3.3. Circular dichroism
3.3.2. Results and discussion
TrpZip 157 exhibited a unique CD spectrum in near- and far-UV due to the interactions between the Trp residues. Two aromatic rings can interact to form a chiral pair of chromophores, which can be seen in the near UV (characteristic bands are observed between 250 and 320 nm),9 which indicated that the pairs of Trp of the peptides were in a chiral environment and in a well-defined tertiary structure. It was apparent that two cross-strand pairs of Trp were stacking with each other in this peptide resulting in bands at 287 and 295 nm. Bands in this region are generally taken as evidence for presence of a fixed and stable tertiary structure in proteins.
Figure 68: Near- and far-UV CD spectra, TrpZip 157 (blue), GG-TrpZip 158 (red), (R,S)-{G∆G}-TrpZip 159 (pink), (S,R)-(R,S)-{G∆G}-TrpZip 160 (green)
The CD spectra were normalised at the most intense peak for both regions (Figure 68). The exciton bands at 214 and 228 nm were observed in the far-UV as well as the two negative bands at 287 and 295nm in the near-UV that were conserved in all the peptides. The interactions between the Trp were maintained and they were in the same environment.
-1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2
250 260 270 280 290 300 310 320
[θ] mdeg
wavelength (nm)
TrpZip GG-TrpZip
(S,R)-{GdG}-TrpZip (R,S)-{GdG}-TrpZip
-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2
185 195 205 215 225 235 245 255
[θ] mdeg
wavelength (nm)
TrpZip GG-TrpZip
(S,R)-{GdG}-TrpZip (R,S)-{GdG}-TrpZip
either by swapping one residue or by replacing a planar peptide bond by a chiral non-planar cyclopropane unit. It was clear that cross-strand Trp pairs were still interacting with each other in the analogues 158, 159 and 160. Because this kind of interaction stabilises the β-hairpin and initiates the formation of it, it was possible to state that the analogues were also forming the β-hairpin. However, it was not clear whether they are as stable as the TrpZip 157.
TrpZip peptide 157 had been reported to exhibit a reversible thermal unfolding. The CD spectra in the near-UV were recorded at 5 °C (blue), prior heating to 80 °C at which stage the peptide will be unfolded, and once it had been cooled back to 5 °C (red) (Figure 69).
a)
b)
c)
d)
Figure 69: Near UV CD of a) TrpZip 157, b) GG-TrpZip 158, c) (S,R)-{G∆G}-TrpZip 160 and d) (R,S)-{G∆G}-TrpZip 159 at 5 °C, prior prior (blue) and following heating to 80 °C and cooling
to 5 °C (red).
Following unfolding, the analogues behaved in a similar manner to the TrpZip 157 and they refolded to give exactly the same structure. The analogues all exhibited reversible thermal unfolding because the curves matched perfectly before and after heating. The near-UV CD spectrum is influenced by the behaviour of the side chains with regard to their position before and after heating. The data show that the interactions between the side chains were not modified after refolding. The analogues and the native peptide exhibited very similar stabilities in terms of their folding, which means they adopt the same preferred and stable
The evidence that the Trp side chains of the analogues were in a similar arrangement to those of the native form of TrpZip 157 prompted a more detailed study of the stability. The melting temperature (Tm) is the temperature at which 50% of the peptide is in an unfolded state. The higher it is, the greater the stability of the peptide. The data were acquired from 5 to 80 °C in increments of 5 °C for each peptide in the near UV (Figure 70). The arrow indicates the trend as a function of increasing temperature.
Figure 70: Thermal denaturation monitored by near-UV CD of TrpZip 157, GG-TrpZip 158, (S,R)-{G∆G}-TrpZip 160 and (R,S)-{G∆G}-TrpZip 159
All peptides followed the same trend and the CD intensities decreased upon heating, which indicates a secondary structural change from the hairpin to a disordered conformation by destabilising the intramolecular interactions. The data suggest a loss of the Trp cross-strand pairs interactions resulting in conformational mobility and a loss of signal. With this data it was possible to plot the thermal denaturation at a specific wavelength, which was chosen to be 295 nm (the most intense negative peak in near-UV, Figure 70), and also the fraction of peptide that is folded as function of the temperature, and thereby establish the stability of the peptides. Both sets of data demonstrated that the three analogues follow the same trend as the native peptide because the slope was similar for all peptides which indicates similar
-15
250 260 270 280 290 300 310 320
[!] mdeg
250 260 270 280 290 300 310 320
[!] mdeg
250 260 270 280 290 300 310 320
[!] mdeg
250 260 270 280 290 300 310 320
[!] mdeg
stability (and consequently Tm). The two graphs allowed the degree of folding and the Tm to be estimated.
Figure 71: Thermal denaturation at 295 nm and fraction folded as function of the temperature of TrpZip 157 (blue), GG-TrpZip 158 (red), (R,S)-{G∆G}-TrpZip 159 (pink),
(S,R)-{G∆G}-TrpZip 160 (green)
-18 -16 -14 -12 -10 -8 -6 -4 -2 0
278 288 298 308 318 328 338 348
[θ] mdeg
Temperature (K)
TrpZip GG-TrpZip
(R,S)-{GdG}-TrpZip (S,R)-{GdG}-TrpZip
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
278 288 298 308 318 328 338 348
Fraction folded
Temperature (K)
TrpZip GG-TrpZip
(R,S)-{GdG}-TrpZip (S,R)-{GdG}-TrpZip
The fraction folded at any given temperature α was calculated by use of the following (equation 1):205
𝜶 = 𝜽𝒕− 𝜽𝑼 𝜽𝑭− 𝜽𝑼
Equation 1: fraction folded equation
Where θt is the observed ellipticity at any temperature, θF is the ellipticity of the fully folded form (chosen to be that at 278K), and θU is the ellipticity of the unfolded form (chosen to be that at 353 K, the highest temperature used). Subsequently, the Tm was calculated from the graph, it corresponds to the temperature where α = 0.5. For the TrpZip native form 157, the Tm was calculated to be 316.8 K (43.7 °C) which is similar to that reported in literature.194 The GG-TrpZip analogue 158 showed slightly higher stability than the native peptide, with a Tm of 319.8 K (46.7 °C) i.e. 3 K higher than the original peptide. The Gly residue that had been used to replace the Asn residue in this analogue gave more flexibility in the turn sequence than that in the native peptide which results in less constraint in the interaction between the side chains of the peptide allowing it to adopt the most favourable and stable conformation. Both (R,S)-159 and (S,R)-160 (pink and green respectively in Figure 71) of the {G∆G} surrogate were incorporated into the peptide. The resulting analogues had similar stability with a Tm differing of less than 1.5 K (respectively 311.6 K/38.5 °C and 312.9 K/39.8 °C). Both of calculated Tm values were lower than for the native peptide and so they are slightly less stable than either the native or GG-TrpZip peptide 158. This outcome is expected because replacement of the peptide bond with a cyclopropane delivers a more constrained system in which there is likely to be a difference in the strength or in the angle of the Trp interactions because the rings don’t have the same degree of liberty to stack in the edge-to-face approach.
The results described above showed that there are stability differences between the various peptides but they adopt very similar conformations. The GG analogue 158 was found to have a higher stability than the native form, whereas the {G∆G} analogues 159 and 160 were found to be slightly less stable, suggesting a change in the interactions between the Trp, or a difference in the free energy of folding of the peptides (in ∆S or ∆H, and therefore in ∆G)
MD calculations were undertaken in order to understand the differences in the interactions (and thus the stability) and if these have an impact on the type of β-turn formed. Dr Drew Thomson has kindly provided all this data as part of a collaboration on this project.