In order to investigate the influence of an artificial membrane skeleton model system on the structure of lipid bilayers, interactions between the model system monomers among each other and attachment to the lipid bilayer surface have to be validated first. Therefore, a wide assortment of methods has been established in the last decades adjusted to the respective experimental requirements. In the following, the spectroscopic methods applied in this thesis will be elucidated.
Temperature-dependent UV (ultra violet) spectroscopy is an invaluable tool for the assessment of nucleobase pairing stability and was initially established for studying DNA and RNA duplex formations.[124,125] Subsequently, this method was also successfully ap-plied for the investigation of PNA/DNA as well as PNA/PNA duplex formation.[126–128]
Nucleobases absorb in a wavelength range of 240–280 nm and during duplex formation of complementary strands, parallel alignment conveyed by hydrogen bonds between the nucleobases occurs. The resultant conformational immobilization and proximity of the aromatic heterocyclic moieties also lead to interjacent hydrophobic π-π-stacking interac-tions. This base stacking decreases the absorbance of the nucleobases and therefore, an increase of absorbance is observed when the strands are separated by heating and destack-ing of the nucleobases occus, which is denoted as hyperchromicity. The sigmoidal curve shape of the resultant temperature-dependent UV absorption plots indicates cooperative dissociation. The temperature at the inflection point is denoted as the melting temper-ature Tm at which 50 % of the duplex is dissociated.[121] Base stacking has also been found in β-PNA duplexes as indicated by the reported melting curves.[24–26] Therefore, with the nucleobases already preorganized in a linear fashion, a tilted structure has been postulated since a distance of about 3.4 Å is needed for efficient base stacking whereas the helical pitch of the 14-helix has been shown to be 5 Å.[117,129]
Circular dichroism (CD) spectroscopy is a well established method for the analysis of secondary structures of peptides as well as oligonucleotides. In principle, the differential absorption of left- and right-handed circularly polarized light by a conformationally fixed chiral chromophore is detected.[130–133] As already stated in Section 2.3, this method has been employed to thoroughly characterize the varying helical conformations of β -peptides.[134,135] Similar to typical α-peptide secondary structures, the β-helices exhibit distinct CD curve shapes in the far UV wavelength range. In the case of a left-handed 14-helix, a global minimum of around 215 nm, a global maximum of around 195 nm as well as a zero crossing between 200 nm and 205 nm can be observed, and in the case of a right-handed 14-helix, the CD curve shape is mirrored horizontally.[23,96,129] For single
24
2.5. Investigation of Interactions
β-PNA strands, the presence of an additional CD signal at the nucleobase absorption band with a maximum of 270 nm due to potential conformational preorganization of the nucleobases was detected.[24–26] Moreover, the relative increase of this maximum upon dimer formation offers the possibility to apply temperature-dependent CD spectroscopy as an alternative method for duplex stability determination.[121,136,137]
Fluorescence spectroscopy is a highly sensitive and straightforward method suitable for a multitude of applications such as colocalization studies, examining fluorescence sensors and interaction studies.[138] Often observed for molecules with expanded aromatic systems (fluorophores), fluorescence is the emission of light hνF when electrons of a fluorophore transition from the electronically excited state S1 to the electronic ground state S0 (Fig-ure 2.19). Prior to this, the electrons are excited from the ground state S0 to a higher vibrational electronic level S1∗ or S2 by the absorption of light hνA followed by internal conversion and vibrational relaxation to the lowest vibrational level in the electronically exited state S1.[138] These relaxation processes lead to a decreased energy of the electrons.
As a consequence, the emitted light is shifted to longer wavelengths in relation to the wavelength of the absorbed light which is denoted as the Stokes shift. Apart from in-ternal conversions in the excited state or vibrational relaxation, the solvent polarity can have an effect on the emission properties. As depicted in Figure 2.19 the excited S1 state causes a change in the electric dipole moment of the fluorophore which causes the solvent dipoles to rearrange before the fluorescent light is emitted. The solvent rearrangement, also termed solvent relaxation, results in a decreased energy level of the fluorophore. This solvatochromic effect can be highly pronounced increasing with higher solvent polarity rendering the corresponding fluorophores environment-sensitive probes e.g. for structural changes of biomolecules, incorporation of transmembrane peptides into lipid bilayers or localization of molecules at the water/lipid interface of membranes.[139–141]
Förster resonance energy transfer (FRET) is a distance-dependent nonradiative en-ergy transfer between two fluorophores, one denoted as the donor and the second as the acceptor. Instead of fluorescence emission, the energy of the donor in state S1 is transferred to an adjacent acceptor by dipole interactions. Therefore, emission of the acceptor can be observed upon excitation of the donor fluorophore whose fluorescence is quenched.[33,138,142] The FRET efficiency EFRET is inversely proportional to the donor-acceptor distance r to the power of six:
EFRET = 1 1 + (Rr
0)6 (2.2)
2. Membrane-Associated Protein Networks & Model Systems
Figure 2.19. Jablonski diagram of a solvatochromic fluorophore (green oval) whose elec-trons are first elevated from the ground state S0 to an excited electronic state S1 by the absorption of light (hνA) leading to an altered dipole mo-ment indicated by the black arrow in the green oval. Subsequently, the sur-rounding solvent molecules reorient according to the changed fluorophore dipole moment in a solvent relaxation process and the S1 energy level is lowered resulting in a red shift of the emitted light (hνF). Reprinted from Trends in biotechnology, 28, G. S. Loving, M. Sainlos, B. Imperiali, Moni-toring protein interactions and dynamics with solvatochromic fluorophores.
73-83, Copyright (2010), with permission from Elsevier.[140]
26
2.5. Investigation of Interactions withR0 being theFörster radius defined as the distance between donor and acceptor at whichEFRET is 50 %. As illustrated in Figure 2.20(a), a steep decrease ofEFRET occurs with increasing distance and therefore, FRET intensity can be employed as a molecular ruler for a distance range of (0.5–2)R0. The Förster radius in turn is dependent on the overlap of donor emission and acceptor excitation spectra (Figure 2.20(b)).[142,143] Be-cause the emission and excitation spectra are fluorophore-specific properties, R0 is a spe-cific value for every donor-acceptor pair. For the donor-acceptor pairs 7-nitrobenz-2-oxa-1,3-diazol-4-yl /5(6)-carboxytetramethylrhodamine (NBD/TAMRA) and NBD/lissamine rhodamine B (NBD/Rhod), which are commmonly selected combinations for membrane associated FRET experiments, R0 values of 5.1 nm and 4.9 nm could be determined, respectively.[144–146]
EFRET
0 0.2 0.4 0.6 0.8 1
r / R0
0 0.5 1 1.5 2 2.5
F1
a)
donor emission acceptor absorption
wavelength
2F1 F22 2
b)
Donor Acceptor
Figure 2.20. Illustration of EFRET as a function of the distance r/R0 with the range of (0.5–2)R0marked in grey (a) as well as a schematic depiction of the spectral overlap shaded in grey between donor emission and acceptor excitation spectra required for FRET (b).[142]
With these tools in hand, the interactions that are required for the designed model system, specifically theβ-PNA/β-PNA interactions as well as β-peptide/membrane inter-actions, can be investigated.