sequence↗

PubMed1990

Synthesis and biophysical characterization of engineered topographic immunogenic determinants with alpha alpha topology.

Model peptides with predetermined secondary, tertiary, and quaternary conformation have been successfully designed, synthesized, and characterized in an attempt to mimic the three-dimensional structure of an antigenic determinant. This work is a continuing effort to map the antigenic structure of the protein antigen lactate dehydrogenase C4 (LDH-C4) to develop a contraceptive vaccine. A putative topographic determinant with alpha alpha topology which associates into four-helix bundles was designed on the basis of the framework model of protein folding. An idealized amphiphilic 18-residue sequence (alpha 1) and a 40-residue alpha alpha fold (alpha 3) have been shown to form stable 4-helix structures in solution with a free energy of association on the order of -20.8 kcal/mol (tetramerization of alpha 1) and -7.8 kcal/mol (dimerization of alpha 3). Both alpha 1 and alpha 3 form stable monolayers at the air-water interface. The CD spectra of Langmuir-Blodgett monolayers are characteristically alpha-helical. Both CD and FTIR spectroscopic studies reval a high degree of secondary structure. The SAXS data strongly suggest that the helices are arranged in a four-helix bundle since the radius of gyration of 17.2 A and the vector distribution function are indicative of a prolate ellipsoid of axial dimensions and molecular weight appropriate for the four-helix bundle. The major contribution to the formation and stabilization of alpha 1 and alpha 3 is believed to be hydrophobic interaction between the amphiphilic alpha-helices. The displayed heptad repeat, helix dipole, ion pairs, and the loop sequence may have also contributed to the overall stability and antiparallel packing of the helices. A detailed structural analysis of a relevant topographic immunogenic determinant will elucidate the nature of antigen-antibody interactions as well as provide insight into protein folding intermediates.

Amino Acid Sequence↗

PubMed1989

Conformational analysis of PKI(5-22)amide, the active inhibitory fragment of the inhibitor protein of the cyclic AMP-dependent protein kinase.

Fourier-transform i.r. spectroscopy, 1H-n.m.r. spectroscopy and X-ray scattering were used to study the conformation and shape of the peptide PKI(5-22)amide, which contains the active site of the inhibitor protein of the cyclic AMP-dependent protein kinase [Cheng, Van Pattern, Smith & Walsh (1985) Biochem. J. 231, 655-661]. The X-ray-scattering solution studies show that the peptide has a compact structure with Rg 0.9 nm (9.0 A) and a linear maximum dimension of 2.5 nm (25A). Compatible with this, Fourier-transform i.r. and n.m.r. determinations indicate that the peptide contains approx. 26% alpha-helix located in the N-terminal one-third of the molecule. This region contains the phenylalanine residue that is one essential recognition determinant for high-affinity binding to the protein kinase catalytic site.

Amides↗

PubMed1989

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.

Animals↗