Dynamical structure of the short multifunctional peptide BP100 in membranes

Abstract

BP100 is a multifunctional membrane-active peptide of only 11 amino acids, with a high antimicrobial activity, an efficient cell-penetrating ability, and low hemolytic side-effects. It forms an amphiphilic α-helix, similar to other antimicrobial peptides like magainin. However, BP100 is very short and thus unlikely to form membrane-spanning pores as proposed for longer peptides as a mechanism of action. We thus studied the conformation, membrane alignment and dynamical behavior of BP100 in lipid bilayers (DMPC/DMPG), using oriented circular dichroism (OCD) and solid-state (19)F and (15)N NMR. According to OCD and (15)N NMR, the BP100 helix is oriented roughly parallel to the membrane surface, but these methods yield no information on the azimuthal alignment angle or the dynamics of the molecule. To address these questions, a systematic (19)F NMR analysis was performed, which was not straightforward for this short peptide. Only a limited number of positions could be (19)F-labeled, all of which are located on one face of the helix, which was found to lead to artifacts in the data analysis. It was nevertheless possible to reconcile the (19)F NMR data with the OCD and (15)N NMR data by using an advanced dynamical model, in which peptide mobility is described by fluctuating tilt and azimuthal angles with Gaussian distributions. (19)F NMR thus confirmed the regular α-helical conformation of BP100, revealed its azimuthal angle, and described its high mobility in the membrane. Furthermore, the very sensitive (19)F NMR experiments showed that the alignment of BP100 does not vary with peptide concentration over a peptide-to-lipid molar ratio from 1:10 to 1:3000.

Membrane Thinning and Thickening Induced by Membrane-Active Amphipathic Peptides

“…Furthermore, both methods are compatible with oriented bilayer samples, which were used for three reasons. Firstly, this way the results of the present study can be directly related with our previous solid-state NMR measurements of the orientation of the selected peptides in mechanically oriented membranes (Salgado et al, 2001; Glaser et al, 2005; Grage et al, 2006; Tremouilhac et al, 2006; Afonin et al, 2008a,b, 2014; Strandberg et al, 2013; Wadhwani et al, 2014; Misiewicz et al, 2015a,b). Secondly, hydrated oriented samples provide a well-defined bilayer structure and morphology, with a uniform environment for the peptides.…”

“…The peptides PGLa (GMASKAGAIAGKIAKVALKAL-amide), Mag2 (GIGKFLHSA KKFGKAFVGEIMNS), BP100 (KKLFKKILKYL-amide), and TisB (MNLVDIAILILK LIVAALQLLDAVLKYLK) were synthesized using standard solid phase Fmoc protocols, and purified with reverse-phase chromatography (HPLC), as described earlier (Steinbrecher et al, 2012; Wadhwani et al, 2014; Strandberg et al, 2015). GS (cyclo-[PVOLf] 2 ) was produced by fermentation of A. migulanus , and extracted and purified with HPLC, as outlined earlier (Berditsch et al, 2015).…”

“…BP100 was originally developed as an antibacterial agent against plant microbes (Badosa et al, 2007) but is also a very effective cell penetrating agent (Eggenberger et al, 2011), forming a short regular α-helix when bound to the membrane (Torcato et al, 2013). Both GS and BP100 bind predominantly in an S-state alignment and possess a relatively high mobility as a consequence of their small size and compact shape (Salgado et al, 2001; Wadhwani et al, 2014; Zamora-Carreras et al, 2016). GS exhibits a low propensity for flipping into the membrane at high peptide:lipid ratio to form a putative oligomeric pore, which is only observed near the lipid phase transition temperature (Afonin et al, 2008b, 2014), whereas BP100 does not seem to undergo any concentration-dependent flip in its alignment (Misiewicz et al, 2015a).…”

Structural characterization of a C-terminally truncated E5 oncoprotein from papillomavirus in lipid bilayers

“…OCD is a powerful tool for studying the orientation of membrane-active peptides and protein segments in membranes (Lange et al, 2007;Bürck et al, 2008;Strandberg et al, 2008;Wadhwani et al, 2008Wadhwani et al, , 2012Wadhwani et al, , 2014Nolandt et al, 2009;Windisch et al, 2010;Heinzmann et al, 2011;Klein et al, 2012;Muhle-Goll et al, 2012;Steinbrecher et al, 2012;Aberle et al, 2014;Fanghänel et al, 2014). For α-helices, the OCD band at 208 nm can be used to estimate the helix tilt angle relative to the membrane normal (Wu et al, 1990).…”

Structure Analysis and Conformational Transitions of the Cell Penetrating Peptide Transportan 10 in the Membrane-Bound State

“…The L - enantiomer has been recently established as a selective NMR label in studies of membrane-bound peptides with simple α-helical or β-pleated conformations [40], [52], [54], [63], [64]. By incorporating D - amino acids into peptides, it has furthermore been possible to modulate their ability to aggregate as β-sheets [39], [65], [66].…”