16 research outputs found

    Coulomb Repulsion in Short Polypeptides

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    Coulomb repulsion between like-charged side chains is presently viewed as a major force that impacts the biological activity of intrinsically disordered polypeptides (IDPs) by determining their spatial dimensions. We investigated short synthetic models of IDPs, purely composed of ionizable amino acid residues and therefore expected to display an extreme structural and dynamic response to pH variation. Two synergistic, custom-made, time-resolved fluorescence methods were applied in tandem to study the structure and dynamics of the acidic and basic hexapeptides Asp<sub>6</sub>, Glu<sub>6</sub>, Arg<sub>6</sub>, Lys<sub>6</sub>, and His<sub>6</sub> between pH 1 and 12. (i) End-to-end distances were obtained from the short-distance Förster resonance energy transfer (sdFRET) from N-terminal 5-fluoro-l-tryptophan (FTrp) to C-terminal Dbo. (ii) End-to-end collision rates were obtained for the same peptides from the collision-induced fluorescence quenching (CIFQ) of Dbo by FTrp. Unexpectedly, the very high increase of charge density at elevated pH had no dynamical or conformational consequence in the anionic chains, neither in the absence nor in the presence of salt, in conflict with the common view and in partial conflict with accompanying molecular dynamics simulations. In contrast, the cationic peptides responded to ionization but with surprising patterns that mirrored the rich individual characteristics of each side chain type. The contrasting results had to be interpreted, by considering salt screening experiments, N-terminal acetylation, and simulations, in terms of an interplay of local dielectric constant and peptide-length dependent side chain charge–charge repulsion, side chain functional group solvation, N-terminal and side chain charge–charge repulsion, and side chain–side chain as well as side chain–backbone interactions. The common picture that emerged is that Coulomb repulsion between water-solvated side chains is efficiently quenched in short peptides as long as side chains are not in direct contact with each other or the main chain

    Diffusion-Enhanced Förster Resonance Energy Transfer and the Effects of External Quenchers and the Donor Quantum Yield

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    The structural and dynamic properties of a flexible peptidic chain codetermine its biological activity. These properties are imprinted in intrachain site-to-site distances as well as in diffusion coefficients of mutual site-to-site motion. Both distance distribution and diffusion determine the extent of Förster resonance energy transfer (FRET) between two chain sites labeled with a FRET donor and acceptor. Both could be obtained from time-resolved FRET measurements if their individual contributions to the FRET efficiency could be systematically varied. Because the FRET diffusion enhancement (FDE) depends on the donor-fluorescence lifetime, it has been proposed that the FDE can be reduced by shortening the donor lifetime through an external quencher. Benefiting from the high diffusion sensitivity of short-distance FRET, we tested this concept experimentally on a (Gly–Ser)<sub>6</sub> segment labeled with the donor/acceptor pair naphthylalanine/2,3-diazabicyclo[2.2.2]­oct-2-ene (NAla/Dbo). Surprisingly, the very effective quencher potassium iodide (KI) had no effect at all on the average donor–acceptor distance, although the donor lifetime was shortened from ca. 36 ns in the absence of KI to ca. 3 ns in the presence of 30 mM KI. We show that the proposed approach had to fail because it is not the experimentally observed but the radiative donor lifetime that controls the FDE. Because of that, any FRET ensemble measurement can easily underestimate diffusion and might be misleading even if it employs the Haas–Steinberg diffusion equation (HSE). An extension of traditional FRET analysis allowed us to evaluate HSE simulations and to corroborate as well as generalize the experimental results. We demonstrate that diffusion-enhanced FRET depends on the radiative donor lifetime as it depends on the diffusion coefficient, a useful symmetry that can directly be applied to distinguish dynamic and structural effects of viscous cosolvents on the polymer chain. We demonstrate that the effective FRET rate and the recovered donor–acceptor distance depend on the quantum yield, most strongly in the absence of diffusion, which has to be accounted for in the interpretation of distance trends monitored by FRET

    Investigation of the voltage-dependence of PorACj in single-channel experiments.

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    <p>A: The purified protein was added to the <i>cis</i>-side of a PC membrane (10 ng/ml) and the reconstitution of channels was followed until about 10 PorACj-channels inserted into the membrane. Then 40 mV were applied to the <i>cis</i>-side of the membrane, and the membrane current was measured as a function of time. The aqueous phase contained 1M KCl; T = 20°C. B: Histogram of 56 closing events of the experiment in A and and similar experiments. The closing events were plotted in a bargraph as a function of the conductance of the closing events. ! M KCl; T = 20°C. Note that the PorACj channels closed in two distinct conductance values of 1 and 2 nS.</p

    Analysis of PorACj purification.

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    <p>(A) Western blot analysis illustrating IMAC purification of his-tagged PorACj protein. The protein was expressed in <i>C. glutamicum</i> ATCC13032 <i>ΔporHΔporA</i> and purified by Ni<sup>2+</sup> affinity from the supernatant of detergent extracted whole cells. CMDIE represents chloroform-methanol treated cells in which the crude protein content was concentrated around 8 fold by diethyl-ether precipitation of pXJK0268His transfected (+) or non-transfected (−) <i>C. glutamicumΔporHΔporA</i> cells. Subsequent to tricine (12%)-SDS-PAGE the gel was blotted on a nitrocellulose membrane and PorACj-His was visualized by Anti-His antibodies and a chemiluminescent reaction. All samples were boiled for 5 minutes in Redmix before loading. (B) Silver stained tricine (16.5%)-SDS-PAGE of Ni<sup>2+</sup>-purified and factor Xa digested PorACj-His protein. Lanes: 1, 3 units of protease Xa (control); 2, 10 ”l of three pooled Ni-NTA elution containing PorACj-His; 3, 10 ”l of protease Xa treated and purified PorACj protein (for details see text). The dot blot immunoassay pictures underneath lanes 2 and 3 show cleavage of the histidine tail using anti-his antibody of 5 ”l of the corresponding protein samples. Before loading all samples were boiled for 5 minutes in Redmix.</p

    Investigation of the voltage-dependence of PorACj in a multi-channel experiment.

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    <p>The purified protein was added to the <i>cis</i>-side of a PC membrane (100 ng/ml) and the reconstitution of channels was followed until equilibrium. Then increasing positive (upper traces) and negative voltages (lower traces) were applied to the <i>cis</i>-side of the membrane, and the membrane current was measured as a function of time. The aqueous phase contained 1 M KCl; T = 20°C.</p

    Analysis of secondary structure of PorACj usinf CD-spectrometry.

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    <p>A: CD spectra of recombinant PorACj (69 ”M) and PorA-His<sub>8</sub> (12 ”M) solubilized in 0.5% Genapol, 100 mM NaCl, 50 mM TrisHCl and 1 mM CaCl<sub>2</sub>, pH 8 measured at room temperature. B: CD-spectra of the same protein samples as in (A). The aqueous solutions of the proteins was supplemented with 4 M urea to destroy the secondary structure of the proteins.</p

    Analysis of PorACj secondary structure.

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    <p>(A) The panel shows the hydrophobicity indices of the individual amino acids of PorACj according to ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075651#pone.0075651-Kyte1" target="_blank">[80]</a>. (B) The secondary structure of PorACj was predicted using a consensus method [83] at the Pole Bioinformatique Lyonnaise network (<a href="http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_seccons.html" target="_blank">http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_seccons.html</a>); the protein was suggested to form α-helices. Amino acid residues arranged on basis of heptameric repeats (a–g) showing distinct separation in a hydrophobic domain supposable surrounded by lipid molecules (dark grey) while the hydrophilic domain (light grey) is suggested to represent the component orientated to the water-filled lumen in the presumed oligomeric PorACj.</p

    Conductance (G) at a given membrane potential (V<sub>m</sub>) divided by the conductance at 10

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    <p> <b>mV (G<sub>0</sub>) expressed as a function of the membrane potential.</b> The symbols represent the mean (± SD) of six measurements, in which pure PorACj protein was added to the <i>cis</i>-side of the membranes. The aqueous phase contained 1 M KCl and 100 ng/ml porin. The membranes were formed from PC/<i>n</i>-decane at a temperature of 20°C.</p

    Study of pore-forming capacity of purified PorACj.

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    <p>(A) Single-channel recording of a PC/<i>n</i>-decane membrane in the presence of pure PorACj. The aqueous phase contained 1 M KCl, pH 6 and 10 ng/ml protein. The applied membrane potential was 20 mV; T = 20<sup>°</sup>C. (B) Histogram of the probability P(G) for the occurrence of a given conductivity unit observed with membranes formed of 1% PC dissolved in <i>n</i>-decane. It was calculated by dividing the number of fluctuations with a given conductance rise by the total number of conductance fluctuations in the presence of pure PorACj. Two frequent conductive units were observed for 295 single events taken from eight individual membranes. The average conductance of the steps corresponding to the left-side maximum was 1.25 nS and that of the right-side maximum was 2.5 nS. The aqueous phase contained 1 M KCl, pH 6 and 10 ng/ml protein, the applied membrane potential was 20 mV, T = 20°C.</p
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