Breaking the Backbone: Central Arginine Residues Induce Membrane Exit and Helix Distortions within a Dynamic Membrane Peptide

Abstract

Transmembrane domains of membrane proteins sometimes contain conserved charged or ionizable residues which may be essential for protein function and regulation. This work examines the molecular interactions of single Arg residues within a highly dynamic transmembrane peptide helix. To this end, we have modified the GW4,20ALP23 (acetyl-GGAW4(AL)7AW20AGA-amide) model peptide framework to incorporate Arg residues near the center of the peptide. Peptide helix formation, orientation and dynamics were analyzed by means of solid-state NMR spectroscopy to monitor specific 2H- or 15N-labeled residues. GW4,20ALP23 itself adopts a tilted orientation within lipid bilayer membranes. Nevertheless, the GW4,20ALP23 helix exhibits moderate to high dynamic averaging of NMR observables, such as 2H quadrupolar splittings or 15N–1H dipolar couplings, due to competition between the interfacial Trp residues on opposing helix faces. Here we examine how the helix dynamics are impacted by the introduction of a single Arg residue at position 12 or 14. Residue R14 restricts the helix to low dynamic averaging and a well-defined tilt that varies inversely with the lipid bilayer thickness. To compensate for the dominance of R14, the competing Trp residues cause partial unwinding of the helix at the C-terminal. By contrast, R12GW4,20ALP23 exits the DOPC bilayer to an interfacial surface-bound location. Interestingly, multiple orientations are exhibited by a single residue, Ala-9. Quadrupolar splittings generated by 2H-labeled residues A3, A5, A7, and A9 do not fit to the α-helical quadrupolar wave plot defined by residues A11, A13, A15, A17, A19, and A21. The discontinuity at residue A9 implicates a helical swivel distortion and an apparent 310-helix involving the N-terminal residues preceding A11. These molecular features suggest that, while arginine residues are prominent factors controlling transmembrane helix dynamics, the influence of interfacial tryptophan residues cannot be ignored.

Flanking aromatic residue competition influences transmembrane peptide helix dynamics

“…The 2 H Ala-quadrupolar splittings were analyzed using a semistatic 'geometric analysis of labeled alanines' ('GALA') method [20], which generates a quadrupolar wave based on the peptide's given orientation with regard to its apparent tilt (s 0 ) from the bilayer normal, apparent rotation (q 0 ) about the helix axis, and an isotropic order parameter (S zz ), as described in detail previously [32]. The experimental 2 H quadrupolar splittings are compared to those calculated by the analysis for each Ala residue in the sequence to determine the average helix orientation with the lowest root mean square deviation (RMSD).…”

“…A value of S zz = 0.88 should therefore be considered an upper limit for an isolated well‐order transmembrane helix. Among the other motions, the extent of rotational averaging about the helix axis has been found to be much more important than the extent of helix ‘wobble’ for distinguishing highly dynamic from minimally dynamic helices [21,22], a finding which has been convincingly confirmed [17,29–32]. In this framework, many transmembrane helices have shown a small wobble στ of about 5°–10°.…”

Thermodynamics-Based Molecular Modeling of α-Helices in Membranes and Micelles

“…At the next step of verification, we compared FMAP 2.0 predictions of TM and non-TM peptide arrangements in lipid bilayers with published experimental data. The test set 6 included synthetic pH-triggered membrane peptides with ionizable residues within hydrophobic α-helices studied by solid-state NMR, − ATR-FTIR spectroscopy, and OCD ,− at different pH values (50 data points for 32 peptides). These peptides were designed to examine the pH-dependent equilibrium between membrane-spanning TM α-helices and surface-bound non-TM states in model PC bilayers .…”

De Novo Designed Hexadecapeptides Synergize Glycopeptide Antibiotics Vancomycin and Teicoplanin against Pathogenic Klebsiella pneumoniae via Disruption of Cell Permeability and Potential

“…In order to improve bioactivity, we further extended the amino acid sequences to 16 residues by introducing more arginine. Arginine has a high frequency of occurrence in antimicrobial peptide sequences because it provides a positive charge like lysine in a neutral environment, therefore enhancing the affinity of peptide molecules to a negatively charged membrane surface. , The cross-arrangement of basic amino acids and hydrophobic amino acids formed the articulated skeleton of our synthetic peptides. Compared to our reported peptide library, these newly designed compounds have a more net positive charge (from +8 to +10), theoretically endowing them a stronger electrostatic attraction with bacterial membrane.…”