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S I Chan

Publications and source records attributed to S I Chan.

At least 109 records · Page 6Linked to original sources
PubMed1984

Chloride binding to the anion transport binding sites of band 3. A 35Cl NMR study.

Band 3 is an integral membrane protein that exchanges anions across the red cell membrane. Due to the abundance and the high turnover rate of the band 3 transport unit, the band 3 system is the most heavily used ion-transport system in a typical vertebrate organism. Here we show that 35Cl NMR enables direct and specific observation of substrate Cl- binding to band 3 transport sites, which are identified by a variety of criteria: (a) the sites are inhibited by 4,4'- dinitrostilbene -2,2'-disulfonate, which is known to inhibit competitively Cl- binding to band 3 transport sites; (b) the sites have affinities for 4,4'- dinitrostilbene -2,2'-disulfonate and Cl- that are quantitatively similar to the known affinities of band 3 transport sites for these anions; and (c) the sites have relative affinities for Cl-, HCO-3, F-, and I- that are quantitatively similar to the known relative affinities of band 3 transport sites for these anions. The 35Cl NMR assay also reveals a class of low affinity Cl- binding sites (KD much greater than 0.5 M) that are not affected by 4,4'- dinitrostilbene -2,2'-disulfonate. These low affinity sites may be responsible for the inhibition of band 3 catalyzed anion exchange that has been previously observed at high [Cl-]. In the following paper the 35Cl NMR assay is used to resolve the band 3 transport sites on opposite sides of the membrane, thereby enabling direct observation of the transmembrane recruitment of transport sites.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1984

Direct observation of the transmembrane recruitment of band 3 transport sites by competitive inhibitors. A 35Cl NMR study.

Numerous models describing anion exchange across the red cell membrane by band 3 have been discussed in literature. These models are readily distinguished from one another by an experiment which tests the ability of band 3 transport sites to be recruited to one side of the membrane. In order to observe directly the transmembrane recruitment of transport sites, we have developed 35Cl NMR techniques that resolve the two transport site populations on opposite sides of the membrane. Using these techniques, we show that the inhibitors 4,4'- dinitrostilbene -2,2'-disulfonate and p- nitrobenzensulfonate each recruit all of the transport sites on both sides of the membrane to the extracellular facing conformation. This result indicates that band 3 has an alternating site transport mechanism: each band 3 transport unit possesses a single functional transport site which is alternately exposed first to one side of the membrane then to the other.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1984

NMR spectroscopic identification of a hexacyanochromate(III) binding site on Pseudomonas azurin.

Studies of redox reactions between the blue copper protein azurin and inorganic reagents have suggested the formation of discrete complexes between the reaction partners prior to the actual electron-transfer step. To get an insight into the structural nature of the complexes formed, we have studied the interaction (i) between oxidized Pseudomonas aeruginosa azurin and the anion Fe(CN)6(3-) and (ii) between reduced azurin and Cr(CN)6(3-). At low ionic strengths, stoichiometric binding of one Fe(CN)6(3-) ion to the oxidized protein is observed. In the high-resolution proton magnetic resonance spectra of the reduced protein, specific broadening of the assigned residues is observed upon titration with the redox-inert Cr(CN)6(3-) ion. Analysis of this paramagnetic spectral broadening in terms of the three-dimensional structure of the protein has led to the proposal that the binding site of the anions lies approximately midway between lysine residues 85 and 92. Evidence in support of this conclusion is provided by parallel studies on Alcaligenes faecalis azurin, which lacks these lysine residues. A similar site on the surface of Pseudomonas azurin has recently been identified by affinity labeling with chromous ions as an electron-transfer locus [ Farver , O., & Pecht , I. (1981) Isr . J. Chem. 21, 13-17]. The results presented here suggest that this region on the protein surface also may be employed by anionic electron-transfer agents.

Alcaligenes↗
PubMed1984

Halide binding by the purified halorhodopsin chromoprotein. II. New chloride-binding sites revealed by 35Cl NMR.

Halorhodopsin is a light-driven chloride pump in the cell membrane of Halobacterium halobium. Recently, a polypeptide of apparent Mr = 20,000 has been purified that contains the halorhodopsin chromophore. Here we use 35Cl NMR to show that the purified chromoprotein possesses two previously unknown classes of chloride-binding sites. One class exhibits a low affinity (KD much greater than 1 M) for chloride and bromide. The second class exhibits a higher affinity (KD = 110 +/- 50 mM) for chloride and also binds other anions according to the affinity series I-, SCN- greater than Br-, NO-3 greater than Cl- greater than F-, citrate. Both classes of NMR site remain intact at pH 11, indicating that the essential positive charges are provided by arginine. Also, both classes are unaffected by bleaching, suggesting that the sites are not in the immediate vicinity of the halorhodopsin chromophore. Although the chromoprotein also appears to contain the chloride-transport site (Steiner, M., Oesterhelt, D., Ariki, M., and Lanyi, J. K. (1984) J. Biol. Chem. 259, 2179-2184), this site was not detected by 35Cl NMR, suggesting that the transport site is in the interior of the protein where it is sampled slowly by chloride in the medium. It is proposed that the purified chromoprotein possesses a channel leading from the medium to the transport site and that the channel contains the high affinity NMR site which facilitates the migration of chloride between the medium and the transport site. We have also used 35Cl NMR to study chloride binding to purified monomeric bacteriorhodopsin; however, this protein contains no detectable chloride-binding sites.

Bacteriorhodopsins↗
PubMed1984

Complexation and phase transfer of nucleic acids by gramicidin S.

A novel interaction between gramicidin S (GrS) and nucleic acids is characterized which, like that between GrS and nucleotides, exploits both the dicationic and amphiphilic properties of the peptide. Complex formation between calf thymus DNA and GrS is demonstrated by (i) phase transfer to CHCl3 of ultrasonically irradiated DNA and (ii) inhibition of phase transfer to CHCl3 of adenosine 5'-triphosphate by either native or ultrasonically irradiated DNA. The stoichiometry of the interaction is 2:1 (DNA-P:GrS), which is consistent with a predominantly electrostatic mode of binding. The apparent affinity of GrS for DNA is considerably higher than it is for free nucleotides. The interaction of the monocationic derivative [2-N delta-acetylornithyl]gramicidin S with calf thymus DNA is considerably weaker. DNA binding by GrS provides a rationale for the lag between germination and RNA synthesis exhibited by wild-type spores of producer strains of Bacillus brevis but not by GrS-negative mutants. On the basis of these results in vitro, a protective role is proposed for GrS in the dormant spore.

Adenosine Triphosphate↗
PubMed1984

Statistical mechanics of lipid membranes. Protein correlation functions and lipid ordering.

An expression is derived for the lipid-mediated intermolecular interaction between protein molecules embedded in a lipid bilayer. It is assumed that protein particles are accommodated by the bilayer, but they distort the lipids in some manner from their equilibrium protein-free configuration. We treat this situation by expanding the free energy density in the plane of the membrane as a Taylor series in some arbitrary parameter and its gradient. Minimization of the total membrane energy for a given particle configuration yields the interparticle interaction energy for that configuration. A test of the model is provided by measurement of the protein-protein pair distribution function from freeze-fracture micrographs of partially aggregated membranes. The measured functions can be simulated by adjustment of two parameters (a) a lipid correlation length that characterizes the distance over which a distortion of the bilayers is transmitted laterally through the bilayer, and (b) a term quantifying the energy of the protein-lipid interaction at the protein-lipid boundary. Correlation lengths obtained by fitting the calculated particle distribution functions to the data are found to be several nanometers. Protein-lipid interaction energies are of the order of a few kT.

Biophysical Phenomena↗
PubMed1983

Complexation and phase transfer of nucleotides by gramicidin S.

Gramicidin S (GrS), an amphiphilic cyclosymmetric decapeptide produced by Bacillus brevis G-B and Nagano, binds nucleotides in water to yield a complex which partitions into organic solvents. The observed phase-transfer efficiencies at a given pH increase in the order AMP less than ADP less than ATP. The lipophilic complexes have well-defined stoichiometries, which were determined to be 1:1 for ADP-GrS at pH 7 and ATP-GrS at pH 3 and 1:2 for ATP-GrS at pH 7. The interaction is primarily ionic, involving coordination of the ornithine N delta H3+ groups of the peptide and the phosphoryl groups of the nucleotide, with little contribution from the nucleoside moiety. Exchange of organic and inorganic phosphates was also found to be mediated by GrS. The nucleotide complexes are sparingly soluble in water and self-associate extensively in CHCl3, most likely by cross-beta-aggregation, to yield large, ribbonlike aggregates which give rise to broad NMR resonances. Structures for the 1:1 and 1:2 complexes are proposed. In the latter, two GrS molecules envelop the nucleotide, orienting their apolar faces externally in opposite directions, while the lateral faces retain considerable polar character and direct aggregation in organic media. The 1:1 complex possesses a single apolar face and is less lipophilic. Binding constants were estimated by simulation of the extraction data. For the 1:1 complexes, K1:1 congruent to 4 X 10(4) M-1 for either ADP or ATP. Phase transfer of the ATP complex at pH 7 could be modeled either by stochastically independent binding to two noninteracting sites on the nucleotide with K1 approximately K2 approximately K1:1 or by a sequential process with K1 approximately K1:1 and K2/K1 less than 100. It is concluded that the apparent selectivity of GrS for ATP over ADP is a consequence of the greater lipophilicity and tendency to aggregate of the 1:2 complex, rather than an intrinsically higher binding affinity for triphosphates. GrS is, to our knowledge, the first peptide known to possess phase-transfer activity toward nucleotides; this is, in addition, the first molecular recognition process in which GrS is demonstrated to participate in vitro at physiologically active concentrations.

Gramicidin↗
PubMed1983

Structure of alamethicin in solution: nuclear magnetic resonance relaxation studies.

An NMR relaxation study at 500 MHz of the icosapeptide antibiotic alamethicin is reported. This study lends further support to the partly helical, partly extended, amphiphilic, and dimeric structure recently proposed for this peptide in methanolic solutions [Banerjee, U., Tsui, F. P., Balasubramanian, T. N., Marshall, G. R., & Chan, S. I. (1983) J. Mol. Biol. 165, 757]. The N-acetyl methyl groups toward the N terminus of alamethicin in this solvent system were found to exhibit unusual NMR relaxation behavior. The decay of the transverse magnetization due to these protons was nonexponential, but the spin-lattice relaxation recovery of the longitudinal magnetization was exponential. In a solution saturated with urea, however, both decays were exponential. These observations are shown to be consistent with the proposed structure. Studies in water yielded qualitatively similar but more complex results. The transverse relaxation times suggest further aggregation in water and indicate that the larger aggregates in water may be made up of the smaller units observed in methanol.

Alamethicin↗
PubMed1983

Structure of Alamethicin in solution. One- and two-dimensional 1H nuclear magnetic resonance studies at 500 MHz.

We report here the 500 MHz 1H nuclear magnetic resonance spectra of Alamethicin, an icosapeptide antibiotic isolated from Trichoderma viride, in methanol, water and methanol/water mixtures. At this frequency, resonances from all the protons are well-resolved in methanol and may be assigned unambiguously. Spectral assignments were made using two-dimensional spin-echo correlated spectroscopy and by spin-decoupling experiments. The amide coupling constants (JNH-alpha CH) facilitated conformational predictions, which were confirmed in part by two-dimensional nuclear Overhauser experiments. On the basis of these data, we propose a secondary structure for Alamethicin that is alpha-helical toward the N terminus and extended beta-sheet at the C-terminal end. This structure is consistent with earlier circular dichroism measurements (McMullen et al., 1971), infrared attenuated total reflection spectroscopy studies (Fringeli & Fringeli, 1979) and proton exchange data (Davis & Gisin, 1981). The proposed structure is a tightly bound dimer, wherein the beta-sheet is stabilized by intermolecular hydrogen-bonds between opposing molecules. An interesting feature of this structure is that it exhibits both a hydrophobic and a hydrophilic surface. This highly amphiphilic nature of the dimer structure may account for the extensive further aggregation of Alamethicin in water. The 1H n.m.r. spectrum of Alamethicin in water is broad, suggesting extensive association. However, spectral assignments and amide coupling constant measurements in water, which were accomplished by titration of methanolic solution of Alamethicin by water, revealed no gross changes in the basic secondary structure of the molecule.

Alamethicin↗
PubMed1983

Pair distribution functions of bacteriorhodopsin and rhodopsin in model bilayers.

The pair distribution functions have been measured from freeze-fracture pictures of bacteriorhodopsin and rhodopsin recombinants with diacyl phosphatidylcholines (PC) of various hydrocarbon chain lengths. Pictures were used of samples that had been frozen from above the phase transition temperature of the lipid. Measured functions were compared with those calculated from two model interparticle potential energy functions, (a) a hard-disk repulsion only, and (b) a hard-disk repulsion plus electrostatic repulsion for a point charge buried in the membrane. The measured functions for bacteriorhodopsin di 12:0 PC, di 14:0 PC, and di 16:0 PC recombinants can be simulated using an interparticle hard-disk repulsion only. Bleached rhodopsin di 12:0 PC and di 18:1 trans-PC recombinants, and dark-adapted rhodopsin di 10:0 PC recombinants yield functions that are better simulated by assuming an additional repulsive interaction. The measured functions resemble those calculated using the hard-disk plus electrostatic repulsion model. The picture of dark-adapted rhodopsin in di 18:1 trans-PC frozen from 20 degrees C shows partial aggregation that is apparent in the measured pair distribution function. This attractive interaction persists even at 37 degrees C, where the measured function shows deviations from the hard-disk repulsive model, indicative of an attractive interparticle interaction. Implications of these results are discussed in terms of protein-lipid interactions.

Bacteriorhodopsins↗
PubMed1982

The nature of CuA in cytochrome c oxidase.

The isolation and purification of yeast cytochrome c oxidase is described. Characterization of the purified protein indicates that it is spectroscopically identical with cytochrome c oxidase isolated from beef heart. Preparations of isotopically substituted yeast cytochrome c oxidase are obtained incorporating [1,3-15N2]histidine or [beta,beta-2H2]cysteine. Electron paramagnetic resonance and electron nuclear double resonance spectra of the isotopically substituted proteins identify unambiguously at least 1 cysteine and 1 histidine as ligands to CuA and suggest that substantial spin density is delocalized onto a cysteine sulfur in the oxidized protein to render the site Cu(I)--S.

Copper↗
PubMed1982

Cholesterol-phospholipid interaction in membranes. 1. Cholestane spin-label studies of phase behavior of cholesterol-phospholipid liposomes.

The effect of cholesterol concentration on the thermotropic phase behavior of aqueous phospholipid multi-bilayers was monitored by means of electron spin resonance spectroscopy (ESR) of a cholestane spin-label (CSL). The spin-label itself induces an additional transition in several different phospholipids, which is attributed to local melting around the spin probe. In contrast, cholesterol prevents its neighboring phospholipids from undergoing fluidization. Small additions of cholesterol affect the position of the probe-induced lipid mobilization curve. The phospholipid main gel-liquid-crystal transition, which is also observed as a separate change in probe mobilization, is not affected by low concentrations of cholesterol. These observations indicate the presence of two phases, a cholesterol-rich phase and a pure phospholipid phase, and indicate that CSL preferentially enters the cholesterol-rich phase. Addition of more than 20 mol % cholesterol abolishes the bulk phospholipid phase. This is evidenced by the disappearance of the gel-liquid-crystal transition as observed by ESR. However, the CSL-induced transition is present at all concentrations of cholesterol and CSL. The behavioral differences between the two sterols caution against using this probe as a direct substitute for cholesterol. However, it remains a useful tool for monitoring the phase behavior of cholesterol-phospholipid bilayer systems.

Cholestanes↗
PubMed1982

Cholesterol-phospholipid interaction in membranes. 2. Stoichiometry and molecular packing of cholesterol-rich domains.

A model for the molecular interaction between cholesterol and phospholipid in bilayer membranes is presented. We propose that cholesterol forms associations with phospholipids with stoichiometries of both 1:1 and 1:2. A hydrogen bond between the beta-OH of cholesterol and the glycerol ester oxygen of a phospholipid is suggested as a likely mechanism for tight binding in a 1:1 complex. A second phospholipid molecule is loosely associated with the complex to form domains of 1:2 stoichiometry, which may coexist with pure phospholipid domains. Interfacial boundary phospholipid separates these two domains. Under conditions in which interfacial phospholipid is maximal, the perturbed phospholipid assumes a composition of 20 mol % cholesterol. To account for the phase behavior and surface properties of cholesterol-lipid membranes, we propose a molecular packing model for linear arrays within the cholesterol-rich domains. In this arrangement, two rows of 1:1 complex run antiparallel with loosely associated phospholipid intercalated between them. The loosely associated phospholipid can pack in the nearly hexagonal manner in which pure crystalline phospholipid is known to pack. The model provides maximal van der Waals contact in the hydrocarbon region of the bilayer and can maintain phospholipids as cholesterol's nearest neighbors at all concentrations up to 50 mol % cholesterol. The model is compatible with the diverse experimental observations compiled by many investigators over the past decade.

Cholesterol↗
PubMed1982

The phospholipid packing arrangement in small bilayer vesicles as revealed by proton magnetic resonance studies at 500 MHz.

Proton magnetic resonance spectra of saturated phospholipids in small unilamellar vesicles has been recorded at 500 MHz on a Bruker WM500 spectrometer. The additional spectral dispersion reveals new structure in the acyl chain resonances. At temperatures near the thermal phase transition, the chain methylene and methyl peaks are split, both showing a broad and a relatively sharp component. Magnetization transfer experiments together with studies in the presence of manganese ions inside or outside the vesicles indicate that the sharp component is to be assigned to the protons from the acyl chains in the inner half of the bilayer and the broad component to chains in the outer monolayer. These experiments demonstrate unambiguously that the extreme surface curvature intrinsic to small unilamellar vesicles induces a profound asymmetry in the packing arrangement of the hydrocarbon chains in the two leaflets of the bilayer and causes the two monolayers to exist in markedly different motional states.

Aged↗
PubMed1981

Reactions of nitric oxide with tree and fungal laccase.

The reactions of nitric oxide (NO) with the oxidized and reduced forms of fungal and tree laccase, as well as with tree laccase depleted in type 2 copper, are reported. The products of the reactions were determined by NMR and mass spectroscopy, whereas the oxidation states of the enzymes were monitored by EPR and optical spectroscopy. All three copper sites in fungal laccase are reduced by NO. In addition, NO forms a specific complex with the reduced type 2 copper. NO similarly reduces all of the copper sites in tree laccase, but it also oxidizes the reduced sites produced by ascorbate or NO reduction. A catalytic cycle is set up in which N2O, NO2-, and various forms of the enzyme are produced. On freezing of fully reduced tree laccase in the presence of NO, the type 1 copper becomes reoxidized. This reaction does not occur with the enzyme depleted in type 2 copper, suggesting that it involves intramolecular electron transfer from the type 1 copper to NO bound to the type 2 copper. When the half-oxidized tree laccase is formed in the presence of NO, a population of molecules exists which exhibits a type 3 EPR signal. A triplet EPR signal is also seen in the same preparation and is attributed to a population of the enzyme molecules in which NO is bound to the reduced copper of a half-oxidized type 3 copper site.

Basidiomycota↗
PubMed1981

Conformations of oxidized cytochrome c oxidase.

Oxidized cytochrome c oxidase is shown to exist in three conformations in addition to the transient "g5" conformation previously reported [Shaw, R. W., Hansen, R. E., & Beinert, H. (1978) J. Biol. Chem. 253, 6637-6640]. The "resting" and "g12" conformations are distinguished by an NO-induced cytochrome a3 electron paramagnetic resonance (EPR) signal and an EPR signal at g' = 12, respectively. The "oxygenated" conformatin exhibits an unusual EPR signal in the presence of fluoride and is identical with the "oxygenated" state first discovered by Okunuki et al. [Okunuki, K., Hagihora, B., Sekuzu, I., & Horio, T. (1958) Proc. Int. Symp. Enzyme Chem., Tokyo, Kyoto, 264]. It is proposed that when the reduced enzyme is reoxidized by dioxygen, the oxidized enzyme first relaxes from the "g5" into the "oxygenated" conformation after which a percentage of the molecules slowly relax into the "g12" conformation. The "resting" conformation is not formed when the enzyme is reoxidized. On the basis of the EPR observations, it is proposed that these various conformations of the oxidized enzyme differ in the structure of the cytochrome a3--Cua3 site. Structures for the cytochrome a3--Cua3 site are proposed for each conformation, and a mechanism by which these conformations undergo interconversion among themselves is described.

Animals↗