J B Findlay

University of Leeds, Leeds, ENG, United Kingdom

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Publications (111)506.98 Total impact

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    ABSTRACT: A simple and rapid protein chemical approach for determining the transmembrane structure of membrane proteins is described. The method involves single substitutions of consecutive amino acid residues, within putative transmembrane segments, to cysteine. This is followed by the analysis of their susceptibility to modification by maleimides with different physico-chemical properties. Fluorescein-5-maleimide (FM), being hydrophilic, modified only residues located in the aqueous environment, while the hydrophobic reagent, benzophenone-4-maleimide (BM) modified residues exposed to the lipid phase. These probes are large enough to cause an increase in the molecular weight of relatively small membrane proteins or polypeptide fragments, which is detectable by SDS-PAGE. Modification by much smaller probes, such as N-ethylmaleimide (NEM), could also be monitored indirectly by the ability to prevent SDS-solubilized protein from being modified with fluorescein-5-maleimide. The approach is demonstrated with the proteolipid complex of the vacuolar H(+)-ATPase expressed in yeast and with the putative Isk K(+)-channel expressed and radiolabelled in E. coli. The advantages of this approach are: (1)it is rapid, easy and inexpensive, (2) detection of the modification of engineered cysteines is simple, (3) it requires only minute quantities of the protein, (4) the protein does not require purification, (5) a broad range of maleimides with different physico-chemical properties can be used, (6) the structure can be investigated under native conditions and does not require protein reconstitution into artificial bilayers.
    Molecular Membrane Biology 07/2009; 13(1):53-60. · 3.13 Impact Factor
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    ABSTRACT: We report the use of thiol chemistry to define specific and reversible disulfide interactions of Cys-substituted NK2 receptor mutants with analogues of neurokinin A (NKA) containing single cysteine substitutions. The NKA analogues were N-biotinylated to facilitate the rapid detection of covalent analogue-receptor interactions utilizing streptavidin reactivity. N-biotinyl-[Tyr1,Cys9]NKA, N-biotinyl-[Tyr1,Cys10]NKA were both found to reversibly disulfide bond to the NK2 receptor mutant Met297 --> Cys. This is consistent with the improved affinities of these particular analogues for the Met297 --> Cys receptor as compared with those for the wild-type and Met297 --> Leu receptors. In our three-dimensional model, Met297 occupies the equivalent position in helix 7 to the retinal binding Lys296 in rhodopsin. Binding of the NK2 receptor antagonist [3H]SR 48968 and of 125I-NKA was used to characterize additional receptor mutants. It seems that the aromatic residues Trp99 (helix 3), His198 (helix 5), Tyr266, His267, and Phe270 play an important role in NKA binding as structural determinants. The existence of overlapping SR 48968 and NKA binding sites is also evident. These data suggest that the peptide binding site of the NK2R is at least in part formed by residues buried deep within the transmembrane bundle and that this intramembranous binding domain may correspond to the binding sites for substantially smaller endogenous GPCR ligands.
    Journal of Biological Chemistry 10/2001; 276(41):37944-9. · 4.65 Impact Factor
  • M A Ali, N Bhogal, C W Fishwick, J B Findlay
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    ABSTRACT: Computer-aided modelling has been used to identify a putative antagonist binding site in the tachykinin NK2 receptor. In order to validate the implied spatial requirements for this region, a series of compounds, based on the potent antagonist GR 149861 have been synthesised and their binding affinities established. Our findings suggest the presence of a large hydrophobic cavity in the putative binding crevice of GR 149861.
    Bioorganic & Medicinal Chemistry Letters 04/2001; 11(6):819-22. · 2.34 Impact Factor
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    ABSTRACT: Cs+ block of GIRK1/GIRK4 expressed in Xenopus oocytes has been investigated. It has been reported that a negatively charged aspartate residue at position 172 in IRK1 is responsible for Cs+ block of the channel. IRK1, a homotetramer, has four aspartate residues at this position. GIRK1/GIRK4 is a heterotetramer and has two aspartate residues at the equivalent position (GIRK1-D173) and, consequently, it should be less sensitive to Cs+. Cs+ caused voltage-dependent block of GIRK1/GIRK4 current (measured with the two-microelectrode voltage-clamp technique). The apparent fraction of the electrical field through which Cs+ moves in order to reach its site of block (delta approximately equals 1.66) is comparable to that in IRK1, suggesting that Cs+ binds to a similar site in the two channels. GIRK1/GIRK4 was less sensitive than IRK1 to Cs+ -the Kd was 3.0-8.5 times greater and at potentials more negative than approximately or = to 130 mV there was voltage-dependent relief of block of GIRK1/GIRK4 (not the case with IRK1). However, the mutations GIRK1-D173A and GIRK1-D173Q increased the sensitivity of the channel to Cs+, while adding a negatively charged aspartate residue to GIRK4 at the equivalent position (GIRK4-N 79D) decreased Cs+ sensitivity. GIRK1-D173 cannot be the site of Cs+ block of GIRK1/GIRK4.
    Pflügers Archiv - European Journal of Physiology 10/2000; 440(5):740-4. · 4.87 Impact Factor
  • M Harrison, B Powell, M E Finbow, J B Findlay
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    ABSTRACT: Proton translocation by the vacuolar H(+)-ATPase is mediated by a multicopy transmembrane protein, the 16-kDa proteolipid. It is proposed to assemble in the membrane as a hexameric complex, with each polypeptide comprising four transmembrane helices. The fourth helix of the proteolipid contains an intramembrane acidic residue (Glu140) which is essential for proton translocation and is reactive toward N,N'-dicyclohexylcarbodiimide (DCCD). Current theoretical models of proton translocation by the vacuolar ATPase require that Glu140 should be protonated and in contact with the membrane lipid. In this study we present direct support for this hypothesis. Modification with the fluorescent DCCD analogue N-(1-pyrenyl)cyclohexylcarbodiimide, coupled to fluorescence quenching studies and bilayer depth measurements using the parallax method, was used to probe the position of Glu140 with respect to the bilayer. Glutamate residues were also introduced mutagenically as targets for the fluorescent probe in order to map additional lipid-accessible sites on the 16-kDa proteolipid. These data are consistent with a structural model of the 16-kDa proteolipid oligomer in which the key functional residue Glu140 and discrete faces of the second and third transmembrane helices of the 16-kDa proteolipid are exposed at the lipid-protein interface.
    Biochemistry 07/2000; 39(25):7531-7. · 3.38 Impact Factor
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    ABSTRACT: Theoretical mechanisms of proton translocation by the vacuolar H(+)-ATPase require that a transmembrane acidic residue of the multicopy 16-kDa proteolipid subunit be exposed at the exterior surface of the membrane sector of the enzyme, contacting the lipid phase. However, structural support for this theoretical mechanism is lacking. To address this, we have used cysteine mutagenesis to produce a molecular model of the 16-kDa proteolipid complex. Transmembrane helical contacts were determined using oxidative cysteine cross-linking, and accessibility of cysteines to the lipid phase was determined by their reactivity to the lipid-soluble probe N-(1-pyrenyl)maleimide. A single model for organization of the four helices of each monomeric proteolipid was the best fit to the experimental data, with helix 1 lining a central pore and helix 2 and helix 3 immediately external to it and forming the principal intermolecular contacts. Helix 4, containing the crucial acidic residue, is peripheral to the complex. The model is consistent not only with theoretical proton transport mechanisms, but has structural similarity to the dodecameric ring complex formed by the related 8-kDa proteolipid of the F(1)F(0)-ATPase. This suggests some commonality between the proton translocating mechanisms of the vacuolar and F(1)F(0)-ATPases.
    Journal of Biological Chemistry 10/1999; 274(36):25461-70. · 4.65 Impact Factor
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    ABSTRACT: The effects of Ba2+, Mg2+, Ca2+ and Na+ as blocking ions were investigated in 90 and 10 mM extracellular K+ solutions on the cloned inward rectifying K+ channel Kir2.1 expressed in Xenopus oocytes. Some data were also obtained using another inward rectifying K+ channel Kir3.1/Kir3.4. The addition of Ba2+ caused a concentration-, voltage- and time-dependent block of both channels. Decreasing the extracellular K+ concentration augmented the block. The data suggest that Ba2+ blocks the channels by binding to a site within the channel pore and that the electrical binding distance, delta, of the site is significantly different for Kir2.1 and Kir3. 1/Kir3.4 (0.38 and 0.22, respectively). Mg2+ and Ca2+ caused an instantaneous concentration- and voltage-dependent block of both channels. With Kir2.1, decreasing the K+ concentration augmented the block. The voltage dependence of the block was less than that of Ba2+ ([delta], 0.1), indicating a more superficial binding site for these ions within the channel pore. The affinity of the channels for Mg2+ and Ca2+ was 1000-fold lower than that for Ba2+. Addition of Na+ resulted in a concentration-, voltage- and time-dependent block of Kir2.1, similar to that observed with Ba2+. The competition between the blocking cations (for Kir2.1: Ba2+, Mg2+, Ca2+; for Kir3. 1/Kir3.4: Ba2+) and extracellular K+ suggests that the binding sites for the blocking cations may be sites to which K+ binds as part of the normal passage of K+ through the channels. It is possible that under normal physiological conditions naturally occurring extracellular cations may partly block the two inward rectifying K+ channels.
    Experimental Physiology 06/1999; 84(3):471-88. · 2.79 Impact Factor
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    ABSTRACT: The C-terminal domain of the voltage-gated potassium channel Kv2.1 is shown to have a role in channel assembly using dominant negative experiments in Xenopus oocytes. Kv2.1 channel polypeptides were co-expressed with a number of polypeptide fragments of the cytosolic C-terminus and the assembly of functional channel homotetramers quantified electrophysiologically using the two electrode voltage clamp technique. Co-expression of C-terminal polypeptides corresponding to the final 440, 318, 220 and 150 amino acid residues of Kv2.1 all resulted in a significant reduction in the functional expression of the full-length channel. A truncated version of Kv2.1 lacking the final 318 amino acids of the C-terminal domain (Kv2. 11-535) exhibited similar electrophysiological properties to the full-length channel. Co-expression with either the 440 or 318 residue polypeptides resulted in a reduction in the activity of the truncated channel. In contrast, the 220 and 150 residue C-terminal fragments had no effect on Kv2.11-535 activity. These data demonstrate that C-terminal interactions are important for driving Kv2.1 channel assembly and that distinct regions of the C-terminal domain may have differential effects on the formation of functional tetramers.
    Biochimica et Biophysica Acta 05/1999; 1418(1):176-84. · 4.66 Impact Factor
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    ABSTRACT: We have studied the effects of agonist and antagonist binding, agonist-induced activation and agonist-induced desensitization of the human tachykinin NK2 receptor mutated at polar residues Asn-51 [in transmembrane helix 1 (TM1)], Asp-79 (TM2) and Asn-303 (TM7), which are highly conserved in the transmembrane domain in the rhodopsin family of G-protein-coupled receptors. Wild-type and mutant receptors were expressed in both COS-1 cells and Xenopus oocytes. The results show that the N51D mutation results in a receptor which, in contrast with the wild-type receptor, is desensitized by the application of a concentration of 1 microM of the partial agonist GR64349, indicating that the mutant is more sensitive to agonist activation than is the wild-type receptor. In addition, we show that, whereas the D79E mutant displayed activation properties similar to those of the wild-type receptor, the D79N and D79A mutants displayed a severely impaired ability to activate the calcium-dependent chloride current. This suggests that it is the negative charge at Asn-79, rather than the ability of this residue to hydrogen-bond, that is critical for the activity of the receptor. Interestingly, the placement of a negative charge at position 303 could compensate for the removal of the negative charge at position 79, since the double mutant D79N/N303D displayed activation properties similar to those of the wild-type receptor. This suggests that these two residues are functionally coupled, and may even be in close proximity in the three-dimensional structure of the human tachykinin NK2 receptor. A three-dimensional model of the receptor displaying this putative interaction is presented.
    Biochemical Journal 05/1999; 339 ( Pt 1):55-61. · 4.65 Impact Factor
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    ABSTRACT: A complete series of analogs of tyrosine modified neurokinin A ([Tyr1]-NKA or [Tyr0]-NKA) has been synthesized by substituting each natural residue with 1-Cys. These analogs were tested for their ability to bind recombinant neurokinin-2 (NK-2) receptor. Substitution of Phe6 with Cys completely abolished binding of the analog to the receptor. Substitution of residues in the carboxyl-terminal region of the peptide (Met10, Leu9, Gly8, Val7) and Asp4 with Cys gave reductions in binding affinity of between 23- and 250-fold. Molecular dynamics simulations of these analogs suggest that changes in peptide structure and flexibility are not large contributors to the losses in receptor binding affinity. Reductions in binding affinity are therefore more confidently ascribed to losses of peptide-receptor interactions.
    Peptides 02/1999; 20(7):795-801. · 2.52 Impact Factor
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    K N Degtyarenko, A C North, J B Findlay
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    ABSTRACT: The PROMISE (prosthetic centres andmetalions in protein activesites) database aims to present comprehensive sequence, structural, functional and bibliographic information on metalloproteins and other complex proteins, with an emphasis on active site structure and function. The database is available on the WorldWide Web at http://bioinf.leeds.ac.uk/promise/
    Nucleic Acids Research 02/1999; 27(1):233-6. · 8.81 Impact Factor
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    ABSTRACT: The large concerted motions in the apo/holo bovine serum retinol-binding protein were studied using molecular dynamics simulation and 'essential dynamics' analysis. Initially, concerted motions were calculated from conformational differences between various crystal structures. The dynamic behaviour of the protein in the configurational space directions, described by these concerted motions, is analysed. This reveals that the large backbone dynamics of the protein is not influenced by the presence of retinol. Study of free retinol dynamics and retinol in the retinol binding site reveals that the protein binds retinol in a favourable conformation, as opposed to what has been previously described for the bovine cellular retinol-binding protein.
    Journal of Computer-Aided Molecular Design 02/1999; 13(1):11-20. · 3.17 Impact Factor
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    ABSTRACT: Molecular dynamics simulations have been performed with the aim of identifying concerted backbone motions in the photoactive yellow protein. Application of the essential dynamics method revealed large, chromophore-linked fluctuations of the protein in the ground state, as well as in a form containing the isomerized chromophore. Various loops become more mobile upon isomerization of the chromophore, including a loop which is part of the PAS domain motif, found in light perception proteins. The hinge points identified in these fluctuations correlate with the positions of evolutionary conserved glycines. The results derived from the simulations directly correlate with available experimental data, provide a framework for understanding the dynamic behaviour of the yellow protein and give clues to subsequent steps in the signal transduction pathway.
    Protein engineering 11/1998; 11(10):873-9.
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    ABSTRACT: Epididymal retinoic acid-binding protein (ERABP) is the major androgen-dependent protein present in the lumen of the epididymis and is thought to be involved in sperm maturation. It displays a high degree of three-dimensional structural similarity to serum retinol-binding protein (RBP). Although both proteins interact with retinoids, RBP exhibits a broad specificity, binding retinol, retinoic acid and retinaldehyde with roughly equal affinities, whereas ERABP is specific for all-trans- and 9-cis-retinoic acids. Consistent with this, the binding pockets of the two proteins are different: in RBP it is predominantly hydrophobic, whereas that for ERABP is amphipathic, with a network of charged residues at the open end of the binding pocket. In order to investigate the roles of these charged residues, Arg-80 and Glu-63 have been mutated to isoleucine. The resultant double mutant, Glu-63-->Ile/Arg-80-->Ile, as well as the wild-type protein, were subsequently expressed in Escherichia coli as fusion proteins, with the streptavidin recognition sequence (Strep) tagged to their C-termini. The expressed proteins were purified in a single step by streptavidin-affinity chromatography and their ligand-binding properties were examined using fluorimetric titrations. Whereas the wild-type ERABP binds only retinoic acid, the double mutant is capable of binding retinol, retinoic acid and retinaldehyde with similar affinities. These observations provide experimental support for the proposition that the charged residues near the open end of the binding pocket are responsible for restricting the specificity of ERABP for retinoic acid. These studies demonstrate that changes in specificity can be engineered into lipocalins.
    Biochemical Journal 09/1998; 334 ( Pt 1):155-60. · 4.65 Impact Factor
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    S Maudsley, J P Gent, J B Findlay, D Donnelly
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    ABSTRACT: 1. Repeated applications of neurokinin A (NKA) to oocytes injected with 25 ng wild-type hNK2 receptor cRNA caused complete attenuation of second and subsequent NKA-induced responses while analogous experiments using repeated applications of GR64349 and [Nle10]NKA(4-10) resulted in no such desensitization. This behaviour has been previously attributed to the ability of the different ligands to stabilize different active conformations of the receptor that have differing susceptibilities to receptor kinases (Nemeth & Chollet. 1995). 2. However, for Xenopus oocytes injected (into the nucleus) with 10 ng wild-type hNK2 receptor cDNA, a single 100 nM concentration of any of the three ligands resulted in complete desensitization to further concentrations. 3. On the other hand, none of the ligands caused any desensitization in oocytes injected with 0.25 ng wild-type hNK2 receptor cRNA. even at concentrations up to 10 microM. 4. The two N-terminally truncated analogues of neurokinin A have a lower efficacy than NKA and it is likely that it is this property which causes the observed differences in desensitization, rather than the formation of alternative active states of the receptor. 5. The peak calcium-dependent chloride current is not a reliable measure of maximal receptor stimulation and efficacy is better measured in this system by studying agonist-induced desensitization. 6. The specific adenylyl cyclase inhibitor SQ22536 can enhance NKA and GR64349-mediated desensitization which suggests that agonist-induced desensitization involves the inhibition of adenylyl cyclase and the subsequent down-regulation of the cyclic AMP-dependent protein kinase, possibly by cross-talk to a second signalling pathway.
    British Journal of Pharmacology 07/1998; 124(4):675-84. · 5.07 Impact Factor
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    ABSTRACT: IsK (minK) protein, in concert with another channel protein KVLQT1, mediates a distinct, slowly activating, voltage-gated potassium current across certain mammalian cell membranes. Site-directed mutational studies have led to the proposal that the single transmembrane segment of IsK participates in the pore of the potassium channel [Takumi, T. (1993) News Physiol. Sci. 8, 175-178]. We present functional and structural studies of a short peptide (K27) with primary structure NH2-1KLEALYILMVLGFFGFFTLGIMLSYI27R-COOH, corresponding to the transmembrane segment of IsK (residues 42-68). When K27 was incorporated, at low concentrations, into phosphatidylethanolamine, black-lipid membranes, single-channel activity was observed, with no strong ion selectivity. IR measurements reveal the peptide has a predominantly helical conformation in the membrane. The atomic resolution structure of the helix has been established by high-resolution 1H NMR spectroscopy studies. These studies were carried out in a solvent comprising 86% v/v 1,1,1,3,3,3-hexafluoro-isopropanol-14% v/v water, in which the IR spectrum of the peptide was found to be very similar to that observed in the bilayer. The NMR studies have established that residues 1-3 are disordered, while residues 4-27 have an alpha-helical conformation, the helix being looser near the termini and more stable in the central region of the molecule. The length (2. 6 nm) of the hydrophobic segment of the helix, residues 7-23, matches the span of the hydrocarbon chains (2.3 +/- 0.25 nm) of fully hydrated bilayers of phosphatidylcholine lipid mixture from egg yolk. The side chains on the helix surface are predominantly hydrophobic, consistent with a transmembrane location of the helix. The ion-channeling activity is believed to stem from long-lived aggregates of these helices. The aggregation is mediated by the pi-pi stacking of phenylalanine aromatic rings of adjacent helices and favorable interactions of the opposing aliphatic-like side chains, such as leucine and methionine, with the lipid chains of the bilayer. This mechanism is in keeping with site-directed mutational studies which suggest that the transmembrane segment of IsK is an integral part of the pore of the potassium channel and has a similar disposition to that in the peptide model system.
    Biochemistry 07/1998; 37(22):8121-31. · 3.38 Impact Factor
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    L V Mello, D M van Aalten, J B Findlay
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    ABSTRACT: The dynamic properties of the alpha-subunit of bovine transducin (Galphat) were studied using molecular dynamics simulations and essential dynamics analyses. The helical domain of transducin seems to move toward the guanosine triphosphate hydrolase (GTPase) domain. Our studies suggest that this movement is facilitated by a hinge bending motion that is centered on residues Gly56 and Gly179 and that this motion may be involved in GDP release and GTP hydrolysis. The dynamic properties of the GTPase domain of Galphat-GDP were compared to those of ras p21 and reveal a significant degree of similarity, indicating common dynamic properties for an equivalent domain in two different proteins.
    Biochemistry 04/1998; 37(9):3137-42. · 3.38 Impact Factor
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    ABSTRACT: The hypothesis that the cellular uptake of retinol involves the specific interaction of a plasma membrane receptor with serum retinol-binding protein (RBP) at the extracellular surface followed by ligand transfer to cytoplasmic cellular retinol-binding protein (CRBP) has been investigated. The experimental system consisted of the [3H]retinol-RBP complex, Escherichia coli-expressed recombinant apo-CRBP containing the 10 amino acid long streptavidin-binding peptide sequence at its C terminus (designated as CRBP-Strep) and permeabilized human placental membranes. [3H]Retinol transfer from RBP to CRBP-Strep was monitored by measuring the radioactivity associated with CRBP-Strep retained by an immobilized streptavidin resin. Using this assay system, we have demonstrated that optimal retinol uptake is achieved with holo-RBP, the membrane receptor and apo-CRBP. The effects are specific: other binding proteins, including beta-lactoglobulin and serum albumin, despite their ability to bind retinol, failed to substitute for either RBP or apo-CRBP. The process is facilitated by membranes containing the native receptor suggesting that this protein is an important component in the transfer mechanism. Taken together, the data suggest that the RBP receptor, through specific interactions with the binding proteins, participates (either directly or via associated proteins) in the mechanism which mediates the transfer of retinol from extracellular RBP to intracellular CRBP.
    Journal of Biological Chemistry 03/1998; 273(6):3336-42. · 4.65 Impact Factor
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    ABSTRACT: Vitamin A is transported in the plasma as retinol bound to a carrier protein, called retinol-binding protein (RBP), which itself forms a complex with the thyroxine-binding protein, known as transthyretin (TTR). This complex exists in equilibrium with free holo-RBP, which can then interact with a specific cell-surface receptor, thereby inducing the release of its retinol to the target cell. Thus, RBP possesses at least three molecular-recogmtion properties it binds retinol and it interacis with both TTR and the cell-surface receptor (for reviews, see refs. 1 and 2).
    Methods in molecular biology (Clifton, N.J.) 02/1998; 89:141-53. · 1.29 Impact Factor
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    ABSTRACT: To study the interaction of retinol-binding protein (RBP) with its plasma carrier, transthyretin (TTR), spectrofluorimetry, and circular dichroism have previously been used. Both these techniques require milligram quantities of the proteins and this sets limitations on the use of these techniques for the study of RBP-TTR interactions using recombinant proteins. The Escherichia coli expression system described in Chapter 11 does not readily produce milligram quantities of RBP for routine use. For this reason, we have developed a highly sensitive method which employs radioiodmated 125I-RBP (unpublished). The method requires only microgram quantities of protein. This chapter describes a method to radioiodinate RBP without loss of activity and protocols for its use in the study of its interaction with TTR.
    Methods in molecular biology (Clifton, N.J.) 02/1998; 89:155-63. · 1.29 Impact Factor

Publication Stats

3k Citations
506.98 Total Impact Points

Institutions

  • 1982–2009
    • University of Leeds
      • School of Biomedical Sciences
      Leeds, ENG, United Kingdom
  • 1999
    • University of Copenhagen
      København, Capital Region, Denmark
    • EMBL-EBI
      Cambridge, England, United Kingdom
  • 1995–1997
    • Max Planck Institute for Biophysical Chemistry
      Göttingen, Lower Saxony, Germany
    • University of East Anglia
      • School of Chemistry
      Norwich, ENG, United Kingdom
  • 1996
    • University of Groningen
      • Groningen Biomolecular Sciences and Biotechnology Institute (GBB)
      Groningen, Groningen, Netherlands
  • 1991
    • Beatson Institute for Cancer Research
      Glasgow, Scotland, United Kingdom
  • 1989
    • The University of Sheffield
      • Department of Molecular Biology and Biotechnology
      Sheffield, ENG, United Kingdom
  • 1988
    • University of Birmingham
      Birmingham, England, United Kingdom
  • 1986
    • John Innes Centre
      Norwich, England, United Kingdom