Search PubMed⌕ Search

Biomedical subjects

S J Perkins

Publications and source records attributed to S J Perkins.

At least 91 records · Page 5Linked to original sources
PubMed1990

Conformational transition between native and reactive center cleaved forms of alpha 1-antitrypsin by Fourier transform infrared spectroscopy and small-angle neutron scattering.

alpha 1-Antitrypsin (alpha 1-AT) is the best-characterized member of the serpin superfamily of plasma proteins. Protease inhibitor members of this family undergo a characteristic reactive-center cleavage during expression of their inhibitory activity. The physical basis of this transition in alpha 1-AT from the stressed native conformation to the more stable reactive center cleaved (split) form was studied by Fourier transform infrared (FT-IR) spectroscopy and neutron scattering. The FT-IR spectra show that, while split alpha 1-AT has three intense well-resolved components associated with the presence of antiparallel beta-sheet and alpha-helix conformations, the amide I band of native alpha 1-AT has only one intense component, associated with the presence of beta-sheet structure. 1H-2H exchange within the polypeptide backbone, studied by FT-IR and NMR spectroscopy, shows that the native form undergoes greater exchange than the split form. Under the same conditions, neutron scattering shows no differences in the radius of gyration RG of the native and the split forms. In contrast, in high concentrations of phosphate approaching those used for crystallization, the native form (unlike the split form) undergoes dimerization. These data indicate that the conformational transition largely involves localized secondary and tertiary structure rearrangements. We propose that the energetically stressed native alpha 1-AT structure is the consequence of a significantly reduced number of hydrogen bonds in secondary structure components and that reactive-site cleavage between Met358 and Ser359 is the key for the development of the fully hydrogen bonded more stable serpin structure.

Fourier Analysis↗
PubMed1990

Molecular modeling of human complement component C4 and its fragments by X-ray and neutron solution scattering.

The solution structures of human complement component C4 and five derived fragments, C4u, C4(a + b), C4b, C4c, and C4d, were analyzed by synchrotron X-ray and neutron scattering. The X-ray radii of gyration RG for C4, C4u, and C4(a + b) in H2O buffers are similar at 5.23-5.28 nm, and likewise the cross-sectional radii of gyration RXS are similar at 2.48-2.52 nm. Molecular mass calculations using X-rays and neutrons show unexpectedly that C4c is dimeric; however, all the other forms are monomeric. C4c2 has an X-ray RG of 5.18 nm and an RXS of 2.89 nm. Neutron contrast variation gives RG values at infinite contrast of 4.87-4.93 nm for C4 and C4u, 4.79 nm for C4b, 4.94 nm for C4c2, and 2.69 nm for C4d. The RXS values at infinite contrast are 2.23-2.25 nm for C4 and C4u, 1.89 nm for C4b, and 2.62 nm for C4c2. These data show that a large conformational change occurs on going from C4 to C4b, but not on going from C4 to C4u, and this is attributed to the presence of the C4a moiety in C4u. Comparisons of the C4 and C4u scattering curves show that these are very similar out to a nominal resolution of 4 nm. Scattering-curve models were developed to account for the neutron scattering curves of C4, C4c2, and C4d in 2H2O buffers. The C4c monomer could be represented by a lamellar ellipsoid of size 8 nm x 2 nm x 18 nm. C4d was found to be 4 nm x 2 nm x 9 nm. The combination of these structures gave good accounts of the neutron data for C4, C4b, and C4c2 to resolutions of 5-6 nm. The C4 model was obtained by placing the long axis of C4d parallel to that of C4c such that the cross section is extended. C4b was best modeled by repositioning C4d relative to C4c such that this cross section becomes more compact. The C4 and C4b models are compared with possible structures for the C1 component of complement to show the importance of the surface accessibility of the protease domains and short consensus repeat domains in C1 for C4 activation.

Blood Donors↗
PubMed1990

Structural homologies of component C5 of human complement with components C3 and C4 by neutron scattering.

The complement component C5 is one of a family of structurally related plasma proteins that includes components C3 and C4. Activation of C5 is the initial step in the formation of the membrane attack complex of complement. Analysis of the solution structure of C5 and comparisons with similar analyses of the structures of C3 and C4 are reported here. Neutron solution scattering gave an Mr for C5 of 201,000, which demonstrates that C5 is monomeric in solution. The radius of gyration RG of C5 at infinite contrast is 4.87 nm and corresponds to an elongated structure. The longest length of C5 was determined to be at least 15-16 nm from three calculations on the basis of the RG, the scattering intensity at zero angle I(0), and the indirect transformation of the scattering curve into real space. Comparison of the RG and contrast variation data and indirect transformations of the scattering curves for C3, C4, and C5 show that these have very similar structures. Comparisons of the C5 scattering curve with Debye small-sphere models previously employed for C4 and C3 show that good curve fits could be obtained. Unlike previous studies that have suggested significant differences, these experiments indicate that, while C5 differs from C3 and C4 in its activation and inactivation pathways, significant structural homology exists between the native proteins, as might be predicted from their high (and similar) sequence homology.

Complement C3↗
PubMed1989

The quaternary structure in solution of human complement subcomponent C1r2C1s2.

C1r2C1s2 is a subcomponent of first component C1 of the complement cascade. Previously two distinct models for its structure have been described, in which C1r2C1s2 is either a linear rod-like assembly of the globular domains found in each of C1s and C1r, or these domains are arranged to form an asymmetric X-shaped structure. These two models were evaluated by using hydrodynamic simulations and neutron scattering. The data on C1s, C1s2 and C1r are readily represented by straight hydrodynamic cylinders, but not C1r2 or C1r2C1s2. Tests of the X-structure for C1r2 and C1r2C1s2 successfully predicted the experimental sedimentation coefficients, thus supporting this model. Neutron scattering analyses on C1s and C1r2 are consistent with a linear structure for C1s, but not for C1r2. An X-shaped structure for C1r2 was found to give a good account of the neutron data at large scattering angles. The total length of the C1s and C1r monomers was determined as 17-20 nm, which is compatible with electron microscopy. On the basis of the known sequences of C1r and C1s, this length is accounted for by a linear arrangement of a serine-proteinase domain (length 4 nm), two short consensus repeat domains (2 x 4 nm), and a globular entity containing the I, II and III domains (4-7 nm).

Complement C1r↗
PubMed1989

Secondary structure in properdin of the complement cascade and related proteins: a study by Fourier transform infrared spectroscopy.

Six structural repeat motifs of 58 amino acids are found in the sequence of both mouse and human properdins. Twelve more examples of the motif are available from the sequences of thrombospondin, the terminal complement components, and the thrombospondin-related anonymous protein. The averaged Robson and Chou-Fasman secondary structure predictions show that there are 57-66% turn and 19-38% beta-sheet structures in the typical repeat motif. The high amount of turn structure is consistent with Gly, Pro, Cys, and Ser being the four most abundant amino acid residues in properdin. Comparisons with sequences found in the circumsporozoite protein from several species of malaria parasites show that their sequences and secondary structures strongly coincide only in a 18-residue segment. Further secondary structure analysis utilized Fourier transform infrared spectroscopy of human properdin in 2H2O buffers. These show a broad amide I band that, after second-derivative and deconvolution calculations, is shown to be composed of several components. Two at 1633 and 1683 cm-1 are strong evidence for beta-sheet structure, although overlap from beta-turns can also contribute. The presence of beta-turn structure is indicated by absorptions at 1662-1675 and 1645 cm-1. The properdin structure contains substantial quantities of beta-sheet and beta-turn structures, which is consistent with the secondary structure predictions and amino acid compositions. The length of the repeat motif is estimated as 3.3-4.3 nm, and an estimated 14-22% of nonexchanged amide protons reside in properdin. This is suggestive of a high degree of solvent accessibility in the structure.

Amino Acid Sequence↗
PubMed1989

Structural changes in oxidised low-density lipoproteins and of the effect of the anti-atherosclerotic drug probucol observed by synchrotron X-ray and neutron solution scattering.

The atherosclerotic properties of low-density lipoproteins (LDL) are thought to be strongly enhanced by oxidation. The lipid-lowering drug probucol reduces the susceptibility of LDL to oxidation. Synchrotron X-ray and high-flux neutron solution scattering curves were used to characterise the structural properties of human LDL, before and after modification by oxidation with Cu2+ and the addition of probucol, in order to evaluate these techniques. Analyses based on Guinier plots, simple two-shell spherical modelling, and the use of cubic splines and indirect transformation show that a 20-h incubation with Cu2+ ions (but not 6 h) causes some of the LDL to associate to form larger aggregated particles. Gel electrophoresis on Cu2+ -oxidised LDL shows a concomitant degradation of the apolipoprotein B-100 as well as the formation of high molecular mass forms. These experiments indicate that the apoprotein B-100 structure has been significantly disrupted by oxidation. The addition of probucol to LDL causes an increase in the polydispersity of LDL, as evidenced by small changes in the Guinier curves and some weakening of the minima in the X-ray scattering curves. No changes in the quasispherical shape of LDL are observed and gel electrophoresis indicates no changes. It is possible that probucol may exert its effect by increasing the range of sizes of LDL and that the lipid-lowering effect of probucol in vivo might be caused by the preferential catabolism of the higher molecular mass forms of LDL thus created.

Apolipoprotein B-100↗
PubMed1989

Immunoglobulin fold and tandem repeat structures in proteoglycan N-terminal domains and link protein.

Detailed primary sequence and secondary structure analyses are reported for the hyaluronate binding region (G1 domain) and link protein of proteoglycan aggregates. These are based on six full or partial sequences from the chicken, pig, human, rat and bovine proteins. Determinations of a full pig and a partial human link protein sequence are reported in the Appendix. Five sequences at the N terminus in both proteins were compared with the structures of 11 variable immunoglobulin (Ig) fold domains for which crystal structures are available. Despite only modest sequence homology, a clear alignment could be proposed. Analysis of this shows that the equivalents of the first and second hypervariable segments are now significantly longer, and both proteins have N-terminal extensions that are up to 23 residues in length. Secondary structure predictions showed that these sequences could be identified with available crystal structures for the variable Ig fold. However the hydrophobic residues involved in interactions between the light and heavy chains in Igs are replaced by hydrophilic charged groups in both proteins. These results imply that both proteins are members of the Ig superfamily, but exhibit structural differences distinct from other members of this superfamily for which crystal structures are known. The proteoglycan tandem repeat (PTR) is a repeat of 99 residues that is found twice in the amino acid sequence of link protein and the proteoglycan G1 domain adjacent to the Ig fold, and also twice in the proteoglycan G2 domain. A total of 16 PTRs was available for analysis. Compositional analyses show that these are positively charged if these originate from link protein, and negatively charged if from the G1 or G2 domains. The 16 Robson secondary structure predictions for the PTRs were averaged to improve the statistics of the prediction, and checked by comparison with Chou-Fasman calculations. A strong alpha-helix prediction was found at residues 13 to 25, and several beta-strands were predicted. The overall content is 18% alpha-helix and 28% beta-sheet, with 44% of the remaining sequence being predicted as turns. These analyses show that both the proteoglycan G1 domain and link protein are constructed from two distinct globular components, which may provide the two functional roles of these proteins in proteoglycan aggregation.

Amino Acid Sequence↗
PubMed1989

Models for the C1 complex determined by physical techniques.

The C1 complex is an association of C1q and C1r2C1s2. Neutron scattering and ultracentrifugation provide a valuable means of understanding the solution structure of the subcomponents and their complex, and these can be supplemented by protein structure prediction techniques. C1q is constructed from six globular heads connected by collagen-like arms. The solution data for C1q show that the arms are of length 14.5 nm and not 11.5 nm as proposed from electron microscopy, the average arm-axis angle is 40 degrees, and that the structure is flexible in solution at the junction of the six arms. The sequences of C1r and C1s show that each is constructed from six protein domains. C1r and C1s are elongated macromolecules of lengths 18-20 nm. Their solution properties are best described as the lengthwise arrangement of a protease domain of diameter 4 nm, two "short consensus repeat" domains, each of length 4 nm, and an N-terminal globular entity of length 6 nm containing the first three protein domains. Solution data on the C1r2 dimer is interpreted as an X-shaped association of the two C1r monomers as proposed from electron microscopy. Six criteria are enumerated for constructing models of C1 from these two structures, and four distinct models for the C1 complex are reviewed. While further evidence is required to make this choice unequivocal, the W-model is favoured. This places each monomer of C1r and C1s on four adjacent arms of C1q, and offers the most reasonable explanation of the known properties of the C1 complex.

Complement C1↗
PubMed1988

A pentameric form of human serum amyloid P component. Crystallization, X-ray diffraction and neutron scattering studies.

Human serum amyloid P component crystallizes from sodium acetate buffer (pH 5.5) in the presence of calcium and polyethylene glycol 6000, at 4 degrees C. The space group is P2(1) and the cell parameters are a = 69.0 A (1 A = 0.1 nm), b = 99.3 A, c = 96.8 A, beta = 96.1. Density considerations supported by neutron scattering and gel filtration experiments indicate that the species crystallized is pentameric. The orientation of the pentamer 5-fold axis is determined and a crystal packing for the discs is proposed.

Crystallization↗
PubMed1988

A study of the structure of human complement component factor H by Fourier transform infrared spectroscopy and secondary structure averaging methods.

Fourier transform infrared spectroscopy was used to investigate the secondary structure of human complement component factor H in H2O and 2H2O buffers. The spectra show a broad amide I band which after second-derivative calculations is shown to be composed of three components at 1645, 1663, and 1685 cm-1 in H2O and at 1638, 1661, and 1680 cm-1 in 2H2O. The frequencies of these components are consistent with the existence of an extensive antiparallel beta-strand secondary structure. The exchange properties of the amide protons of factor H as measured in 2H2O buffers are rapid and lead to an estimate of NH proton nonexchange that is comparable with those for small globular proteins. Human factor H is constructed from a linear sequence of 20 short consensus repeats with a mean of 61 residues in each one. To investigate the secondary structure further, secondary structure predictions were carried out on the basis of an alignment scheme for 101 sequences for these repeats as found in human factor H and 12 other proteins. These predictions were averaged in order to improve the reliability of the calculations. Both the Robson and the Chou-Fasman methods indicate significant beta-structural contents. Residues 21-51 in the 61-residue repeat show a clear prediction of four strands of beta-structure and four beta-turns. A structural model based on antiparallel beta-strands in the secondary structure is proposed and discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Molecular modelling of human complement component C3 and its fragments by solution scattering.

Solution scattering experiments using both X-rays and neutrons are reported for human complement component C3 and up to six other glycoprotein fragments that are derived from C3. The X-ray and neutron molecular masses and neutron matchpoints are in agreement with the known primary sequence of C3. The X-ray radius of gyration RG of C3 is 5.2 nm and is similar for the related forms C3u, C3(a + b) and C3b. The X-ray cross-sectional radius of gyration RXS of C3b is however less than that of C3, C3u and C3(a + b). The major fragments of C3b, namely C3c and C3dg, were studied. The RG of C3c is 4.7 nm and for C3dg is 2.9 nm. C3c and C3dg do not interact when they coexist in solution in equimolar amounts. When C3u is cleaved into iC3u, the RG of iC3u increases to 5.9 nm and its RXS decreases, showing that C3c and C3dg behave as independent entities within the parent glycoprotein. Analyses of the neutron RG and RXS values by contrast variation techniques confirm the X-ray analyses, and show no evidence for significant hydrophobic or hydrophilic domains within C3 or any of its fragments. Shape analyses show that C3, C3c and C3dg are elongated particles. Debye models were developed using the scattering curve out to Q = 1.6 nm-1. These show that C3 and C3c resemble oblate ellipsoids while C3dg resembles a prolate ellipsoid. C3dg lies on the long edge of C3c within C3. The dimensions of the models are 18 nm X 2 nm X 10 nm for C3, 18 nm X 2 nm X 7 nm for C3c and 10 nm X 2 nm X 3 nm for C3dg. These models are compatible with analyses of the scattering curve RG and RXS values, data from sedimentation coefficients, and images of C3 and C3c seen by electron microscopy.

Complement C3↗
PubMed1986

Protein volumes and hydration effects. The calculations of partial specific volumes, neutron scattering matchpoints and 280-nm absorption coefficients for proteins and glycoproteins from amino acid sequences.

Amino acid sequences, carbohydrate compositions and residue volumes are used to compare critically calculations of partial specific volumes v, neutron scattering matchpoints and 280-nm absorption coefficients with experimental v values for proteins and glycoproteins. The v values that are obtained from amino acid densitometry underestimate experimental v values by 0.01-0.02 ml/g while the v values from crystallographic volumes overestimate the experimental v values by 0.04-0.05 ml/g. An intermediate consensus volume set of amino-acid-residue volumes is proposed in order to predict experimental v values using sequence information. The method is extended to carbohydrates and glycoproteins. Neutron scattering matchpoints can be calculated from crystallographic residue volumes on the basis of the non-exchange of 10% of the main-chain NH protons. Crystallographic results on protein-bound water are used to account for the experimental values of v and matchpoints. Finally, 280-nm absorption coefficients, A1%, 1 cm 280, of 5-27 are found to be well predicted by the Wetlaufer procedure based on the totals of Trp, Tyr and Cys residues. Average errors are +/- 0.7, and the experimental A(1%,1cm)280 values can be larger than the predicted values by 3%.

Amino Acid Sequence↗
PubMed1986

Unusual ultrastructure of complement-component-C4b-binding protein of human complement by synchrotron X-ray scattering and hydrodynamic analysis.

Solution X-ray-scattering experiments with the use of synchrotron radiation on the human complement-component-C4b-binding protein showed that its RG is 13 nm and that its Mr is 550,000. From the known primary amino acid sequence and estimated carbohydrate content, C4b-binding protein is inferred to have a total of 7.4 +/- 1 subunits. Heptameric computer models for C4b-binding protein were based on the X-ray-scattering curve to a resolution of 6.4 nm, and literature values for sedimentation coefficients and electron-microscopy images. The macromolecule was represented by a bundle of seven arms held together at the C-terminal end and spaced out by a base containing 23% of C4b-binding protein by volume. If the overall length of each arm is assumed to be 33 nm as seen in electron microscopy, the solution data indicate an average arm-axis angle of 5-10 degrees. The seven arms of C4b-binding protein are found to be close together, in distinction to the splayed-out images seen in electron micrographs.

Carrier Proteins↗