Simultaneous Analysis of Secondary Structure and Light Scattering from Circular Dichroism Titrations: Application to Vectofusin-1.

Louic S Vermeer,Burkhard Bechinger,David Fenard,Anne Galy,Michel Schoup,Arnaud Marquette

doi:10.1038/srep39450

Abstract

Circular Dichroism data are often decomposed into their constituent spectra to quantify the secondary structure of peptides or proteins but the estimation of the secondary structure content fails when light scattering leads to spectral distortion. If peptide-induced liposome self-association occurs, subtracting control curves cannot correct for this. We show that if the cause of the light scattering is independent from the peptide structural changes, the CD spectra can be corrected using principal component analysis (PCA). The light scattering itself is analysed and found to be in good agreement with backscattering experiments. This method therefore allows to simultaneously follow structural changes related to peptide-liposome binding as well as peptide induced liposome self-association. We apply this method to study the structural changes and liposome binding of vectofusin-1, a transduction enhancing peptide used in lentivirus based gene therapy. Vectofusin-1 binds to POPC/POPS liposomes, causing a reversal of the negative liposome charge at high peptide concentrations. When the peptide charges exactly neutralise the lipid charges on both leaflets reversible liposome self-association occurs. These results are in good agreement with biological observations and provide further insight into the conditions required for efficent transduction enhancement.

Highlights

Peptide-induced liposome self-association which is in good agreement with biological observations on this transduction enhancing peptide used in gene therapy[15,16]
Because the peptide structural changes have no effect on the signal intensity at this wavelength, it depends on the light scattering only
principal component analysis (PCA) and singular value decomposition (SVD) have been used extensively to decompose CD spectra into their structural constituents[6] and are well established methods for the analysis of spectroscopic data[14], but we found no reports where PCA or other decomposition methods are used to correct for light scattering

Summary

Introduction

Peptide-induced liposome self-association which is in good agreement with biological observations on this transduction enhancing peptide used in gene therapy[15,16]. Besides the case demonstrated here we have observed distortions in CD spectra due to light scattering in a multitude of studies and expect spectral decomposition for analysis and removal of light scattering to be widely applicable, for example in studies of antimicrobial peptides interacting with liposomes[17] or entire bacteria[18], studies of peptide self-association in the presence of phosphate or other divalent ions[19], investigations of nucleic-acid delivery peptides that cause DNA condensation[20], the interaction of transduction enhancing peptides with liposomes or intact viruses, the characterisation and screening of peptide and protein samples for x-ray crystallography, and the optimisation of bicelle and nanodisc samples for NMR spectroscopy. Studies of liposome-induced fibril formation by amyloid and amyloid-like proteins and peptides may benefit, as long as the light scattering is not correlated with the structural changes

Methods

Results

Discussion

Conclusion