The first group of the peptides represent imino-rich triplets within the native sequence, peptides P1-P3. The peptide P1 is a simple GPO repeat, with isotopic enrichment of Gly and Pro in the middle of the sequence, as highlighted in Table 6.1. This enrichment will show environments and dynamics of a very well-ordered triple helix. Peptides P2 and P3 are identical with hydroxyproline in the eighteen-position substituted by Pro in all three polypeptide chains, giving a GPP insertion in the middle of the sequence. This change was introduced partly due to challenges associated with obtaining isotopic enrichment of Hyp. X-Ray diffraction structures of GPO and GPP-rich synthetic model collagen peptides suggest that
114 changing a single O for P should have at most only a very small effect on the triple helix conformation (PDB: 1V4F25 and 1A3J195).
The two-dimensional 15N chemical shift – T1 relaxation time correlation contour plots of the P-type peptides were obtained for all imino rich peptides that were further used to extract the relaxation constants. In Figure 6.2. (a) only plots for measurements performed at 290 K are shown as an example; the extracted slices from the ILT contour plot at the highlighted chemical shifts are shown in Figure 6.2. (b).
In the case of the proline 15N relaxation, it was not possible to record spectra at sufficiently long 𝜏 delays to obtain full decay of the signal (Appendix B). In all experiments (at all temperatures) the signal intensity of the proline 15N decayed to half the initial intensity, and glycine 15N to almost zero. The longest relaxation delay used in the measurements was 120 s. If there are components that have significantly longer relaxation constants than 120 s, these will not appear in the T1 relaxation spectra. Similarly, the T1 spectra will not show components that have faster relaxation than the shortest 𝜏 delay used, which was 0.0001s; however, that would imply much faster relaxation than is expected, so this is not a big restriction. The 1H relaxation in these samples is of the order of tenths of seconds, so a recycle delay of 2 s was used in all experiments between scans (i.e. 5 times larger than the proton T1 relaxation).
115
Figure 6.2. (a) Contour plots of relaxation time constant T1 versus 15N chemical shift of peptides (i) P1,
(ii) P2 and (iii) P3 at 290 K. Above each contour plot, 1D 15N NMR spectra of the corresponding peptide
are given. Signals that are not corresponding to the chemical shifts for the collagen triple helix are crossed out and are not considered for 15N T1 analysis. (b) Slice from the contour plot extracted from
highlighted cross-section with the dashes in (a); proline (left) and glycine signals (right) compared.
The contour plots show that there is a distribution of T1 values across each 15N signal, that looks like broadening and stretching of the 2D signals. The broadening in the T1 dimension can be a result of a sub-optimal S/N ratio in the 15N chemical shift spectra or it can reflect a real distribution of the T1 values, i.e. T1 depends on the isotropic 15N chemical shift. The distribution of the relaxation constant values is highly plausible because these samples are lyophilised, and they will show a distribution of the 15N chemical shifts; i.e. line-broadening of the 15N signals for glycine and proline in the chemical shift dimension likely reflect slightly different molecular conformations which may in principle be associated with different relaxation times. However, it is not possible to conclude from the ILT contour plots whether the T1 distributions are due to heterogeneity or an artefact from poor S/N ratio. Furthermore, the artefacts could arise due to the proton spin diffusion that affects the 15N signal intensity
116 from the initial NMR experiment (Section 6.3.1.). Thus, the weighted average of the resulting distributions of the 15N T1 relaxation constants of each separate chemical shift signal (per residue) from the ILT contour plot were calculated and summarised in Table 6.2.
In the contour plots shown in Figure 6.2., there are signals observed with chemical shifts at 100 and 134 ppm, which cannot be assigned to glycine or proline residues being in a triple helical structure, respectively. These signals are crossed out in the contour plots and are not considered for the relaxation of the triple helical peptides. These signals could be either artefacts due to poor S/N ratio, or denatured polypeptide chains. Furthermore, the 15N T1 relaxation experiments were performed three times in total, and only consistent signals were reported and analysed.
Table 6.2. The table below shows 15N T1 relaxation constants estimated at temperatures of 250, 270,
290, 300 and 310 K for glycine and proline residues in P-type peptides after ILT processing. Errors of T1 are taken as a half-width of the signal intensity for the ILT calculated relaxation constants. At some
temperatures, the nitrogen nuclei in the glycine residues show two separate signals with significant intensities.
P1: (GPO)5GPO(GPO)5
P2: (GPO)5GPPGPO(GPO)4
P3: (GPO)5GPPGPO(GPO)4
Peptide and Residue iso / ppm 250 K 270 K 290 K 300 K 310 K
P1 Pro 129 190 ± 17 167± 16 182 ± 34 124 ± 14 87 ± 8 Gly 106 95 ± 11 81 ± 9 71 ± 10 47 ± 6 43 ± 7 P2 Pro 131 391 ± 83 190± 18 180 ± 18 198 ± 18 167± 16 Gly 105 - - - - 100 ± 11 104 74 ± 8 43 ± 5 68 ± 9 67.9 ± 10 14 ± 2 P3 Pro 129 157 ± 30 130 ± 11 136 ± 11 121 ± 14 100.0 ± 9.0 Gly 106 19 ± 3 - 15 ± 2 - - 105 68 ± 21 42 ± 9 65 ± 10 51 ± 8 44 ± 9 103 - - - 35 ± 5 -
The most apparent feature from the relaxation data of imino-rich peptides is that the T1 constants are very different for the nitrogen nuclei in proline and glycine residues, with glycine showing faster relaxation compared to proline. This is expected due to the protonation on the glycine nitrogen that introduces much stronger 1H-15N heteronuclear dipolar coupling that helps to drive the relaxation. Furthermore, a bimodal distribution of the relaxation time constants is observed for glycine residues in peptides P2 and P3. However, this behaviour is not consistent for all temperatures.
Comparing the T1 constants across all P-peptide samples there are constant patterns observed. Overall, 15N T1 values decrease with increasing temperature. However, there are outliers observed for glycine nitrogen in peptides P2 and P3 between temperatures of 250 and
117 290 K. The T1 value is significantly lower at 270 K than at 250K and 290K, around the freezing temperature of water. The same change in T1 at ca. 270 K is also observed for the proline nitrogen in the peptide P1. These abrupt changes in the T1 around the water freezing temperatures will be addressed further in Section 6.5. Significant differences of T1 values between peptides P1 and P3 are observed at low temperatures, below 290 K. In these two peptides the isotopic enrichment is the same, however, peptide P3 contains a GPP insertion. The difference in the relaxation constants between P1 and P3 suggests that there might be a difference in the molecular motion for the two peptides. They are expected to have highly similar molecular structures and therefore, nuclear spin interaction strengths for their respective 15N. The key differences in molecular motion or possibly the structure of water around the peptides can account for differences in 15N T1. This will be discussed more fully below.