Large scale conformational transitions in β‐structural motif of gramicidin A: kinetic analysis based on CD and FT‐IR data

Abstract

Gramicidin A (gA) is a polypeptide antibiotic, which forms dimeric channels specific for monovalent cations in artificial and biological membranes. It is a polymorphic molecule that adopts a unique variety of helical conformations, including antiparallel double-stranded ↑↓β5.6 or ↑↓β7.2 helices (number of residues per turn) and a single-stranded β6.3 helix (the 'channel form'). The behavior of gA-Cs(+) complex in the micelles of TX-100 was studied in this work. Transfer of the complex into the micelles activates a cascade of sequential conformational transitions monitored by CD and FT-IR spectroscopy: [Formula: see text] At the first step after Cs(+) removal, the RH ↑↓β5.6 helix is formed, which has been discussed so far only hypothetically. Kinetics of the transitions was measured, and the activation parameters were determined. The activation energies of the ↑↓β5.6 → β-helical monomer transition in dioxane and dioxane/water solutions were also measured for comparison. The presence of water raises the transition rate constant ~10(3) times but does not lead to crucial fall of the activation energy. All activation energies were found in the 20-25 kcal/mol range, i.e. much lower than would be expected for unwinding of the double helix (when 28 H-bonds are broken simultaneously). These results can be accounted for in the light of local unfolding (or 'cracking') model for large scale conformational transitions developed by the P. G.Wolynes team [Miyashita O, Onuchic JN, Wolynes PG. Proc. Natl. Acad. Sci. USA 2003; 100: 12570-12575.].

Distinguishing gramicidin D conformers through two-dimensional infrared spectroscopy of vibrational excitons

“…These preparations were adapted from literature preparations 14,16 to be compatible with IR spectroscopy, specifically, increasing the concentration of peptide and preparing the samples in deuterated solvents.…”

“…Both the SDS and Triton X-100 solubilized samples were heated for 6 h at 50 • C. This is necessary for the Triton X-100 sample to ensure the DH form is entirely in the left-handed β helix with 5.5 residues per turn, as several conformations are initially formed. 16 The sample should be completely in the left-handed 5.5 residues per turn conformer after 1 h. The SDS sample was heated only so both samples were treated consistently.…”

“…They are the Helical Dimer (HD), a headto-head dimer composed of two β helices which have parallel β-sheet contacts in the monomer unit 14 and antiparallel β-sheet contacts at the interface, and the Dimeric Helix (DH), a doublestranded helix with antiparallel β-sheet hydrogen bonding contacts. 15 Though these structures can be distinguished with Circular Dichroism (CD) spectroscopy, 16 it has also been observed that differences in the amide I (carbonyl) stretch infrared (IR) spectra can distinguish between the different conformations. 12,17 This offers the possibility of studying gramicidin D in a diverse range of lipid structures, including those where artifacts arising from scattering in CD are known.…”

Rational Design, Synthesis, and Biological Evaluation of Lactam‐Bridged Gramicidin A Analogues: Discovery of a Low‐Hemolytic Antibacterial Peptide

“…[19] In both membrane systems, the natural product GA provided two positive peaks, one around 220 nm and the other around 240 nm, which are known to arise from the b 6.3 -helical structure (Figure 2 a,b). [21] Notably, the difference of one methylene unit between 3 and 4 had a large impact on the organization of the three-dimensional structures of these molecules. In contrast, the positive peaks at 220 and 240 nm were enhanced in the spectra of macrolactam 3 in both membrane systems (Figure 2 c,d).…”

“…These data suggest that 3 adopts the stable b 6.3 -helical conformation, whereas the b 6.3 helix of 4 is either somewhat distorted or is in equilibrium with other conformations. [21] Notably, the difference of one methylene unit between 3 and 4 had a large impact on the organization of the three-dimensional structures of these molecules.…”