Intrinsic Tyrosine Fluorescence as a Tool To Study the Interaction of the Shaker B “Ball” Peptide with Anionic Membranes

Abstract

Steady-state and time-resolved fluorescence from the single tyrosine in the inactivating peptide of the Shaker B potassium channel (ShB peptide) and in a noninactivating peptide mutant, ShB-L7E, has been used to characterize their interaction with anionic phospholipid membranes, a model target mimicking features of the inactivation site on the channel protein. Partition coefficients derived from steady-state anisotropy indicate that both peptides show a high affinity for anionic vesicles, being higher in ShB than in ShB-L7E. Moreover, differential quenching by lipophilic spin-labeled probes and fluorescence energy transfer using trans-parinaric acid as the acceptor confirm that the ShB peptide inserts deep into the membrane, while the ShB-L7E peptide remains near the membrane surface. The rotational mobility of tyrosine in membrane-embedded ShB, examined from the decay of fluorescence anisotropy, can be described by two different rotational correlation times and a residual constant value. The short correlation time corresponds to fast rotation reporting on local tyrosine mobility. The long rotational correlation time and the high residual anisotropy suggest that the ShB peptide diffuses in a viscous and anisotropic medium compatible with the aliphatic region of a lipid bilayer and support the hypothesis that the peptide inserts into it as a monomer, to configure an intramolecular -hairpin structure. Assuming that this hairpin structure behaves like a rigid body, we have estimated its dimensions and rotational dynamics, and a model for the peptide inserted into the bilayer has been proposed.The inactivating Shaker B (ShB) 1 peptide comprises the 20 N-terminal amino acids (H 2 N-MAAVAGLYGLGED-RQHRKKQ) of each subunit in the Shaker B potassium channel, the so-called inactivating "ball", responsible for inducing fast, N-type inactivation in this and many other related or unrelated channels (1-6). Fast inactivation implies the physical occlusion of the channel's cytoplasmic mouth (7), with the ball peptide acting as an open channel blocker. The site on the channel where the ball peptide interacts consists of a hydrophobic protein pocket, separated from the cytoplasm by a region with a negative surface potential (8,9). Such channel domains can be partly mimicked by anionic phospholipid membranes, which also contain a hydrophobic region (the hydrophobic bilayer) and a negatively charged surface. Thus, anionic phospholipid vesicles have been used as model targets to gain insight into the molecular events in which the ShB peptide might be involved during channel inactivation (10-14). Fourier transform IR spectroscopy, differential scanning calorimetry, and steady-state fluorescence studies of fluorophore-labeled ShB peptide suggest that the ShB peptide (a) binds to the vesicle surface with high affinity, (b) readily adopts a strongly hydrogen-bonded intramolecular -hairpin structure, and (c) becomes inserted into the hydrophobic bilayer in a monomeric form. In contrast, a noninactivating mutant peptide ShB-L7E ...

Membrane Interaction of the Glycosyltransferase WaaG

“…At 40% POPG and in E. coli inner-membrane-like vesicles, the anisotropy was 0.11 5 0.01 and 0.10 5 0.01, respectively. These values are similar to the anisotropy that was determined for the 20-residue ShB peptide upon interaction with anionic LUVs (hri ¼ 0:12050:003) by Poveda and coworkers (44). The distances of the fluorophores from the center of the bilayer at 16 mol % quencher concentration calculated with Eq.…”

“…This implies that under the conditions used in this study, all of MIR-WaaG is bound. Moreover, the K D is similar to what has been reported for peripheral membrane proteins (29) or membrane-associated peptides (44). Our data clearly suggest that binding is driven by anionically charged lipids, but we cannot directly determine whether specific (cationic) amino acid residues are involved in this interaction, or whether there is a preference for the peptide to interact with PG or CL.…”

“…Peptide-membrane interactions are also evident from an increase in fluorescence anisotropy as well as from an increase in quenching efficiency in the presence of a lipophilic quencher or, conversely, a decrease in quenching efficiency in the presence of a hydrophilic quencher. To quantify this interaction, we determined the dissociation constant and found that the affinity of MIR-WaaG to E. coli inner-membrane LUVs is high (K D ¼ 35 mM) (44). This implies that under the conditions used in this study, all of MIR-WaaG is bound.…”

Structural insights into RNA recognition by the alternative-splicing regulator muscleblind-like MBNL1

“…1c), rendering it suitable for fluorescence studies 40 . Tyr188 and Tyr224 stack against the side chains of zinc-coordinated His204 and His238, and Tyr236 intercalates between the guanine and the cytosine bases in the complex.…”

Influence of Lysine Nε-Trimethylation and Lipid Composition on the Membrane Activity of the Cecropin A-Melittin Hybrid Peptide CA(1−7)M(2−9)

“…44 An excitation wavelength of 300 nm instead of 282 nm was used because the fundamental anisotropy of the indole chromophore in a glass state reaches its highest value (r 0 ∼ 0.3) at this wavelength. 45 Determination of Partition Constants.…”