Fluorescence Technologies for Monitoring Interactions Between Biological Molecules In Vitro - Progress in Molecular Biology and Translational Science - page 134

in a single fashion. Moreover, with regard to biological diversity, cellular
membrane composition depends on the cellular type and can vary with
the environment or with the physiological state. 81 Conception of a universal
cellular membrane model is consequently too complicated. In order to cir-
cumvent this limitation, step-by-step approaches have been developed with
artificial membranes. Liposomes, that is, small, large, and giant unilamellar
vesicles (SUV, LUV, and GUV), have been developed to provide reliable
models. 82 The combination of these models with fluorescent probes enables
accurate monitoring of molecule/membrane interactions.
The first basic approach to investigate membrane insertion by fluores-
cence consists in monitoring the intrinsic tryptophan fluorescence of pep-
tides or proteins in the presence of liposomes. The effect of membranes
on the fluorescence emission of tryptophan provides information on the
protein or peptide localization in the phospholipid bilayer by sensing envi-
ronmental changes. Among the water-soluble membrane-active proteins,
the channel-forming family of Colicins has been largely studied. These pro-
teins are able to spontaneously insert into negatively charged membranes
from aqueous media at low pH. A lack of change in the tryptophan fluores-
cence spectrum upon insertion indicates that their hydrophobic environ-
ment is preserved while becoming accessible to lipids, suggesting that
membrane insertion only induces modifications in the relative positioning
of the helices. 83 In contrast, monitoring fluorescence of four tryptophans
of phospholipases indicates a change in the environment of one or more
tryptophan residues, suggesting enzyme/membrane interactions. 84 How-
ever, the solvatochromism of tryptophan was mainly studied for
membrane-active peptides. 59,85-88 For example, comparison of the
membrane insertion of two analogous carrier peptides enabled the
identification of slightly different behaviors ( Fig. 4.14 ). The liposome
effect on tryptophan fluorescence of the CPP MPG- a and MPG- b was
compared. Depending on the nature of the phospholipid, headgroups, that is,
neutral or negatively charged, different results were found. Addition of
negatively charged phospholipids to a solution of MPG- b promotes a blue
shift with maximum fluorescence (from 348 to 328 nm) associated with an
enhancement of fluorescence intensity (about twofold). This behavior is
characteristic of a tryptophan moving from a polar to a nonpolar
environment. On the contrary, neutral lipids containing vesicles do not
induce any modification of the fluorescence spectrum, suggesting no
insertion of peptides into neutral phospholipid bilayers. For MPG- a ,while
negatively charged vesicles induce a similar blue shift, neutral lipids promote