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J B Findlay

Publications and source records attributed to J B Findlay.

At least 37 records · Page 2Linked to original sources
PubMed1998

Conformation and ion-channeling activity of a 27-residue peptide modeled on the single-transmembrane segment of the IsK (minK) protein.

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.

Amino Acid Sequence↗
PubMed1998

Dynamic properties of the guanine nucleotide binding protein alpha subunit and comparison of its guanosine triphosphate hydrolase domain with that of ras p21.

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.

Animals↗
PubMed1998

Inhibition of cromolyn-induced phosphorylation of a 78-kDa protein by phorbol esters in rat peritoneal mast cells.

Disodium cromoglycate (cromolyn) is a well documented inhibitor of immunologically-induced histamine release from rat peritoneal mast cells and has been shown to stimulate the phosphorylation of a mast cell protein of apparent molecular mass 78,000 Da (78 kDa), an event which may be involved in terminating secretion. Here we aimed to determine the role of the ubiquitous enzyme, protein kinase C, in the phosphorylating activity of cromolyn by examining the effects of phorbol esters (activators of protein kinase C) on protein phosphorylation in [32P]orthophosphate loaded rat peritoneal mast cells. Protein kinase C-activating phorbol esters such as 12-O-tetradecanoyl phorbol-13-acetate (TPA) and 4beta-phorbol 12,13-dibutyrate (PdBu) were found to potently inhibit cromolyn-induced phosphorylation when added to mast cells simultaneously with cromolyn (IC50 22 and 79 nM respectively). 4Alpha-phorbol 12,13-didecanoate (PdD), a phorbol ester which does not activate protein kinase C, had no effect on cromolyn-induced phosphorylation. Addition of TPA to mast cells previously exposed to cromolyn for 60 sec (i.e. when 78-kDa protein phosphorylation is maximal) also caused a very rapid dephosphorylation of the 78-kDa protein. Phosphorylation of the 78-kDa protein can also be induced by dibutyryl cyclic GMP and this action was similarly inhibited by TPA and PdBu. Cromolyn inhibited secretion induced by anti-IgE, but not by TPA, and thus inhibition of secretion by cromolyn is further correlated to its phosphorylation of the 78-kDa protein. The data suggest that the inhibitory action of cromolyn on mast cell secretion and phosphorylation of the 78-kDa protein are not mediated through a phorbol ester-sensitive protein kinase C, but more likely that such an enzyme could be involved in regulating dephosphorylation of the 78-kDa protein. Further explanations for this novel dephosphorylating activity of phorbol esters are discussed.

Animals↗
PubMed1998

The transfer of retinol from serum retinol-binding protein to cellular retinol-binding protein is mediated by a membrane receptor.

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.

Humans↗
PubMed1998

The relationship between the agonist-induced activation and desensitization of the human tachykinin NK2 receptor expressed in Xenopus oocytes.

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.

Adenylyl Cyclase Inhibitors↗
PubMed1998

Concerted motions in the photoactive yellow protein.

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.

Amino Acid Sequence↗
PubMed1997

A single-residue deletion alters the lipid selectivity of a K+ channel-associated peptide in the beta-conformation: spin label electron spin resonance studies.

Lipid-peptide interactions with the 27-residue peptide of sequence KLEALYILMVLGFFGFFTLGIMLSYIR reconstituted as beta-sheet assemblies in dimyristoylphosphatidylcholine bilayers have been studied by electron spin resonance (ESR) spectroscopy with spin-labeled lipids. The peptide corresponds to residues 42-68 of the IsK voltage-gated K+ channel protein and contains the single putative transmembrane span of this protein. Lipid-peptide interactions give rise to a second component in the ESR spectra of lipids spin-labeled on the 14C atom of the chain that corresponds to restriction of the lipid mobility by direct interaction with the peptide assemblies. From the dependence on the lipid/peptide ratio, the stoichiometry of lipid interaction is found to be about two phospholipids/peptide monomer. The sequence of selectivity for lipid association with the peptide assemblies is in the order phosphatidic acid > stearic acid = phosphatidylserine > phosphatidylglycerol = phosphatidylcholine. Comparison with previous data for a corresponding 26-residue mutant peptide with a single deletion of the apolar residue Leu2 (Horvath et al., 1995. Biochemistry 34:3893-3898), indicates a very similar mode of membrane incorporation for native and mutant peptides, but a strongly modified pattern and degree of specificity for the interaction with negatively charged lipids. The latter is interpreted in terms of the relative orientations of the charged amino acid side chains in the beta-sheet assemblies of the native and deletion-mutant peptides.

Amino Acid Sequence↗
PubMed1997

Protein dynamics derived from clusters of crystal structures.

A method is presented to mathematically extract concerted structural transitions in proteins from collections of crystal structures. The "essential dynamics" procedure is used to filter out small-amplitude fluctuations from such a set of structures; the remaining large conformational changes describe motions such as those important for the uptake/release of substrate/ligand and in catalytic reactions. The method is applied to sets of x-ray structures for a number of proteins, and the results are compared with the results from essential dynamics as applied to molecular dynamics simulations of those proteins. A significant degree of similarity is found, thereby providing a direct experimental basis for the application of such simulations to the description of large concerted motions in proteins.

Animals↗
PubMed1997

Engineering protein mechanics: inhibition of concerted motions of the cellular retinol binding protein by site-directed mutagenesis.

Recently we reported on the dynamic properties of the cellular retinol binding protein, a member of the fatty acid binding protein family. A few conserved glycines were identified as important for producing the conformational changes necessary for the uptake and release of retinol. Here, we describe a multidisciplinary analysis of a genetically engineered mutation of one of these glycines (Gly67), designed to inhibit an observed hinge bending motion. The correctly folded mutant protein is unable to bind retinol. Analysis of the molecular dynamics simulations of the mutant and wild type protein using the essential dynamics method shows that the mutation indeed inhibits the hinge bending motions which are important for retinol binding.

Binding Sites↗
PubMed1997

Comparison of ras-p21 bound to GDP and GTP: differences in protein and ligand dynamics.

This paper documents the first essential dynamics analysis of ras protein ligands and of the protein itself, showing important features of their dynamic properties. Essential dynamics analysis of 300 ps of full solvent molecular dynamics simulations revealed differences in structure and dynamics between GDP- and GTP-bound forms of H-ras-p21. Regions in the protein which exhibited a structural shift correspond to the switch regions described previously. Differences in dynamics between H-ras-p21 GDP and H-ras-p21 GTP may be related to interactions of ras with GAP and its receptor and effector. Molecular dynamics of free GDP (in the absence of protein) were performed in water for 2 ns and analysed using essential dynamics. The conformations of GDP and GTP when bound to the protein were compared with free GDP, revealing that the ligands bind to the protein in an energetically unfavourable conformation. GDP and GTP molecules from various other protein crystal structures were also analysed. These ligands adopt similar conformations to those seen in H-ras-p21.

Guanosine Diphosphate↗
PubMed1996

Peptides modeled on the transmembrane region of the slow voltage-gated IsK potassium channel: structural characterization of peptide assemblies in the beta-strand conformation.

A 27-residue peptide, having a sequence corresponding to the transmembrane domain of the IsK protein with slow voltage-gated potassium channel activity, has been incorporated into synthetic saturated-chain phospholipid membranes. The peptide-lipid complexes have been characterized by attenuated-total-reflection Fourier-transform-infrared spectroscopy (ATR-FTIR), spin-label electron spin resonance (ESR) spectroscopy, 31P and 2H nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry, and low-angle X-ray diffraction. From FTIR spectroscopy, it is found that, when reconstituted into membranes by dialysis from 2-chloroethanol, the peptide has a predominantly beta-strand secondary structure in which the peptide backbone is oriented at an angle of approximately 56 degrees relative to the membrane normal in dry films of phosphatidylcholines. Hydration of the dry film in the gel phase does not appear to affect the orientation of the peptide backbone, and a relatively small change in orientation occurs when the bilayer undergoes the transition to the fluid phase. The ESR and NMR spectra from spin-labeled and 2H-labeled phospholipids, respectively, indicate that the incorporated peptide restricts the rotational motion of the lipids, without appreciably affecting the chain order, in a way similar to that found for integral membrane proteins. The characteristic two-component ESR spectra from spin-labeled lipids further indicate a selectivity in the interaction of anionic phospholipids with the peptide. The motional restriction of the chains of the spin-labeled phosphatidylcholine and the reduction in the enthalpy of the lipid chain-melting transition indicate that, on average, approximately two to three phospholipid molecules interact directly with each peptide monomer, which is consistent with a limited degree of aggregation of the beta-sheet structures. Both 31P NMR spectroscopy and X-ray diffraction indicate that the lipid-peptide complexes have a lamellar structure up to the highest peptide concentration studied (Rp = 0.2). The surface area occupied by lipid molecules (ca. 30 A2 per chain) in the peptide complexes, deduced from the lamellar repeat spacings at defined water content, is very similar to that in pure fluid lipid bilayers, consistent with the 2H NMR results. The additional membrane surface area contributed by the peptide is approximately 112 A2 per monomer. This large value for the peptide area in the fluid bilayer is consistent with the ATR studies of dry peptide/lipid films which suggest that the long axis of the beta-strand is strongly tilted with respect to the bilayer normal (56 degrees in the dry film).

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1996

Conformational analysis of retinoids and restriction of their dynamics by retinoid-binding proteins.

An exhaustive sampling of the configurational space of all-trans retinol using a 0.1 microsecond molecular-dynamics simulation is presented. The essential dynamics technique is used to describe the conformational changes in retinol using only three degrees of freedom. The different conformational states of retinol are analysed, and differences in free energy are calculated. The essential dynamics description allows a detailed comparison of free retinol and retinoids bound to retinoid-binding proteins and opens new possibilities in the small-molecule docking field. The dynamics of retinoids when complexed with their binding proteins are restricted, and they are forced into strained conformations. A "spring' model for retinoid binding is proposed. This model is extended to a hypothesis for retinoid binding to visual pigments and bacteriorhodopsin.

Computer Simulation↗
PubMed1996

Interaction of dibutyltin-3-hydroxyflavone bromide with the 16 kDa proteolipid indicates the disposition of proton translocation sites of the vacuolar ATPase.

The organotin complex dibutyltin-3-hydroxyflavone bromide [Bu2Sn(of)Br] has been shown to bind to the 16 kDa proteolipid of Nephrops norvegicus, either in the form of the native protein or after heterologous expression in Saccharomyces and assembly into a hybrid vacuolar H(+)-ATPase. Titration of Bu2Sn(of)Br against the 16 kDa proteolipid results in a marked fluorescence enhancement, consistent with binding to a single affinity site on the protein. Vacuolar ATPase-dependent ATP hydrolysis was also inhibited by Bu2Sn(of)Br, with the inhibition constant correlating well with dissociation constants determined for binding of Bu2Sn(of)Br complex to the proteolipid. The fluorescence enhancement produced by interaction of probe with proteolipid can be back-titrated by dicyclohexylcarbodiimide (DCCD), which covalently modifies Glu140 on helix-4 of the polypeptide. Expression of a mutant proteolipid in which Glu140 was changed to a glycine resulted in assembly of a vacuolar ATPase which was inactive in proton pumping and which had reduced ATPase activity. Co-expression studies with this mutant and wild-type proteolipids suggest that proton pumping can only occur in a vacuolar ATPase containing exclusively wild-type proteolipid. The fluorescent enhancement of affinity of Bu2Sn(of)Br for the mutant proteolipid was not significantly altered, with the organotin complex having no effect on residual ATPase activity. Interaction of the probe with mutant proteolipid was unaffected by DCCD. These data suggest an overlap in the binding sites of organotin and DCCD, and have implications for the organization and structure of proton-translocating pathways in the facuolar H(+)-ATPase.

Adenosine Triphosphate↗