CAPÍTULO IV: ANÁLISIS DE RESULTADOS
4.2 MAPA PROCESOS ACTUALES DE LA EMPRESA
For 13C ssNMR experiments, cross polarisation (CP) was used in order to transfer
magnetisation from highly abundant protons with a high gyromagnetic ratio to less abundant carbon atoms which have a lower gyromagnetic ratio, in order to improve sensitivity. The experimental parameter in the pulse program that relates to the transfer of this magnetisation is the so called contact time. The build-up of magnetisation for each individual carbon is dependent on the proton dipolar coupling network to which it belongs and the degree of protonation of each carbon as well as molecular kinetics such as methyl group rotation or aromatic ring flips (THC)(Kolodziejski and Klinowski 2002).
The larger the number of protons, the stronger the dipolar coupling and therefore the faster the cross polarisation transfer. Molecular motions that occur on the microsecond time scale may average the dipolar coupling used to transfer magnetisation resulting in a decay in build-up intensity. For example protonated carbons, with reduced mobility, build-up at a faster rate, methyl groups which have higher rotational mobility and build up more slowly Additionally, at longer CP contact times, more magnetisation is allowed to be transferred to the carbon atoms, therefore improving sensitivity of the experiment, up until a point at which the protons begin to relax as a function of T1ρ. As a result
different peaks will cross polarise and build up at different rates. Therefore when choosing the optimal CP contact time, the entire spectrum as a whole has to be considered and a compromised contact time chosen based upon the overall intensity of resonances of interest from labelled amino acids within the sample. In order to optimise signal to noise, a range of 1D 13C CP MAS spectra of BPV E5
FY were recorded with
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Figure 5.10 1D proton-decoupled 13C CP-MAS spectra of singly labelled BPV E5FY at increasing CP contact time
Spectra recorded at 600 MHz with 11 kHz MAS at 258 K (-15 ºC). A 2.5 µsec 1H 90º with a 2.5
second recycle delay was used with 100 kHz SPINAL-64 proton decoupling during acquisition for 512 scans. The cross polarisation (CP) contact time was increased from 100 to 1500 µs between each experiment as indicated alongside 1D spectra.
As can be seen from the 1D 13C spectra obtained in Figure 5.10 and the graph of
plotted signal intensities at increasing CP contact time (Figure 5.11) using shorter contact times, resulted in improved signal for phenylalanine (Cδ 1,2/Cε 1,2/Cζ) and tyrosine (Cδ 1,2) aromatic ring carbons. As the contact time was increased, from 100 to 250 µs the intensity of these resonances increased, but beyond 250 µs the intensity of these aromatic carbon resonances decreased at a fast rate as a function of increasing contact time. A similar trend was also seen for tyrosine Cε 1,2 carbon atoms, although the initial starting intensity was much lower and the rate at which the signal intensity decreased was much slower. For the resonance relating to phenylalanine and tyrosine CO carbonyl carbons, which have no directly-bound 1H atom, the opposite trend was
P a g e| 155 the CP contact time was increased the signal intensity increased up until 1000 µs, at which point the signal intensity remained the same upon further increase to 1500 µs. For tyrosine Cζ, signal was only observed at contact times of 750 µs and above, before which no distinguishable peak was detected. Increasing the contact time resulted in an increase in the signal intensity for this peak between 750 and 1000 µs after which there was little increase in signal.
A CP contact time of 750 µs was selected for use in all further 1D and 2D 13C
experiments, so as to ensure that all inter-helical cross peaks were observable between the predicted interacting atoms of each labelled amino acid.
Figure 5.11 Graph of 1D 13C resonance signal intensity at increasing cross polarisation (CP) contact times
Absolute intensity of assigned phenylalanine and tyrosine aromatic ring and carbonyl carbon resonances, plotted against increasing cross polarisation (CP) contact time. Resonance intensities were obtained from 1D 13C spectra (as shown in Figure 5.10) recorded at
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5.3.4
2D
13C-
13C ssNMR of BPV E5 labelled at phenylalanine & tyrosine
(BPV E5
FY)
Following optimisation of ssNMR experimental parameters to obtain optimal signal intensity, 2D 13C-13C DARR correlation spectra were recorded at short and long mixing
times (20 and 400 ms) to probe any possible inter-helical, through-space interactions
between the two labelled amino acids in the BPV EFFY ssNMR sample.
A 2D 13C-13C DARR spectrum obtained using a 50 ms mixing time is shown in Figure
5.12, and a number of off diagonal cross peaks were observed. Although experimental
parameters were optimised in order to achieve optimal signal to noise and to increase signal in the region of interest in the 13C spectrum, in moving to higher field we
observed a decrease in signal to noise compared to spectra recorded at 500 MHz. Therefore, the intensity of these cross peaks was much lower than that observed in our previous BPV E5LF sample at lower field (500 MHz). As observed in the 1D 13C spectrum
(Figure 5.7), there was considerable overlap between signals obtained for phenylalanine and tyrosine, making assignment of individual cross peaks difficult. From the 2D spectrum recorded at short mixing time, intra-residue cross peaks were observed between; phenylalanine Cα – Cβ, Cα – (Cδ 1/2, Cε 1/2, Cζ), Cα – CO, Cβ – (Cδ 1/2, Cε 1/2, Cζ), Cβ – CO, and (Cδ 1/2, Cε 1/2, Cζ) – CO, no intra-residue cross peaks to phenylalanine Cγ were observed. For labelled tyrosine; Cα – Cβ, Cα – (Cδ 1/2, Cγ), Cα – CO, Cβ – (Cδ 1/2, Cγ), Cβ – CO, (Cδ 1/2, Cγ) – Cε, (Cδ 1/2, Cγ) – CO intra-residue cross peaks were observed.
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Figure 5.12 50 ms 2D 13C-13C DARR spectrum of singly labelled BPV E5YF in DMPC liposomes
2D 13C-13C DARR correlation spectrum of singly labelled BPV E5
YF, acquired in 48 hours with a
mixing time of 50 ms. Spectrum was recorded at 600 MHz with 11 kHz MAS at 258 K (-15 ºC) for 148 scans. Cross peaks are labelled according to amino acid spin system, with cross peaks arising from phenylalanine 28 in dark blue and tyrosine 31 in orange.
Increasing the DARR mixing time from 50 ms to 400 ms (Figure 5.15) yielded only two additional intra-residue cross peaks, namely phenylalanine Cγ – Cα and tyrosine (Cδ 1/2, Cγ) to Cζ. As with our BPV E5LF sample, no inter-helical cross peaks
were observed at long mixing times between labelled phenylalanine and tyrosine, with all cross peaks observed arising from intra-residue correlations between carbon atoms within each of the two labelled amino acids.
From the CHI molecular model of the BPV E5 homodimer, it was expected that side chains of phenylalanine 28 and tyrosine 31 would be in close proximity to each other at the homodimer interface, with the coupling between phenylalanine Cε1 and tyrosine Cε1 predicted to have the shortest average inter-helical distance of 3.64 Å. Additional inter-helical couplings between phenylalanine Cδ1, Cε1 and Cζ to tyrosine
P a g e| 158 Cδ1, Cε1 and Cζ, as detailed in Table 3.1, (Chapter 3) were all predicted to have inter- helical couplings with inter-atomic distances shorter than 4.5 Å. Based upon the results obtained with singly labelled GpA, coupling over these distances should be detectable at long mixing times. Only the coupling between phenylalanine Cζ and tyrosine Cζ was predicted to have an inter-helical distance (4.81 Å) near the limits of the observable range.
Figure 5.13 400 ms 2D 13C-13C DARR spectrum of singly labelled BPV E5YF in DMPC liposomes
2D 13C-13C DARR correlation spectrum of singly labelled BPV E5
YF, acquired in 48 hours with a
mixing time of 400 ms. Spectrum was recorded at 600 MHz with 11 kHz MAS at 258 K (-15 ºC) for 512 scans. Cross peaks are labelled according to amino acid spin system, with cross peaks arising from phenylalanine 28 in dark blue and tyrosine 31 in orange.
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Figure 5.14 50 vs 400 ms 2D 13C-13C DARR spectrum of singly labelled BPV E5YF in DMPC liposomes
2D 13C-13C DARR correlation spectrum of singly labelled BPV E5
YF, with a mixing time of 50
ms (red) and 400 ms (black). Spectra was recorded at 600 MHz with 11 kHz MAS at 258 K (-15 ºC). Cross peaks are labelled according to amino acid spin system, with cross peaks arising from phenylalanine 28 in dark blue and tyrosine 31 in orange.
As seen from both 1D and 2D 13C-13C experiments recorded on BPV E5 YF, the
aromatic region of the 13C spectrum was unresolvable and appeared as a single broad
peak, consisting of resonances from both phenylalanine and tyrosine carbon signals. This greatly hampered any chances of being able to identify any inter-helical cross peaks, due to spectral overlap of resonances, making potential intra and inter-helical cross peaks extremely difficult to differentiate between. Although the Cε and Cζ carbon atoms of tyrosine had distinct chemical shifts (115.7 and 156.1 ppm respectively) that would theoretically allow for any inter-helical couplings between phenylalanine and tyrosine to be observed, the similarity in chemical shift between phenylalanine Cδ 1/2 and tyrosine Cδ 1/2 made it impossible to differentiate between a cross peak arising from couplings between phenylalanine Cδ 1/2 to tyrosine Cε and tyrosine Cδ 1/2 to Cε.
P a g e| 160 Therefore it was not possible to confidently state that any observed cross peaks arising between the two were solely due to an inter-helical coupling rather than an intra- residue coupling, even when using singly labelled peptides. As such, since the 2D 13C-13C
DARR spectra of BPV E5FY at both short and long mixing times yielded only intra-residue
couplings and no cross peaks arising from possible inter-helical couplings, we were unable to find any evidence of inter-helical interactions through dipolar couplings between labelled phenylalanine 28 and tyrosine 31 at the BPV E5 homodimer interface.