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S J Perkins

Publications and source records attributed to S J Perkins.

At least 55 records · Page 3Linked to original sources
PubMed1996

Time-course studies by synchrotron X-ray solution scattering of the structure of human low-density lipoprotein during Cu(2+)-induced oxidation in relation to changes in lipid composition.

Low-density lipoproteins (LDLs) in plasma are constructed from a single molecule of apolipoprotein B-100 (apoB) (M(r) 512,000) in association with lipid [approximate M(r) (2-3) x 10(6)]. LDL oxidation is an important process in the development of atherosclerosis, and can be imitated by the addition of Cu2+ ions. Synchrotron X-ray scattering of LDL yields curves without radiation damage effects at concentrations close to physiological. The radius of gyration RG for preparations of LDL from different donors ranged between 12.1 and 16.0 nm, with a mean of 13.9 nm. At 4 degrees C, the distance distribution curve P(r) indicated a maximum dimension of 25-27 nm for LDL, a peak at 19.5 nm which corresponds to a surface shell of protein and phospholipid head groups in LDL, and submaxima between 1.7 and 13.5 nm, which correspond to an ordered lipid core in LDL. LDL from different donors exhibited distinct P(r) curves. For oxidation studies of LDL by X-rays, data are best obtained at 4 degrees C at a concentration of > or = 2 mg of LDL protein/ml together with controls based on non-oxidized LDL. LDL oxidation (2 mg of apoB/ml) was studied at 37 degrees C in the presence of 6.4, 25.6 and 51.2 mu of Cu2+/g of apoB. Large changes in P(r) were reproducibly observed in the inter-particle distance range between 13 and 16 nm shortly after initiation of oxidation. This corresponds to the phospholipid hydrocarbon in LDL, which has either increased in electron density during oxidation or become increasingly disordered. After 25 h, the structural changes subsequently spread to regions of the P(r) curves assigned to surface apoB and the central core of cholesteryl esters and triacyl-glycerols. Lipid analyses were carried out under the same solution conditions. The alpha-tocopherol and beta-carotene antioxidant contents of LDL were consumed within 1-2 h. Analyses of the formation of thiobarbituric acid-reactive substances and lipid hydroperoxides indicated that arachidonic acid was preferentially oxidized before the maximal formation of lipid hydroperoxides at 8-12 h after initiation of oxidation. High-performance TLC showed that phosphatidylcholine was continuously converted into lysophosphatidylcholine during oxidation, which is consistent with the early changes in the X-ray P(r) curves. The neutral core lipids became modified only after 12-15 h of oxidation. The combination of X-ray scattering structural analyses with biochemical analyses shows that the oxidation of LDL first affects the outer shell of surface phospholipid, then it spreads towards damage of apoB and the internal neutral lipid core of LDL.

Antioxidants↗
PubMed1996

Predicted alpha-helix/beta-sheet secondary structures for the zinc-binding motifs of human papillomavirus E7 and E6 proteins by consensus prediction averaging and spectroscopic studies of E7.

The E7 and E6 proteins are the main oncoproteins of human papillomavirus types 16 and 18 (HPV-16 and HPV-18), and possess unknown protein structures. E7 interacts with the cellular tumour-suppressor protein pRB and contains a zinc-binding site with two Cys-Xaa2-Cys motifs spaced 29 or 30 residues apart. E6 interacts with another cellular tumour-suppressor protein p53 and contains two zinc-binding sites, each with two Cys-Xaa2-Cys motifs at a similar spacing of 29 or 30 residues. By using the GOR I/III, Chou-Fasman, SAPIENS and PHD methods, the effectiveness of consensus secondary structure predictions on zinc-finger proteins was first tested with sequences for 160 transcription factors and 72 nuclear hormone receptors. These contain Cys2His2 and Cys2Cys2 zinc-binding regions respectively, and possess known atomic structures. Despite the zinc- and DNA-binding properties of these protein folds, the major alpha-helix structures in both zinc-binding regions were correctly identified. Thus validated, the use of these prediction methods with 47 E7 sequences indicated four well-defined alpha-helix (alpha) and beta-sheet (beta) secondary structure elements in the order beta beta alpha beta in the zinc-binding region of E7 at its C-terminus. The prediction was tested by Fourier transform infrared spectroscopy of recombinant HPV-16 E7 in H2O and 2H2O buffers. Quantitative integration showed that E7 contained similar amounts of alpha-helix and beta-sheet structures, in good agreement with the averaged prediction of alpha-helix and beta-sheet structures in E7 and also with previous circular dichroism studies. Protein fold recognition analyses predicted that the structure of the zinc-binding region in E7 was similar to a beta beta alpha beta motif found in the structure of Protein G. This is consistent with the E7 structure predictions, despite the low sequence similarities with E7. This predicted motif is able to position four Cys residues in proximity to a zinc atom. A model for the zinc-binding motif of E7 was constructed by combining the Protein G coordinates with those for the zinc-binding site in transcription factor TFIIS. Similar analyses for the two zinc-binding motifs in E6 showed that they have different alpha/beta secondary structures from that in E7. When compared with 12 other zinc-binding proteins, these results show that E7 and E6 are predicted to possess novel types of zinc-binding structure.

Amino Acid Sequence↗
PubMed1996

Assessment of protein fold predictions from sequence information: the predicted alpha/beta doubly wound fold of the von Willebrand factor type A domain is similar to its crystal structure.

The fold of the von Willebrand Factor type A domain (vWF-A) was predicted to be similar to an alpha/beta doubly wound fold in the GTP-binding domain of ras-p21, despite the lack of sequence or functional similarity. This was subsequently confirmed by the vWF-A crystal structure from complement receptor type 3. The prediction is now reviewed. The vWF-A secondary structure was predicted with 62 to 75% accuracy and 12 of the 13 secondary structure elements were identified correctly. Accessibility predictions were 69 to 71% accurate. The fold recognition analysis was confirmed, but was much improved by averaging the results from 70 complete vWF-A sequences. The related folds of ras-P21 and flavodoxin scored highly. In addition, both the mapping of the predicted vWF-A secondary structure elements with those in 12 known alpha/beta folds and two Asp residues at the C-terminal ends of two adjacent beta-strands matched well with ras-p21 and flavodoxin. The predicted Mg(2+)-binding site, two disulphide bridges and the secondary structure topology were largely accurate. The exception is the reversal of a beta-hairpin at one end of the central beta-sheet. We conclude that non-homologous folds with dissimilar functions can be predicted from sequence data with reasonable accuracy, and that the accuracy in this case was principally limited at the periphery of the fold.

Amino Acid Sequence↗
PubMed1996

Extended glycoprotein structure of the seven domains in human carcinoembryonic antigen by X-ray and neutron solution scattering and an automated curve fitting procedure: implications for cellular adhesion.

Carcinoembryonic antigen (CEA) is one of the most widely used cell-surface tumour markers for tumour monitoring and for targeting by antibodies. It is heavily glycosylated (50% carbohydrate) and a monomer is constructed from one V-type and six C2-type fold domains of the immunoglobulin superfamily. The solution arrangement at low resolution of the seven domains in CEA cleaved from its membrane anchor was determined by X-ray and neutron scattering. Guinier analyses showed that the X-ray radius of gyration RG of CEA was 8.0 nm. The length of CEA was 27 to 33 nm, and is consistent with an extended arrangement of seven domains. The X-ray cross-sectional radius of gyration RXS was 2.1 nm, and is consistent with extended carbohydrate structures in CEA. The neutron data gave CEA a relative molecular mass of 150,000, in agreement with a value of 152,500 from composition data, and validated the X-ray analyses. The CEA scattering curves were analysed using an automated computer modelling procedure based on the crystal structure of CD2. The V-type and C2-type domains in CD2 were separated, and the C2-type domain was duplicated five times to create a linear seven-domain starting model for CEA. A total of 28 complex-type oligosaccharide chains in extended conformations were added to this model. By fixing the six interdomain orientations to be the same, three-parameter searches of the rotational orientations between the seven domains gave 4056 possible CEA models. The best curve fits from these corresponded to a family of zig-zag models. The long axis of each domain was set at 160(+/-25) degrees relative to its neighbour, and the two perpendicular axes were orientated at 10(+/-30) degrees and -5(+/-35) degrees. Interestingly, the curve fit from this model is within error of that calculated from a CEA model generated directly from the CD2 crystal structure by the superposition of adjacent domains. Zig-zag models of this type imply that the protein face of the GFCC' beta-sheet in neighbouring CEA domains lie on alternate sides of the CEA structure. Such a model has implications for the adhesion interactions between CEA molecules on adjacent cells or for the antibody targeting of CEA.

Amino Acid Sequence↗
PubMed1996

The protein fold of the hyaluronate-binding proteoglycan tandem repeat domain of link protein, aggrecan and CD44 is similar to that of the C-type lectin superfamily.

Link protein and aggrecan of the extracellular matrix each contain two proteoglycan tandem repeat (PTR) domains that interact with hyaluronate. Consensus secondary structure predictions for 59 PTR sequences and 129 C-type lectin sequences give similar patterns of two alpha-helices and up to seven beta-strands. Protein fold recognition analyses show that the 59 PTR sequences are highly compatible with the C-type lectin crystal structure. The predicted fold consists of a conserved motif formed from an antiparallel beta-sheet flanked by two alpha-helices, the motif being attached to two distinct types of beta-sheet region in the two superfamilies. Arg9 or Lys11 on an exposed loop and up to three other Arg residues in the beta-sheet region are conserved and may form part of a hyaluronate binding site.

Aggrecans↗
PubMed1995

Bent domain structure of recombinant human IgE-Fc in solution by X-ray and neutron scattering in conjunction with an automated curve fitting procedure.

Human immunoglobulin E (IgE) consists of 14 domains, each with the characteristic immunoglobulin fold structure. Compared with the 12-domain structure of immunoglobulin G (IgG), IgE has an additional pair of domains (C epsilon 2) in the Fc region in place of the hinge of IgG. The crystal structure of the 4-domain Fc fragment of IgG is known, but not that of the 6-domain Fc fragment of IgE (IgE-Fc). In order to elucidate the position of the C epsilon 2 domains in the domain structure of IgE-Fc, IgE-Fc was studied by synchrotron X-ray and pulsed neutron scattering. The upper limit on the X-ray radius of gyration RG which determines macromolecular elongation was determined to be 3.52 +/- 0.14 nm. That for the neutron RG (measured in 100% 2H2O buffers) was 3.53 +/- 0.05 nm. The X-ray and neutron cross-sectional radii of gyration were 1.89 +/- 0.05 and 1.56 +/- 0.09 nm, respectively. The scattering curves were modeled on the basis of a previously-predicted model for IgE-Fc (Helm, B. A., Ling, Y., Teale, C., Padlan, E. A., & Brüggemann, M. (1991) Eur. J. Immunol. 21, 1543-1548). The extended arrangement of domains in that model resulted in poor agreement with experimental data. Interactive and automated procedures for the fitting of crystallographically-derived domain models to scattering data were developed. Each pair of C epsilon 2, C epsilon 3, and C epsilon 4 domains was translated and rotated relative to the remaining structure in a comprehensive five-parameter search of more than 37,000 models. Substantially improved agreement between the experimental and calculated scattering curves was obtained. Bent models for IgE-Fc in which the C epsilon 2 domain pair is rotated by at least 40-50 degrees from its position in the previously predicted linear IgE model consistently gave the best agreement with the X-ray and neutron scattering curves. Such a structure for the Fc fragment accounts in part for the bent structure previously proposed for intact human IgE, which is important for understanding the interaction between IgE and its receptors.

Computer Simulation↗
PubMed1995

Factor VIIa and the extracellular domains of human tissue factor form a compact complex: a study by X-ray and neutron solution scattering.

The four-domain structure of human factor VIIa and the two-domain structure of tissue factor form a tight complex to initiate blood coagulation. By solution scattering, the mean X-ray and neutron radii of gyration RG (which determine macro-molecular elongation) were found to be 3.25 nm, 2.13 nm and 3.14 nm (+/- 0.13 nm) for factor VIIa, the extracellular region of tissue factor and their complex in that order. The mean cross-sectional radii of gyration RXS were 1.33 nm, 0.56 nm and 1.42 nm (+/- 0.13 nm) in that order. The mean lengths were 10.3 nm, 7.7 nm and 10.2 nm in that order. The data show that, in solution, the free proteins have extended domain structures, and the complex is formed by a compact side-by-side alignment of the two proteins along their long axes. The high binding affinity of tissue factor for factor VIIa may thus be accounted for by the occurrence of many intermolecular contacts in the complex.

Animals↗
PubMed1995

Demonstration by pulsed neutron scattering that the arrangement of the Fab and Fc fragments in the overall structures of bovine IgG1 and IgG2 in solution is similar.

The bovine IgG1 and IgG2 isotypes exhibit large differences in effector functions. To examine the structural basis for this, the 12-domain structures of IgG1 and IgG2 were investigated by pulsed neutron scattering using a recently developed camera LOQ. This method reports on the average relative disposition in solution of the Fab and Fc fragments in IgG. The radii of gyration (RG) were found to be similar at 5.64 and 5.71 nm for IgG1 and IgG2 respectively in 100% 2H2O buffers. The two cross-sectional radii of gyration (RXS) were also similar at 2.38-2.41 and 0.98-1.02 nm. Similar values were obtained for porcine IgG. Both bovine IgG1 and IgG2 possess similar overall solution structures, despite sequence differences at the hinge region at the centre of their structures. An automated computer survey of possible IgG structures was developed, in which coordinates for the two Fab fragments were displaced in a two-dimensional plane relative to those of the Fc fragment in 0.25 nm steps. The scattering curves calculated from these structures were found to be sensitive to relative displacements of the three fragments, but not on their rotational orientation about their longest axes. Good agreement with the solution scattering data was obtained with a planar IgG model in which the C-terminus of the CH1 domain of Fab was 3.6 nm from the N-terminus of Fc in both IgG1 and IgG2, with a precision of 0.7 nm. Energy refinement showed that this spatial separation is compatible with the hinge sequences of bovine IgG1 and IgG2. The results show that multidomain protein structures can be modelled using LOQ data, and that a long hinge sequence does not necessarily reflect a large distance between Fab and Fc. The steric accessibility of Fc sites for interactions with cell-surface Fc receptors and C1q of complement is shown to be generally similar for IgG1 and IgG2, and the difference in effector function between IgG1 and IgG2 is probably based on deletions in the IgG2 hinge sequence.

Amino Acid Sequence↗
PubMed1995

Beta-sheet secondary structure of an LDL receptor domain from complement factor I by consensus structure predictions and spectroscopy.

Low density lipoprotein receptor domains (LDLrs) represent a large cell surface receptor superfamily of consensus length 39 residues. Alignment of 194 sequences indicated highly conserved Cys and Asp/Glu residues, and a consensus secondary structure with three beta-strands was predicted. Sequence threading against known protein folds indicated consistency with small beta-sheet proteins. Complement factor I contains two LDLrs, and the second of these was successfully expressed using a bacterial pGEX system. FT-IR spectroscopy on this indicated a small amount of beta-sheet together with turns and loops. LDLr is proposed to have a beta-sheet structure in which the five biologically important Asp/Glu residues are located on an exposed loop.

Amino Acid Sequence↗
PubMed1995

Characterization of the structure of polydisperse human low-density lipoprotein by neutron scattering.

Low-density lipoproteins (LDL) in plasma are constructed from a single molecule of apolipoprotein B-100 (M(r) 512000) in association with lipid (approximate M(r) 2-3 x 10(6)). The gross structure was studied using an updated pulsed-neutron camera LOQ with an area detector to establish the basis for the interpretation of structural changes seen during dynamic studies of LDL oxidation. Neutron-scattering data for LDL in 100% 2H2O buffers emphasize their external appearance. Guinier analysis on a continuous-flux neutron camera D17 revealed pronounced concentration-dependences in the radius of gyration, RG, and the intensity of forward scattering, I(0) (equivalent to the M(r) of LDL) between 0.5 and 11 mg of LDL protein/ml. LDL preparations from different donors gave different RG values. When extrapolated to zero concentration, RG values ranged between 8.3 and 10.6 nm and were linearly correlated with M(r), which is consistent with a spherical structure. The distance-distribution function P(r) in real space showed a single maximum at 9.1-10.9 nm, which is just under half the observed maximum dimension of 23.1 +/- 1.2 nm expected for a spherical structure. The neutron radial-density function p(r) exhibited a plateau of high and featureless density at the centre of LDL. LDL can be modelled by a polydisperse assembly of spheres with two internal densities and a mean radius close to 10.0 nm in a normal distribution of radii with a standard deviation of 2.0 nm. The data are consistent with recent electron-microscopy and ultracentrifugation data.(ABSTRACT TRUNCATED AT 250 WORDS)

Centrifugation, Zonal↗
PubMed1995

Time-course studies by neutron solution scattering and biochemical assays of the aggregation of human low-density lipoprotein during Cu(2+)-induced oxidation.

The oxidative modification of low-density lipoproteins (LDL) is recognized to be a key event in the development of atherosclerotic plaques on artery walls. The characteristics of LDL oxidized by cells of the artery wall can be imitated by the addition of Cu2+ ions to initiate lipid peroxidation in LDL. Neutron scattering of LDL in 2H2O buffers enables the time course of changes in the gross structure of LDL during oxidation to be continuously monitored under conditions close to physiological. Oxidation of LDL [2 mg of apolipoprotein B (apoB) protein/ml] was studied in the presence of 6.4, 25.6 and 51.2 mumol of Cu2+/g of apoB by incubation at 37 degrees C for up to 70 h. Neutron Guinier analyses showed that the radius of gyration RG (indicative of size) and the forward-scattered intensity at zero angle I(0) (indicative of M(r)) continuously increased during oxidation, indicating that LDL had aggregated. Both the rate of aggregation and the change in RG and I(0) values after 10 and 50 h increased with Cu2+ concentration. Distance-distribution functions P(r) showed that, within 4 h, the maximum dimension of LDL increased from 23 to 55 nm. The P(r) curves of oxidatively modified LDL exhibited two peaks at 10-12 nm and 26 nm. The 10-12 nm peak corresponds to native LDL, and the 26 nm peak is assigned to the initial formation of LDL dimers and trimers and their progression to form higher oligomers. The growth of the 26 nm peak depended on Cu2+ concentration. Particle-size-distribution functions Dv(r) suggested that the polydisperse spherical structure of LDL ceased to exist after 30 h, at which point the LDL samples underwent a phase separation. Related, but not identical, changes in the I(Q) and P(r) curves were observed when native LDL was self-aggregated by brief vortexing. Parallel assessment of LDL protein modification by SDS/PAGE showed increased aggregation and degradation of apoB with increased Cu2+ concentrations, and that the main apoB protein band had diminished after 2-8 h, depending on the amount of Cu2+ added. The uptake and degradation of oxidized 125I-labelled LDL by mouse peritoneal macrophages occurred maximally within the first 10 h, and increased in proportion to the Cu2+ concentration. ApoB protein broke down within the first 10 h of oxidation, and this is the period when scavenger receptors on macrophages can recognize and internalize oxidized LDL. Within 10 h, the protein-lipid interactions responsible for the spherical LDL structure became destabilized by protein fragmentation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Characterization of a complement receptor 2 (CR2, CD21) ligand binding site for C3. An initial model of ligand interaction with two linked short consensus repeat modules.

Human CR2 (CD21, EBV receptor) is an approximately 145-kDa receptor and a member of the regulators of complement activation gene family. Regulators of complement activation proteins are characterized by the presence of repeating motifs of 60 to 70 amino acids that are designated short consensus repeats (SCR). CR2 serves as a receptor for four distinct ligands. Three of these ligands (complement C3, gp350/220 of EBV, and CD23) interact with the amino terminal 2 of 16 SCR (SCR 1 and 2). Previous studies have determined that at least four sites are important in allowing CR2 to efficiently bind EBV. Two of these sites are also important for binding mAb OKB7, a reagent that blocks both EBV and iC3b/C3dg binding to CR2. We have identified and characterized important sites of iC3b ligand binding by utilizing human-mouse CR2 chimeras, a rat anti-mouse CR2 mAb designated 4E3 that blocks receptor binding to C3, and human CR2-derived peptides. In addition to demonstrating an important role for the same sequence in SCR 1 that is part of the mAb OKB7 and EBV binding site, we have identified a new region within SCR 2 that interacts with C3. These results, when compared with a model of a dual SCR solution structure derived from human factor H SCR, predict that two distinct largely surface-exposed sites on CR2 interact with iC3b. A relative twist of 130 degrees about the long axis of the second SCR in this model would be necessary for these sites to form a single patch for iC3b binding on CR2.

Amino Acid Sequence↗
PubMed1995

Hydrodynamic studies of a complex between the Fc fragment of human IgE and a soluble fragment of the Fc epsilon RI alpha chain.

The interaction between immunoglobulin E (IgE) and its high-affinity receptor Fc epsilon RI is central to allergic disease. The binding site for Fc epsilon RI lies in the third constant region domain of the epsilon heavy chain of IgE (C epsilon 3). Identical epitopes on the two C epsilon 3 domains in the IgE-Fc are predicted to be on opposite sides of the structure, and therefore each could bind independently to a receptor molecule. Titrations, however, reveal that the IgE-Fc forms an equimolar complex with a soluble fragment of the Fc epsilon RI alpha chain (sFc epsilon RI alpha), and the molecular weight of the complex, as determined by sedimentation equilibrium, confirms this stoichiometry. The measured sedimentation coefficients of the two ligands are in good agreement with computed values for a compact IgE-Fc and an elongated sFc epsilon RI alpha structure. The calculated sedimentation coefficients for possible models of a 1:1 complex lead to exclusion of all highly extended geometries for the complex. Possible explanations for the paradoxical stoichiometry of the IgE-Fc/sFc epsilon RI alpha complex, in terms of the curved shape of IgE, a conformational change in IgE when the receptor binds, and steric interference between two molecules of Fc epsilon RI binding to identical sites, are discussed.

Carbohydrate Sequence↗
PubMed1995

The protein fold of the von Willebrand factor type A domain is predicted to be similar to the open twisted beta-sheet flanked by alpha-helices found in human ras-p21.

The von Willebrand Factor type A domain is the prototype for a protein superfamily. It possesses no significant sequence similarity to any known protein structure. Secondary structure predictions indicate a largely alternating pattern of six alpha-helices and six beta-strands. A protein fold for this domain is proposed to correspond to a doubly-wound open twisted beta-sheet structure flanked by alpha-helices. Close agreement was found with the GTP-binding domain of human ras-p21, provided that an extra alpha-helix was inserted. The structure of the predicted fold showed high compatibility with the proximate location of two Mg(2+)-binding Asp residues, two disulphide-bridged Cys residues, and other known functional attributes of this domain.

Amino Acid Sequence↗
PubMed1994

Beta-sheet secondary structure of the trimeric globular domain of C1q of complement and collagen types VIII and X by Fourier-transform infrared spectroscopy and averaged structure predictions.

C1q plays a key role in the recognition of immune complexes, thereby initiating the classical pathway of complement activation. Although the triple-helix conformation of its N-terminal segment is well established, the secondary structure of the trimeric globular C-terminal domain is as yet unknown. The secondary structures of human C1q and C1q stalks and pepsin-extracted human collagen types I, III and IV (with no significant non-collagen-like structure) were studied by Fourier-transform i.r. spectroscopy in 2H2O buffers. After second-derivative calculation to resolve the fine structure of the broad amide I band, the Fourier-transform i.r. spectrum of C1q showed two major bands, one at 1637 cm-1, which is a characteristic frequency for beta-sheets, and one at 1661 cm-1. Both major bands were also detected for Clq in H2O buffers. Only the second major band was observed at 1655 cm-1 in pepsin-digested C1q which contains primarily the N-terminal triple-helix region. The Fourier-transform i.r. spectra of collagen in 2H2O also showed a major band at 1659 cm-1 (and minor bands at 1632 cm-1 and 1682 cm-1). It is concluded that the C1q globular heads contain primarily beta-sheet structure. The C-terminal domains of C1q show approximately 25% sequence identity with the non-collagen-like C-terminal regions of the short-chain collagen types VIII and X. To complement the Fourier-transform-i.r. spectroscopic data, averaged Robson and Chou-Fasman structure predictions on 15 similar sequences for the globular domains of C1q and collagen types VIII and X were performed. These showed a clear pattern of ten beta-strands interspersed by beta-turns and /or loops. Residues thought to be important for C1q-immune complex interactions with IgG and IgM were predicted to be at a surface-exposed loop. Sequence insertions and deletions, glycosylation sites, the free cysteine residue and RGD recognition sequences were also predicted to be at surface-exposed positions.

Amino Acid Sequence↗
PubMed1994

The secondary structure of the von Willebrand factor type A domain in factor B of human complement by Fourier transform infrared spectroscopy. Its occurrence in collagen types VI, VII, XII and XIV, the integrins and other proteins by averaged structure predictions.

The type A domain of the von Willebrand Factor is found also in the complement proteins factor B (FB), C2, CR3 and CR4, the integrins, collagen types VI, VII, XII and XIV, and other proteins. FB is a component of the alternative pathway of the complement system of immune defence, and is cleaved into the fragments Bb and Ba during complement activation. Bb contains a von Willebrand Factor type A (vWF) domain of unknown secondary structure and a serine proteinase (SP) domain, whereas Ba contains three short consensus repeat/complement control protein (SCR/CCP) domains. Fourier transform infrared (FT-IR) spectroscopy on a recombinant vWF domain and on FB and its Bb and Ba fragments shows a broad amide I band. In H2O buffer, second derivative spectra of the amide I band show subcomponents at 1654 to 1657 cm-1, which is typical of alpha-helix, and at 1676 to 1685 cm-1 and 1636 to 1637 cm-1, which are typical of beta-strand. alpha-Helix was detected in the vWF domain, the Bb fragment and FB, and the proportion of alpha-helix present decreased in that order. This shows that the vWF domain contains appreciable amounts of alpha-helix, while the SP and SCR/CCP domains are almost entirely beta-sheet in their secondary structures. Quantitative integration of the vWF FT-IR spectrum showed that this contained 31% alpha-helix and 36% beta-sheet. In 2H2O buffer, the alpha-helix content in the vWF domain is sensitive to the solvent, while the beta-sheet content is less so. An alignment of 75 vWF type A sequences from 25 proteins was used for averaged secondary structure predictions of the total length of 206 residues by the Robson and Chou-Fasman methods. In support of the FT-IR analysis, a total of at least five well-predicted alpha-helices (35% of residues) and at least five well-predicted beta-strands (21% of residues) were identified by both predictive methods, all of which were interspersed by regions of coil or turn conformations. Eight of the ten predicted alpha-helices and beta-strands form an alternating arrangement with each other. Since the predicted alpha-helices are mostly amphipathic, and since the alpha-helix FT-IR band is sensitive to solvent, the alpha-helices are inferred to be on the protein surface.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗