Biomedical subjects

D G Norman

Publications and source records attributed to D G Norman.

10 recordsLinked to original sources

PubMed1992

Activity, disulphide mapping and structural modelling of the fifth domain of human beta 2-glycoprotein I.

Complexes formed by the interaction of negatively charged phospholipids and beta 2-glycoprotein I (beta 2-I) are the target of autoantibodies in systemic lupus erythematosus. The highly positively charged fifth (C-terminal) domain of human beta 2-I was produced as a fusion protein in an Escherichia coli expression system and was shown to bind to the negatively charged phospholipid, cardiolipin, almost as well as the intact protein. In an attempt to define the 3D structure of this domain, the disulphide linkage pattern was determined and shown to be Cys 1-4, Cys 2-5 and Cys 3-6 in contradiction to an earlier report. In the light of this information, the sequence of the fifth domain of beta 2 I (beta 2-I-5) is readily aligned with that of the 16th repeat of factor H, of which the 3D structure is known, and a model of beta 2I-5 has been built by homology. On the basis of the model we suggest residues that might be the target of profitable site-directed mutagenesis in structure-function studies.

Amino Acid Sequence↗

PubMed1992

Solution structure of the fifth repeat of factor H: a second example of the complement control protein module.

Modules which share the same consensus sequence are assumed to have common structural features, at the secondary and tertiary level. In order to test the extent of such similarities, it is necessary to examine the structures of several examples from each module family. Recently, the first three-dimensional structure of a complement control protein (CCP) module (the 16th repeat of human factor H, H16) was determined using a combination of two-dimensional NMR and simulated annealing [Norman, D.G., Barlow, P.N., Baron, M., Day, A.J., Sim, R.B., & Campbell, I.D. (1991) J. Mol. Biol. 219, 717-725]. Using the same techniques, the three-dimensional structure of a second CCP module (the 5th repeat of human factor H, H5) has now been determined. The primary sequence of H5 contains 17 residues which are identical and in equivalent position to those in H16. Thirteen of these 17 are part of the consensus sequence. The similarities between the secondary structure of H5 and that of H16 are extensive. This implies that the consensus sequence dictates a particular secondary structure. The tertiary structure of H5, a compact hydrophobic core wrapped in beta-strand and sheet, bears much overall resemblance to that of H16. However, there is a deletion in the first strand of H5, and an insertion in a loop, resulting in slightly shorter overall length. This is associated with a rearrangement of residues within the hydrophobic core. The side chain of the highly conserved Tyr29, which occupies a central position within the core of H16, lies on the periphery of the core of H5.

Amino Acid Sequence↗

PubMed1992

The three-dimensional structure of the first EGF-like module of human factor IX: comparison with EGF and TGF-alpha.

The three-dimensional structure of the first epidermal growth factor (EGF)-like module from human factor IX has been determined in solution using two-dimensional nuclear magnetic resonance (in the absence of calcium and at pH 4.5). The structure was found to resemble closely that of EGF and the homologous transforming growth factor-alpha (TGF-alpha). Residues 60-65 form an antiparallel beta-sheet with residues 68-73. In the C-terminal subdomain a type II beta-turn is found between residues 74 and 77 and a five-residue turn is found between residues 79 and 83. Glu 78 and Leu 84 pair in an antiparallel beta-sheet conformation. In the N-terminal region a loop is found between residues 50 and 55 such that the side chains of both are positioned above the face of the beta-sheet. Residues 56-60 form a turn that leads into the first strand of the beta-sheet. Whereas the global fold closely resembles that of EGF, the N-terminal residues of the module (46-49) do not form a beta-strand but are ill-defined in the structure, probably due to the local flexibility of this region. The structure is discussed with reference to recent site-directed mutagenesis data, which have identified certain conserved residues as ligands for calcium.

Amino Acid Sequence↗

PubMed1991

Solution structure of pardaxin P-2.

Pardaxin is a mucosal secretion of the Pacific sole Pardachirus pavoninus that exhibits unusual shark repellent and surfactant properties [Thompson, S. A., Tachibana, K., Nakanishi, K., & Kubota, I. (1986) Science 233, 341-343]. This 33 amino acid polypeptide folds into ordered structures in trifluoroethanol-water solution and in micelles but adopts a random-coiled structure in water solution. The complete proton NMR spectrum of pardaxin P-2 has been assigned in CF3CD2OD/H2O (1:1) solution, and the three-dimensional structure has been elucidated with distance restrained molecular dynamics calculations. It is demonstrated that peptide segments within the 7-11 and 14-26 residue stretches are helical while residues at the C- and N-terminus exist predominantly in extended conformations in solution. The dipeptide 12-13 segment connecting the two helices exists as a bend or a hinge allowing the two helices to be oriented in a L-shaped configuration. These studies establish that pardaxin P-2 adopts a novel amphiphilic helix (7-11)-bend (12-13)-helix (14-26) motif with Pro-13 forming the focal point of the turn or bend between the two helices.

Amino Acid Sequence↗

PubMed1991

Three-dimensional structure of a complement control protein module in solution.

The complement control protein (CCP) modules (also known as short consensus repeats) are defined by a consensus sequence within a stretch of about 60 amino acid residues. These modules have been identified more than 140 times in over 20 proteins, including 12 proteins of the complement system. The solution structure of the 16th CCP module from human complement factor H has been determined by a combination of 2-dimensional nuclear magnetic resonance spectroscopy and restrained simulated annealing. In all, 548 structurally important nuclear Overhauser enhancement cross-peaks were quantified as distance restraints and, together with 41 experimentally measured angle restraints, were incorporated into a simulated annealing protocol to determine a family of closely related structures that satisfied the experimental observations. The CCP structure is shown to be based on a beta-sandwich arrangement; one face made up of three beta-strands hydrogen-bonded to form a triple-stranded region at its centre and the other face formed from two separate beta-strands. Both faces of the molecule contribute highly conserved hydrophobic side-chains to a compact core. The regions between the beta-strands are composed of both well-defined turns and less well-defined loops. Analysis of CCP sequence alignments, in light of the determined structure, reveals a high degree of conservation amongst residues of obvious structural importance, while almost all insertions, deletions or replacements observed in the known sequences are found in the less well-defined loop regions. On the basis of these observations it is postulated that models of other CCP modules that are based on the structure presented here will be accurate. Certain families of CCP modules differ from the consensus in that they contain extra cysteine residues. As a test of structural consensus, the extra disulphide bridges are shown to be easily accommodated within the determined CCP model.

Amino Acid Sequence↗

PubMed1991

Secondary structure of a complement control protein module by two-dimensional 1H NMR.

The complement control protein (CCP) module (also known as the short consensus repeat) is a consensus sequence of about 60 amino acid residues which is thought to fold independently. It occurs over 140 times in more than 20 extracellular mosaic proteins including 12 proteins of the complement cascade. An isolated CCP module, the 16th repeat from human complement factor H, has been expressed in a yeast vector and shown to fold with the same pattern of disulfide bond formation as is seen in the native protein. Two-dimensional 600-MHz 1H NMR spectra of this module have been recorded at pH 3.3 and 6.0 and analyzed to permit determination of secondary structure in solution. The CCP module comprises two predominantly extended segments (Glu1-His13 and Ala17-Glu27), two segments of double-stranded antiparallel beta-sheet (Gly14-Val16 paired with Tyr31-Cys33 and Gly38-Asp40 paired with Ser57-Ile59), and a short piece of triple-stranded beta-sheet (Glu27-Thr30, Ile44-Leu48, and Lys51-Ser53). Turns occur at Asp22, Gly36, and Glu50, while Gly41-Ala43 appear to form a looped-out segment or bulge. This structure is compared with a secondary structure prediction made on the basis of an alignment scheme of 101 sequences for CCP modules [Perkins, S. J., Haris, P. I., Sim, R. B., & Chapman, D. (1988) Biochemistry 27, 4004-4012]--the experimentally determined secondary structure bears an overall resemblance to the predicted one but differs in the number and position of turns. Some of those amino acid residues which are highly conserved throughout the range of CCP modules appear to play a role in stabilizing the global fold.

Amino Acid Sequence↗

PubMed1991

Protein modules.

As the database of protein sequences grows it is becoming apparent that many proteins are constructed from relatively few modular units that appear many times. Determination of the three-dimensional structure of such modules by NMR has been possible due to their production in relatively large quantities by recombinant DNA techniques. The knowledge gained about the structure of individual modules can then be used to predict their properties in a variety of intact proteins.

Amino Acid Sequence↗

PubMed1990

Conformational transitions in thymidine bulge-containing deoxytridecanucleotide duplexes. Role of flanking sequence and temperature in modulating the equilibrium between looped out and stacked thymidine bulge states.

Structural features at extra thymidine bulge sites in DNA duplexes have been elucidated from a two-dimensional NMR analysis of through-bond and through-space connectivities in the otherwise self-complementary d(C-C-G-T-G-A-A-T-T-C-C-G-G) (GTG 13-mer) and d(C-C-G-G-A-A-T-T-C-T-C-G-G) (CTC 13-mer) duplexes in aqueous solution. These studies establish that the extra thymidine flanked by guanosines in the GTG 13-mer duplex is in a conformational equilibrium between looped out and stacked states. The looped-out state is favored at low temperature (0 degrees C), whereas the equilibrium shifts in favor of the stacked state at elevated temperatures (35 degrees C) prior to the onset of the duplex-strand transition. By contrast, the extra thymidine flanked by cytidines in the CTC 13-mer duplex is looped out independent of temperature in the duplex state. Our results demonstrate that temperature and flanking sequence modulate the equilibrium between looped-out and stacked conformations of single base thymidine bulges in DNA oligomer duplexes.

Base Sequence↗

PubMed1989

Conformation of adenosine bulge-containing deoxytridecanucleotide duplexes in solution. Extra adenosine stacks into duplex independent of flanking sequence and temperature.

Structural features at the extra adenosine bulge sites in DNA duplexes have been elucidated from an NMR analysis of two-dimensional through space and through bond connectivities in the self-complementary d(C-C-G-G-A-A-T-T-C-A-C-G-G) (CAC 13-mer) and d(C-C-G-A-G-A-A-T-T-C-C-G-G) (GAG 13-mer) duplexes in aqueous solution. These studies establish that the extra adenosine stacks into the helix at all temperatures below the onset of the melting transition in solution, and the results are independent of whether the extra adenosine is flanked by cytidines (CAC 13-mer) or guanosines (GAG 13-mer). The NMR parameters establish that the extra adenosine can be accommodated into the helix with the flanking base pairs adopting a wedge-shaped orientation. The resulting perturbation extends out to the C10-G11 phosphodiester backbone adjacent to the bulge segment in both the CAC 13-mer and GAG 13-mer duplexes.

Adenosine↗

PubMed1989

Conformational transitions in cytidine bulge-containing deoxytridecanucleotide duplexes: extra cytidine equilibrates between looped out (low temperature) and stacked (elevated temperature) conformations in solution.

High-resolution homonuclear and heteronuclear two-dimensional NMR studies have been carried out on the self-complementary d(C-C-G-C-G-A-A-T-T-C-C-G-G) duplex (designated GCG 13-mer) in aqueous solution. This sequence contains an extra cytidine located between residues G3 and G4 on each strand of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) and correlated (COSY and relay COSY) spectra for the GCG 13-mer duplex in H2O and D2O solution. The extra cytidine at the bulge site (designated CX) results in more pronounced changes in the NOE distance connectivities for the G3-CX-G4 segment centered about the CX residue compared to the C9-C10 segment on the partner strand opposite the CX residue for the GCG 13-mer duplex at 25 degrees C. The cross-peak intensities in the short mixing time NOESY spectrum also establish that all glycosidic torsion angles including that of CX are anti in the GCG 13-mer duplex at 25 degrees C. The observed chemical shift changes for the CX base protons and the G3pCX phosphorus resonance with temperature between 0 and 40 degrees C demonstrate a temperature-dependent conformational equilibrium in the premelting transition region. The NOE and chemical shift parameters establish that the predominant conformation at low temperature (0 degree C) has the extra cytidine looped out of the helix with the flanking G3.C10 and G4.C9 base pairs stacked on each other. These results support conclusions based on earlier one-dimensional NMR studies of extra cytidine containing complementary duplexes in aqueous solution [Morden, K. M., Chu, Y. G., Martin, F. H., & Tinoco, I., Jr. (1983) Biochemistry 22, 5557-5563. Woodson, S. A., & Crothers, D. M. (1987) Biochemistry 26, 904-912]. By contrast, the chemical shift and NOE parameters demonstrate that the conformational equilibrium shifts toward a structure with a stacked extra cytidine on raising the temperature to 40 degrees C prior to the helix-coil melting transition. The most downfield shifted phosphorus resonance in the GCG 13-mer duplex has been assigned to the phosphate in the C2-G3 step, and this observation demonstrates that the perturbation in the phosphodiester backbone extends to regions removed from the (G3-CX-G4).(C9-C10) bulge site.

Base Composition↗