Location and Conformation of the LKα14 Peptide in Water/Ethanol Mixtures

Abstract

It is widely recognized that solvation is one of the major factors determining structure and functionality of proteins and long peptides, however it is a formidable challenge to address it both experimentally and computationally. For this reason, simple peptides are used to study fundamental aspects of solvation. It is well established that alcohols can change the peptide conformation and tuning of the alcohol content in solution can dramatically affect folding and, as a consequence, the function of the peptide. In this work, we focus on the leucine and lysine based LKα14 peptide designed to adopt an α-helical conformation at an apolar-polar interface. We investigate LKα14 peptide's bulk and interfacial behavior in water/ethanol mixtures combining a suite of experimental techniques (namely, circular dichroism and nuclear magnetic resonance spectroscopy for the bulk solution, surface pressure measurements and vibrational sum frequency generation spectroscopy for the air-solution interface) with molecular dynamics simulations. We observe that ethanol highly affects both the peptide location and conformation. At low ethanol content LKα14 lacks a clear secondary structure in bulk and shows a clear preference to reside at the air-solution interface. When the ethanol content in solution increases, the peptide's interfacial affinity is markedly reduced and the peptide approaches a stable α-helical conformation in bulk facilitated by the amphiphilic nature of the ethanol molecules.

Figure 1

(a) Surface pressure curves for the LKα14 peptide in different H₂O/EtOH mixtures with f_EtOH = (red) 0, (orange) 0.1, (green) 0.2, (purple) 1. The peptide was injected at t = 0. The surface pressure was set to zero before the peptide injection and the curves are vertically offset for clarity. (b) Partial densities of the LKα14 peptide and the solvent components obtained from MD simulations for f_EtOH= (top) 0, (middle) 0.45, and (bottom) 1. (c) Fraction of LKα14 peptide molecules located in (filled circles) bulk or (empty circles) at the air–solution interface for different EtOH volume fractions, f_EtOH, according to MD simulations.

Figure 2

¹H¹H-TOCSY NMR spectra showing the crosspeak region of the backbone amide protons with the H_α and side-chain protons of LKα14 (a) in fully deuterated ethanol and (b) in water (H₂O/D₂O = 9:1). (c) CD spectra for different H₂O/EtOH mixtures; the different colors indicate f_EtOH = (red) 0, (orange) 0.1, (green) 0.2, (blue) 0.5, and (purple) 1 solutions. (d) Probabilities of different secondary structural motifs in bulk solution for different mixtures as obtained from MD simulations: (green) α-helix, (blue) random coil, and (black) other structures.

Figure 3

(a) SFG spectra in the amide I region acquired after equilibration (∼30 min) following the peptide injection in different mixtures. The spectra in this region in absence of LKα14 are shown for (black dashed line) H₂O and (black dotted line) EtOH as reference. (b) Relative probability of different secondary structures (empty circles) at the interface and (filled circles) in bulk for different mixtures as obtained from MD simulations.

Figure 4

Average tilt angle between the helices and the interfacial plane in pure (a) H₂O and (b) EtOH: black lines indicate the angle while the colored lines the peptide distribution. (c) Average angle (filled circles) in bulk and (empty circles) at the interface for different f_EtOH. For a random distribution the angle averages at 45 deg.