Distance measurements by fluorescence quenching

The results of the fluorescence measurements (see Appendix C) confirmed the incorporation of the synthesised β-peptides in the model membranes. To receive more detailed information, further methods have to be performed. Based on the tryptophan fluorescence and the interaction with other molecules the penetration depth of tryptophan can be calculated.^[22–25]

The basis for the penetration depth of tryptophan is the fluorescence interference by quenchers. Quencher molecules do not influence the absorption behaviour of a fluorophore, though the quantum yield will be affected.^[114,115] The modified fluorescence behaviour is induced by a spin-orbit interaction what results in a (S1→T1)-crossing (intersystem crossing, ISC, see Figure 3.1). This transition is reflected by a nonradiative transfer of energy from the fluorophore to a quencher molecule.[24,114,115,163]

For this transfer it is necessary that the molecules are located close to each other. Depending on the type of quenching mechanism it can be distinguished between static and dynamic fluorescence quenching.[24,114,115,163]

In the following, a static fluorescence quenching for the transmembrane peptide-bounded tryptophan mechanism is postulated.^[20] In the static fluorescence quenching mechanism the fluorophore and the quencher form a complex [FQ]

which absorbs the light radiation (h) like a quencher free fluorophore. The excited state [FQ]^* relaxes in contrast to the quencher free fluorophore without fluorescence but through the emission of heat (). Based on this concept by increasing the amount of quencher, the number of available fluorophore will be decreased. In Equation 3.1, the processes for a quencher free process is given, whereas in Equation 3.2 the process for the static fluorescence quenching mechanism is displayed.[24,114,115,163]

3.1

3.2

Due to the peptide-bound position of the tryptophan, the imposed requirements on the quencher are versatile. Requirements of the quencher molecules are membrane-permeability, a definite distance to the centre of the membrane and no change of the membrane properties. Here, especially lipid-bound quencher molecules have proven to be useful. As quencher unit, bromine^[23] atoms or DOXYL^[20] groups are incorporated into the acyl chains.

29 In the following, the radical DOXYL was used as static quencher molecule for tryptophan.

DOXYL can be covalently bound at defined positions of steric acid or 1-palmitoyl-2-stearoyl-(X-DOXYL)-sn-glycero-3-phosphocholine (16:0-X DOXYL PC) lipids (see Figure 3.9).^[164]

Figure 3.9 1-Palmitoyl-2-stearoyl-(5-doxyl)-sn-glycero-3-phosphocholine (16:0-5 DOXYL PC).

Because of the quencher’s specific localisation in the lipid membrane, it is possible to establish a distance correlation between the quencher and fluorophore. This method is called the parallax method. By the variation of the quencher mole fraction DOXYL and the resulting change in florescence intensity, the position of tryptophan can be calculated using the two-dimensional Perrin equation (see Equation 3.3).[24,114,115,163]

The critical quenching radius R₀ and lateral distance between fluorophore and quencher X is presumed as a constant for the parallax calculation. The distance of the fluorophore and quencher is described by Z (see Figure 3.10). For the graphic analysis, the natural logarithm of tryptophan fluorescence in the present of quencher (I) divided by tryptophan fluorescence in the absence of quencher (I0) was plotted against DOXYL. The resulting slope of the straight line is in direct proportion to . The comparison of different graphic analysis with various DOXYL-labelled lipids has a known distance to the lipid bilayer centre (Q) and allows the calculation of the distance of tryptophan to the lipid bilayer centre (P).

Figure 3.10 Graphical visualisation of the geometric parameters which are necessary for the calculation of the distance Trp/lipid bilayer centre (P): lateral distance between fluorophore and quencher (X), distance of the fluorophore and quencher (Z), distance of the quencher and lipid bilayer centre (Q).^[20] Adapted with permission from L. A. Chung et al, Biochemistry 1992, 31, 6608–6616. Copyright (1992) American Chemical Society.

3.3

DOXYL Trp

lipid bilayer centre

Q P

Z

X

30

Measurements

For the parallax measurements different LUV compositions were prepared. The used films were perpetrated with a constant lipid concentration (n = 0.3 · 10^-6 mol) of DOPC or a combination of DOPC and the corresponding DOXYL-labelled lipid. As DOXYL-labelled lipid 16:0-10 DOXYL PC and 16:0-16 DOXYL PC were used. The peptide/lipid concentration was 1:200 and the vesicles were rehydrated with ultrapure water.

In Figure 3.11 a section of the results from the parallax measurement of Pep 1 with 16:0-16 DOXYL PC (full results in Appendix C) is illustrated. The normalised fluorescents spectra show a strong dependence of the fluorescence intensity to the mole fraction of the quencher molecule DOXYL. Due to the spatial proximity, this behaviour was expected. The graphic display of ln(I/I0) against DOXYL creates a balance line with a negative slope. In a comparative manner to Figure 3.11, Figure 3.12 shows the correlation of Pep 1 and 16:0-10 DOXYL PC (full results in Appendix C). Here, the fluorescence intensity increased in small mole fraction of the 16:0-10 DOXYL PC. This behaviour was also observed during several measurements under the same conditions. Therefore, it is not possible to draw a balance line with a negative slope, which would be necessary for a meaningful evaluation of the distance measurements.

a) b) lipid/quencher ratio. b) Fluorescence depth quenching curves derived from the corresponding fluorescence spectra (LUVs, P/L-ratio 1:200, T = 25 °C).

The measured peptides Pep 1 and Pep 2 display in general an expected course for the different DOXYL-labelled lipids (see Appendix C). Peptide Pep 1 with the central tryptophan tends to show a stronger quenching effect at 16:0-10 DOXYL PC and 16:0-16 DOXYL PC than peptide Pep 2, with the peripheral tryptophan. Furthermore, the quenching effect from Pep 1 at higher molar fractions DOXYL of 16:0-16 DOXYL PC is greater than at 16:0-10 DOXYL PC. However, the measurements indicated fluorescence

31 properties of peptides as described, which were unexpected. Based on these results, various lipid-peptide interactions might be responsible. An influence of the fluorophore or the fluorophore quencher complexes [FQ] is possible, which would result in changed fluorescence behaviour. It is known that fluorescence parameters, like wavelength maximum, fluorescence intensity, fluorescence lifetimes and band shape are influenced by the fluorophore’s environment.[16–19,165] lipid/quencher ratio. b) Fluorescence depth quenching curves derived from the corresponding fluorescence spectra (LUVs, P/L-ratio 1:200, T = 25 °C).

Possible reasons for this could be peptide aggregations or other lipid-peptide interactions (see Figure 2.6). A further source of error in this measurement might be the vesicle preparation. Although the vesicles were made several times, it could be that the incorporation of 16:0-10 DOXYL PC has led to a modified peptide insertion.

Based on results of ROST et al. with comparable peptides, where a heavy atom labelled peptide was incorporated into the bilayer and the exact position could been proven by X-ray measurements,^[38] it is known that the synthesised peptides are transmembrane peptides. However, in this measurement the labelling with non-commercially available heavy atoms is necessary. The parallax method can be carried out with natural amino acids or with commercially available fluorophores. In further parallax measurements, a change in the experimental setup, such as a subsequent insertion of the peptides in the membrane, should be done.

32

3.3 Cyanide-labelled β³-amino acid as molecular sensor for surface-enhanced