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Simon J Clark,
Liam A Ridge,
Andrew P Herbert,
Svetlana Hakobyan,
Barbara Mulloy,
Rachel Lennon,
Reinhard Würzner,
B Paul Morgan, Dusan Uhrín,
Paul N Bishop,
Anthony J Day
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ABSTRACT: Complement factor H (CFH) regulates complement activation in host tissues through its recognition of polyanions, which mediate CFH binding to host cell surfaces and extracellular matrix, promoting the deactivation of deposited C3b. These polyanions include heparan sulfate (HS), a glycosaminoglycan with a highly diverse range of structures, for which two regions of CFH (CCP6-8 and CCP19-20) have been implicated in HS binding. Mutations/polymorphisms within these glycosaminoglycan-binding sites have been associated with age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome. In this study, we demonstrate that CFH has tissue-specific binding properties mediated through its two HS-binding regions. Our data show that the CCP6-8 region of CFH binds more strongly to heparin (a highly sulfated form of HS) than CCP19-20, and that their sulfate specificities are different. Furthermore, the HS binding site in CCP6-8, which is affected by the AMD-associated Y402H polymorphism, plays the principal role in host tissue recognition in the human eye, whereas the CCP19-20 region makes the major contribution to the binding of CFH in the human kidney. This helps provide a biochemical explanation for the genetic basis of tissue-specific diseases such as AMD and atypical hemolytic uremic syndrome, and leads to a better understanding of the pathogenic mechanisms for these diseases of complement dysregulation.
The Journal of Immunology 01/2013; · 5.79 Impact Factor
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ABSTRACT: Human β-defensin 2 (HBD2) is a member of the defensin family of antimicrobial peptides that plays important roles in the innate and adaptive immune system of both vertebrates and invertebrates. In addition to their direct bactericidal action, defensins are also involved in chemotaxis and Toll-like receptor activation. In analogy to chemokine/glycosaminoglycan (GAG) interactions, GAG-defensin complexes are likely to play an important role in chemotaxis and in presenting defensins to their receptors. Using a gel mobility shift assay, we found that HBD2 bound to a range of GAGs including heparin/heparan sulfate (HS), dermatan sulfate (DS), and chondroitin sulfate. We used NMR spectroscopy of (15)N-labeled HBD2 to map the binding sites for two GAG model compounds, a heparin/HS pentasaccharide (fondaparinux sodium; FX) and enzymatically prepared DS hexasaccharide (DSdp6). We identified a number of basic amino acids that form a common ligand binding site, which indicated that these interactions are predominantly electrostatic. The dissociation constant of the [DSdp6-HBD2] complex was determined by NMR spectroscopy to be 5 ± 5 μM. Binding of FX could not be quantified because of slow exchange on the NMR chemical shift time scale. FX was found to induce HBD2 dimerization as evidenced by the analysis of diffusion coefficients, (15)N relaxation, and nESI-MS measurements. The formation of FX-bridged HBD2 dimers exhibited features of a cooperative binding mechanism. In contrast, the complex with DSdp6 was found to be mostly monomeric.
Biochemistry 11/2010; 49(49):10486-95. · 3.42 Impact Factor
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ABSTRACT: We have used the interaction between module 7 of complement factor H (CFH approximately 7) and a fully sulfated heparin tetrasaccharide to exemplify a new approach for studying contributions of basic side chains to the formation of glycosaminoglycan (GAG)-protein complexes. We first employed HISQC and H(2)CN NMR experiments to monitor the side-chain resonances of lysines and arginines in (15)N, (13)C-labeled protein during titrations with a fully sulfated heparin tetrasaccharide under physiological conditions. Under identical conditions and using (15)N-labeled protein, we then cross-linked tetrasaccharide to CFH approximately 7 and confirmed the 1:1 stoichiometry by FT-ICR-MS. We subsequently characterized this covalent protein-GAG conjugate by NMR and further MS techniques. MALDI-TOF MS identified protein fragments obtained via trypsin digestion or chemical fragmentation, yielding information concerning the site of GAG attachment. Combining MS and NMR data allowed us to identify the side chain of K405 as the point of attachment of the cross-linked heparin oligosaccharide to CFH approximately 7. On the basis of the analysis of NMR and MS data of the noncovalent and cross-linked CFH approximately 7-tetrasaccharide complexes, we conclude that the K446 side chain is not essential for binding the tetrasaccharide, despite the large chemical shift perturbations of its backbone amide (15)N and (1)H resonances during titrations. We show that R444 provides the most important charge-charge interaction within a C-terminal heparin-binding subsite of CFH approximately 7 whereas side chains of R404, K405, and K388 are the predominant contributors to an N-terminal binding subsite located in the immediate vicinity of residue 402, which is implicated in age-related macular degeneration (AMD).
Journal of the American Chemical Society 05/2010; 132(18):6374-81. · 9.91 Impact Factor
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ABSTRACT: Plasmids, conjugative transposons and phage frequently encode anti-restriction proteins to enhance their chances of entering a new bacterial host that is highly likely to contain a Type I DNA restriction and modification (RM) system. The RM system usually destroys the invading DNA. Some of the anti-restriction proteins are DNA mimics and bind to the RM enzyme to prevent it binding to DNA. In this article, we characterize ArdB anti-restriction proteins and their close homologues, the KlcA proteins from a range of mobile genetic elements; including an ArdB encoded on a pathogenicity island from uropathogenic Escherichia coli and a KlcA from an IncP-1b plasmid, pBP136 isolated from Bordetella pertussis. We show that all the ArdB and KlcA act as anti-restriction proteins and inhibit the four main families of Type I RM systems in vivo, but fail to block the restriction endonuclease activity of the archetypal Type I RM enzyme, EcoKI, in vitro indicating that the action of ArdB is indirect and very different from that of the DNA mimics. We also present the structure determined by NMR spectroscopy of the pBP136 KlcA protein. The structure shows a novel protein fold and it is clearly not a DNA structural mimic.
Nucleic Acids Research 12/2009; 38(5):1723-37. · 8.03 Impact Factor
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ABSTRACT: The solution conformation of a fully sulfated heparin-derived tetrasaccharide, I, was studied in the presence of a 4-fold excess of Ca(2+). Proton-proton and proton-carbon residual dipolar couplings (RDCs) were measured in a neutral aligning medium. The order parameters of two rigid hexosamine rings of I were determined separately using singular value decomposition and ab initio structures of disaccharide fragments of I. The order parameters were very similar implying that a common order tensor can be used to analyze the structure of I. Using one order tensor, RDCs of both hexosamine rings were used as restraints in molecular dynamics simulations. RDCs of the inner iduronic acid were calculated for every point of the molecular dynamics trajectory. The fitting of the calculated RDCs of the two forms of the iduronic acid to the experimental values yielded a population of (1)C(4) and (2)S(o) conformers of iduronic acid that agreed well with the analysis based on proton-proton scalar coupling constants. The glycosidic linkage torsion angles in RDC-restrained molecular dynamics (MD) structures of I are consistent with the interglycosidic three-bond proton-carbon coupling constants. These structures also show that the shape of heparin is not affected dramatically by the conformational flexibility of the iduronic acid ring. This is in line with conclusions of previous studies based on MD simulations and the analysis of (1)H-(1)H NOEs. Our work therefore demonstrates the effectiveness of RDCs in the conformational analysis of glycosaminoglycans.
Glycobiology 08/2009; 19(11):1185-96. · 3.58 Impact Factor
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ABSTRACT: Factor H (fH) is essential for complement homeostasis in fluid-phase and on surfaces. Its two C-terminal domains (CCP 19-20) anchor fH to self-surfaces where it prevents C3b amplification in a process requiring its N-terminal four domains. In atypical hemolytic uremic syndrome (aHUS), mutations clustering toward the C terminus of fH may disrupt interactions with surface-associated C3b or polyanions and thereby diminish the ability of fH to regulate complement. To test this, we compared a recombinant protein encompassing CCP 19-20 with 16 mutants. The mutations had only very limited and localized effects on protein structure. Although we found four aHUS-linked fH mutations that decreased binding to C3b and/or to heparin (a model compound for cell surface polyanionic carbohydrates), we identified five aHUS-associated mutants with increased affinity for either or both ligands. Strikingly, these variable affinities for the individual ligands did not correlate with the extent to which all the aHUS-associated mutants were found to be impaired in a more physiological assay that measured their ability to inhibit cell surface complement functions of full-length fH. Taken together, our data suggest that disruption of a complex fH-self-surface recognition process, involving a balance of affinities for protein and physiological carbohydrate ligands, predisposes to aHUS.
The Journal of Immunology 07/2009; 182(11):7009-18. · 5.79 Impact Factor
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ABSTRACT: The carboxy terminus of human complement component C7 comprises two Factor I-like Modules (FIMs) which are essential for formation of the Membrane Attack Complex, the terminal pathway of the innate immune system. C7-FIMs is a 16.9 kDa, recombinant, disulphide-rich, protein encompassing this C-terminal domain. Using conventional triple resonance experiments 93% of the (1)H, (15)N and (13)C assignment has been achieved, accounting for all assignment apart from a flexible N-terminus cloning artefact and an undefined loop. The chemical shifts have been deposited in the BioMagResBank; Accession No. 15996.
Biomolecular NMR Assignments 07/2009; 3(1):49-52. · 0.72 Impact Factor
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Thomas Vargues,
Gareth J Morrison,
Emily S Seo,
David J Clarke,
Helen L Fielder,
Julien Bennani,
Uday Pathania,
Fiona Kilanowski,
Julia R Dorin,
John R W Govan,
C Logan Mackay, Dusan Uhrín,
Dominic J Campopiano
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ABSTRACT: Human beta-defensin 2 (HBD2) has been shown to interact with pathogenic bacteria and components of the mammalian innate and adaptive immune response. We describe a quick and reliable method for the production of HBD2 in Escherichia coli. HBD2 was expressed as an insoluble fusion, chemically cleaved and oxidised to give a single, folded HBD2 beta-isoform. The purified peptide was analysed by high resolution mass spectrometry, displayed a well-dispersed (1)H NMR spectrum, was a chemoattractant to HEK293 cells expressing CCR6 and acted as an antimicrobial agent against E. coli, P. aeruginosa, C. albicans and S. aureus.
Protein and Peptide Letters 02/2009; 16(6):668-76. · 1.94 Impact Factor
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ABSTRACT: beta-Defensins are a family of cationic peptides that contain six invariant cysteine residues that form characteristic disulfide bonds between Cys(1)-Cys(5), Cys(2)-Cys(4) and Cys(3)-Cys(6). They have been shown to act as potent antimicrobial agents and chemokines. Human beta-defensin 2 (HBD2) was first isolated from psoriatic skin lesions and the structure of this peptide has been solved by X-ray crystallography and NMR spectroscopy both of which are consistent with a fold that contains an N-terminal alpha-helix and three antiparallel beta-strands. Here, we report the expression and purification of the first isotopically labelled beta-defensin ((15)N HBD2) with 100% incorporation of (15)N using a recombinant Escherichia coli method. Multidimensional NMR spectroscopy experiments: 2D (1)H-(15)N HSQC, 3D HSQC-TOCSY and 3D HSQC-NOESY allows for the assignment of resonances with no overlapping or ambiguous peaks. This isotopically labelled peptide is highly suitable for studying the interactions between HBD2 and a range of components from both the mammalian immune system and bacterial pathogens.
Protein Expression and Purification 01/2009; 65(2):179-84. · 1.59 Impact Factor
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ABSTRACT: Human complement factor H, consisting of 20 complement control protein (CCP) modules, is an abundant plasma glycoprotein. It prevents C3b amplification on self surfaces bearing certain polyanionic carbohydrates, while complement activation progresses on most other, mainly foreign, surfaces. Herein, locations of binding sites for polyanions and C3b are reexamined rigorously by overexpressing factor H segments, structural validation, and binding assays. As anticipated, constructs corresponding to CCPs 7-8 and 19-20 bind well in heparin-affinity chromatography. However, CCPs 8-9, previously reported to bind glycosaminoglycans, bind neither to heparin resin nor to heparin fragments in gel-mobility shift assays. Introduction of nonnative residues N-terminal to a construct containing CCPs 8-9, identical to those in proteins used in the previous report, converted this module pair to an artificially heparin-binding one. The module pair CCPs 12-13 does not bind heparin appreciably, notwithstanding previous suggestions to the contrary. We further checked CCPs 10-12, 11-14, 13-15, 10-15, and 8-15 for ability to bind heparin but found very low affinity or none. As expected, constructs corresponding to CCPs 1-4 and 19-20 bind C3b amine coupled to a CM5 chip (K(d)s of 14 and 3.5 microM, respectively) or a C1 chip (K(d)s of 10 and 4.5 microM, respectively). Constructs CCPs 7-8 and 6-8 exhibit measurable affinities for C3b according to surface plasmon resonance, although they are weak compared with CCPs 19-20. Contrary to expectations, none of several constructs encompassing modules from CCP 9 to 15 exhibited significant C3b binding in this assay. Thus, we propose a new functional map of factor H.
The Journal of Immunology 09/2008; 181(4):2610-9. · 5.79 Impact Factor
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ABSTRACT: The chemical functionalization of glycosaminoglycans is very challenging due to their structural heterogeneity and polyanionic character; but as an enabling technology it promises rich rewards in terms of the structural and biological data it will afford. This review surveys the known methods for the preparation of glycosaminoglycan oligosaccharides and conditions for the selective functionalization of both the reducing and non-reducing ends. The synthetic merits of each approach are discussed, together with the structural modification of the glycosaminoglycan oligosaccharide which they confer. Recent applications of this methodology are highlighted, including introduction of functional labels for gel mobility shift assays and NMR studies of glycosaminoglycan-protein complexes, and synthesis of immobilised glycosaminoglycan arrays.
Molecular BioSystems 07/2008; 4(6):481-95. · 3.53 Impact Factor
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ABSTRACT: Factor H is a regulatory glycoprotein of the complement system. We expressed the three N-terminal complement control protein modules of human factor H (FH1-3) and confirmed FH1-3 to be the minimal unit with cofactor activity for C3b proteolysis by factor I. We reconstructed FH1-3 from NMR-derived structures of FH1-2 and FH2-3 revealing an approximately 105-A-long rod-like arrangement of the modules. In structural comparisons with other C3b-engaging proteins, factor H module 3 most closely resembles factor B module 3, consistent with factor H competing with factor B for binding C3b. Factor H modules 1, 2, and 3 each has a similar backbone structure to first, second, and third modules, respectively, of functional sites in decay accelerating factor and complement receptor type 1; the equivalent intermodular tilt and twist angles are also broadly similar. Resemblance between molecular surfaces is closest for first modules but absent in the case of second modules. Substitution of buried Val-62 with Ile (a factor H single nucleotide polymorphism potentially protective for age-related macular degeneration and dense deposit disease) causes rearrangements within the module 1 core and increases thermal stability but does not disturb the interface with module 2. Replacement of partially exposed (in module 1) Arg-53 by His (an atypical hemolytic uremic syndrome-linked mutation) did not impair structural integrity at 37 degrees C, but this FH1-2 mutant was less stable at higher temperatures; furthermore, chemical shift differences indicated potential for small structural changes at the module 1-2 interface.
Journal of Biological Chemistry 05/2008; 283(14):9475-87. · 4.77 Impact Factor
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ABSTRACT: The sensitivity of cryoprobes, which are rapidly becoming available, have brought about the possibility of measurement of (13)C, (13)C coupling constants at the natural abundance of (13)C using tens rather than hundreds of milligrams of compounds. This relatively recent development lays the foundation for a more routine use of the (13)C, (13)C long-range coupling constants in the conformational analysis of molecules. We have designed novel (1)H-detected INADEQUATE experiments optimized for long-range (13)C, (13)C correlations and the measurement of long-range coupling constants. These experiments incorporate refocusing of (1)J(CH) coupling constants prior to the formation of DQ coherences and (1)H-decoupling during the long carbon-carbon evolution intervals. Such modifications significantly enhance their performance over (1)H-detected INADEQUATE experiments currently in use for mapping the one-bond (13)C, (13)C correlations. (1)H or (13)C polarization is used a starting point in long-range correlation (1)H-detected IPAP DEPT-INADEQUATE or RINEPT-INADEQUATE experiments. These correlation experiments were modified yielding in-phase (IP) or antiphase (AP) (13)C, (13)C doublets in F(1). Procedures were developed for their editing yielding accurate values of small (13)C, (13)C coupling constants. The methods are illustrated using mono- and disaccharide samples and compared with related (13)C-detected experiments by means of the measurement of interglycosidic (13)C, (13)C coupling constants of a disaccharide.
Journal of Magnetic Resonance 03/2008; 190(2):171-82. · 2.14 Impact Factor
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Beverly E Prosser,
Steven Johnson,
Pietro Roversi,
Andrew P Herbert,
Bärbel S Blaum,
Jess Tyrrell,
Thomas A Jowitt,
Simon J Clark,
Edward Tarelli, Dusan Uhrín,
Paul N Barlow,
Robert B Sim,
Anthony J Day,
Susan M Lea
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ABSTRACT: Nearly 50 million people worldwide suffer from age-related macular degeneration (AMD), which causes severe loss of central vision. A single-nucleotide polymorphism in the gene for the complement regulator factor H (FH), which causes a Tyr-to-His substitution at position 402, is linked to approximately 50% of attributable risks for AMD. We present the crystal structure of the region of FH containing the polymorphic amino acid His402 in complex with an analogue of the glycosaminoglycans (GAGs) that localize the complement regulator on the cell surface. The structure demonstrates direct coordination of ligand by the disease-associated polymorphic residue, providing a molecular explanation of the genetic observation. This glycan-binding site occupies the center of an extended interaction groove on the regulator's surface, implying multivalent binding of sulfated GAGs. This finding is confirmed by structure-based site-directed mutagenesis, nuclear magnetic resonance-monitored binding experiments performed for both H402 and Y402 variants with this and another model GAG, and analysis of an extended GAG-FH complex.
Journal of Experimental Medicine 11/2007; 204(10):2277-83. · 13.85 Impact Factor
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ABSTRACT: The sensitivity of cryoprobes, which are rapidly becoming available, means that the measurement of coupling constants involving 13C, 13C pairs at the natural abundance of 13C can now, in principle, be done by using tens rather then hundreds of milligrams of compounds. However, a robust method that would yield reliable values of small long-range carbon--carbon coupling constants is still missing. In this Communication, we describe a novel 13C-detected incredible natural-abundance double-quantum transfer experiment (INADEQUATE) experiment for simultaneous correlation of one-bond and long-range 13C- 13C pairs and the measurement of both types of coupling constants in 13C natural abundance samples. This method yields accurate values of one-bond and long-range coupling constants by manipulation of pure phase in-phase (IP) and antiphase (AP) doublets, and is referred to as 13C-detected IPAP-INADEQUATE. It is illustrated by the measurement of interglycosidic (3)J(CCOC) coupling constants in a disaccharide molecule providing important information about the conformation of the glycosidic linkage. Owing to the simplicity of INADEQUATE spectra the carbon-carbon coupling constants are particularly suitable for studies of partially oriented molecules through the measurement of carbon-carbon residual dipolar couplings (RDCs). An example of this approach is presented. We expect the method to find a variety of applications in the conformational analysis of small molecules, determination of diastereoisomers and enantiomers, and studies of molecules in aligned media.
Magnetic Resonance in Chemistry 09/2007; 45(8):628-33. · 1.44 Impact Factor
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ABSTRACT: Efficient functionalisation of the non-reducing end of uronic acid derivatives and glycosaminoglycan-derived disaccharides using peptide coupling has been achieved, mediated by the water-soluble agent DMT-MM.
Chemical Communications 08/2007; · 6.17 Impact Factor
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ABSTRACT: A common single nucleotide polymorphism in the factor H gene predisposes to age-related macular degeneration. Factor H blocks the alternative pathway of complement on self-surfaces bearing specific polyanions, including the glycosaminoglycan chains of proteoglycans. Factor H also binds C-reactive protein, potentially contributing to noninflammatory apoptotic processes. The at risk sequence contains His (rather than Tyr) at position 402 (384 in the mature protein), in the seventh of the 20 complement control protein (CCP) modules (CCP7) of factor H. We expressed both His(402) and Tyr(402) variants of CCP7, CCP7,8, and CCP6-8. We determined structures of His(402) and Tyr(402) CCP7 and showed them to be nearly identical. The side chains of His/Tyr(402) have similar, solvent-exposed orientations far from interfaces with CCP6 and -8. Tyr(402) CCP7 bound significantly more tightly than His(402) CCP7 to a heparin affinity column as well as to defined-length sulfated heparin oligosaccharides employed in gel mobility shift assays. This observation is consistent with the position of the 402 side chain on the edge of one of two glycosaminoglycan-binding surface patches on CCP7 that we inferred on the basis of chemical shift perturbation studies with a sulfated heparin tetrasaccharide. According to surface plasmon resonance measurements, Tyr(402) CCP6-8 binds significantly more tightly than His(402) CCP6-8 to immobilized C-reactive protein. The data support a causal link between H402Y and age-related macular degeneration in which variation at position 402 modulates the response of factor H to age-related changes in the glycosaminoglycan composition and apoptotic activity of the macula.
Journal of Biological Chemistry 07/2007; 282(26):18960-8. · 4.77 Impact Factor
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ABSTRACT: Residual dipolar coupling constants (RDCs) are being increasingly applied to elucidate the configuration and conformation of small organic molecules, peptides and oligosaccharides. In this paper we describe a set of robust 1D NMR methods for accurate and precise measurement of proton-proton RDCs of small and medium size molecules. The performance of these techniques is not impeded by the presence of overlapping and broad (1)H multiplets that are typically observed for such molecules in weakly aligned media. The use of these techniques provides access to a large pool of proton-proton RDCs opening new avenues for the solution structure elucidation of medium size molecules by NMR. The techniques are illustrated on the determination of the alignment tensor of the reducing monosaccharide ring of cellobiose and the determination of the relative configuration of sodium cholate.
ChemPhysChem 07/2007; 8(8):1228-35. · 3.41 Impact Factor
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ABSTRACT: Mutations and polymorphisms in the regulator of complement activation, factor H, have been linked to atypical hemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis, and age-related macular degeneration. Many aHUS patients carry mutations in the two C-terminal modules of factor H, which normally confer upon this abundant 155-kDa plasma glycoprotein its ability to selectively bind self-surfaces and prevent them from inappropriately triggering the complement cascade via the alternative pathway. In the current study, the three-dimensional solution structure of the C-terminal module pair of factor H has been determined. A binding site for a fully sulfated heparin-derived tetrasaccharide has been delineated using chemical shift mapping and the C3d/C3b-binding site inferred from sequence comparisons and computational docking. The resultant information allows assessment of the likely consequences of aHUS-associated amino acid substitutions in this critical region of factor H. It is striking that, excepting those likely to perturb the three-dimensional structure, aHUS-associated missense mutations congregate in the polyanion-binding site delineated in this study, thus potentially disrupting a vital mechanism for control of complement on self-surfaces in the microvasculature of the kidney. It is intriguing that a single nucleotide polymorphism predisposing to age-related macular degeneration occupies another region of factor H that harbors a polyanion-binding site.
Journal of Biological Chemistry 07/2006; 281(24):16512-20. · 4.77 Impact Factor
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ABSTRACT: We have developed new 2D and 3D experiments for the measurement of C(alpha)-H(alpha) residual dipolar coupling constants in (13)C and (15)N labelled proteins. Two experiments, 2D (HNCO)-(J-CA)NH and 3D (HN)CO-(J-CA)NH, sample the C(alpha)-H(alpha) splitting by means of C(alpha) magnetization, while 2D (J-HACACO)NH and 3D J-HA(CACO)NH use H(alpha) magnetization to achieve a similar result. In the 2D experiments the coupling evolution is superimposed on the evolution of the (15)N chemical shifts and the IPAP principle is used to obtain (1)H-(15)N HSQC-like spectra from which the splitting is determined. The use of a third dimension in 3D experiments reduces spectral overlap to the point where use of an IPAP scheme may not be necessary. The length of the sampling interval in the J-dimension of these experiments is dictated solely by the relaxation properties of C(alpha) or H(alpha) nuclei. This was made possible by the use of C(alpha) selective pulses in combination with either a DPFGSE or modified BIRD pulses. Inclusion of these pulse sequence elements in the J-evolution periods removes unwanted spin-spin interactions. This allows prolonged sampling periods ( approximately 25 ms) yielding higher precision C(alpha)-H(alpha) splitting determination than is achievable with existing frequency based methods.
Journal of Magnetic Resonance 06/2006; 180(1):127-36. · 2.14 Impact Factor
