The molecular mechanism by which peptide antibiotics (also referred as cationic antimicrobial peptides-CAMPs) penetrate through the bacterial wall barrier, interact with, and disrupt their membrane is complex. It depends mainly on the peptide properties (structure, length, charge and hydrophobicity), on the characteristics of the cell wall matrix and the membrane itself.
Here, we present two fluorescence spectroscopic techniques, one for tracking the interaction of CAMPs with membranes, and the other for evaluating the ability of a peptide to cross the bacterial cell-wall and reach the membrane. The fluorescence approach is relatively simple, highly sensitive, non-invasive and allows time-scale investigation. It can be applied to lipid vesicles or intact bacteria. For membrane model systems such as liposomes, it allows to determine the binding kinetics of a peptide to vesicle and to assess the depth of penetration. By using bacterial strains carrying different mutations in their cell wall components, but not in their membrane, we can investigate how a specific element may affect the cell wall permeability to CAMPs (Saar-Dover et al., 2012).
In order to track the peptide-membrane interaction we conjugate a lipid environmentally sensitive NBD (7-nitrobenz-2-oxa-1, 3-diazole-4-yl) fluorophore to peptides. NBD fluorescence can increase up to approximately 10-fold upon interaction with membranes. Its high excitation wavelength (467 nm) and the high quantum yield reduce significantly the contribution of light scattering. NBD-labeled peptides exhibit fluorescence emission maxima around 540 nm in hydrophilic solution (Shai, 1999). However, upon interaction with lipid component such as the bacterial membrane, relocation of the NBD group into a more hydrophobic environment results in an increase in its fluorescence intensity and a blue shift of the emission maxima (Chattopadhyay and London, 1987). The first property is used to determine the binding constant of the peptide to the membrane. The second property is exploited to evaluate the depth of penetration (Merklinger et al., 2012; Zhao and Kinnunen, 2002). Here, we will focus on how to determine the binding constant. The advantage of the NBD moiety conjugation is that it allows the use of experimental conditions in which the lipid: peptide molar ratio range from < 100:1 up to > 15,000:1. The addition of NBD does not change the biological function of most of the peptide, as was found for different antimicrobial peptides such as paradaxin (Rapaport and Shai, 1992), dermaseptins (Pouny et al., 1992), cecropins (Gazit et al., 1994) and cathelicidin LL-37 (Oren et al., 1999). However, pre-examination must be done for each newly investigated peptide.
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