3.3 Liposome-embedded CitApc
4.3.2 CitApc R93A in DMPC
As the phase transition point for DMPC is at 24◦C compared to the melting temperature of asolectin membranes below 0◦C, initial experiments on DMPC-embedded CitApc R93A were carried out at 25◦C. At these high temperatures the N-C-magnetisation transfer was very inecient, thus later spectra were recorded at temperatures between 6◦C and 10◦C, increasing the magnetisation transfer by 70 % (see gure 3.14). Overall, spectra above
4. Discussion 67
and below the melting point of DMPC are comparable; the isolated domains are readily assignable based on liquid-state data in both cases (see gure 4.9).
As discussed in section 3.3, the percentage of transferable assignments from liquid- to solid-state spectra is higher for PASp than for PASc in all cases. This is in part caused by a generally lower signal/noise ratio for PASc, as can be visualised for isolated Cα-Cβ -peaks in 3D-NCACB and NCOCACB spectra (see gure 3.18). In total, 25 PASc residues in citrate-free and 26 PASc residues in citrate-bound CitApc R93A could be assigned in 3D spectra. The number of unassigned peaks in 3D spectra does not account for the missing assignments based on liquid-state data (see table 4.1), especially if the missing assignments of transmembrane helices, which likely also contribute to unassigned peaks, are taken into account.
Table 4.1: Assignments of PAS domains in CitApc R93A.
citrate-free CitApc R93A
PASp spectrum residues assigned residues missing unassigned peaks*
PDSD 93 35
-NCACB 70 58 14
NCOCACB 23 105 14
PASc spectrum residues assigned residues missing
PDSD 59 50
NCACB 24 85
NCOCACB 2 108
citrate-bound CitApc R93A
PASp spectrum residues assigned residues missing unassigned peaks
PDSD 106 22
-NCACB 101 27 26
NCOCACB 90 38 8
PASc spectrum residues assigned residues missing
PDSD 40 69
NCACB 21 88
NCOCACB 5 104
* due to spectral crowding, it is impossible to specify a number of
* unassigned peaks for PDSD spectra.
68 4. Discussion
Figure 4.9: Spectra of CitApc R93A at dierent temperatures. PDSD-spectra of CitApc R93A were recorded above and below the phase transition temperature of DMPC.
Proton 1D spectra were used for discrimination between the liquid crystalline and solid phases of DMPC.
4. Discussion 69
It is therefore likely that the discrepancy between liquid-state assignments and solid-state peak positions for certain parts of the PAS domains, most notably PASc (59 % and 45 % assigned in citrate-free and citrate-bound state, respectively), is related to increased mobility of the invisible regions. INEPT spectra of CitApc R93A in both signalling states only contain peaks of lipid headgroups, so rapid exchange of unstructured protein regions can be ruled out. Also, no additional peaks appear in INEPT-spectra of the citrate-bound state. The non-assignable parts of PASp and PASc are therefore likely undergoing dynamic motions on a time scale that is not detectable with the acquired solid-state experiments.
The available assignments of PASp and PASc can be utilised to identify structural rearrangements in the two signalling states. Structural dierences can be easily tracked in PASp by analysing Cα-Cβ-secondary chemical shift changes upon citrate binding to CitApc R93A (see gure 4.10). The secondary structure reorganisation observed in liquid state (see section 4.1.1) can be reproduced in membrane-embedded solid state samples.
It can thus be assumed that the changes observed for the individual PASp domain are comparable to the structural switching in context of the transmembrane helices.
70 4. Discussion
Figure 4.10: Cα-Cβ-secondary chemical shift dierences of PASp in membrane-embedded CitApc between the ON- and OFF-state. A: Dierences between citrate-free and citrate-bound PASp R93A. Like in the isolated domains, the whole binding pocket undergoes structural reorganisation. Citrate is shown as cyan sticks for citrate-bound PASp. B: Comparison of citrate-citrate-bound PASp R93A and citrate-citrate-bound wild-type PASp. The citrate binding pocket is structurally conserved, indicating the same confor-mation of both citrate-bound states. Dierences are seen around the mutation site R93A highlighted in green. C: Comparison of citrate-free PASp R93A and citrate-bound wild-type PASp reproduces dierences observed between the ON- and OFF-states of PASp R93A.
Of note is the secondary chemical shift change of leucine 154 at the end of the C -terminal β-strand in PASp, which switches from extended conformation in citrate-free CitApc R93A to helical in citrate-bound samples of both CitApc wild-type and R93A (see gure 4.11). As solid-state assignments of residues succeeding leucine 154 are missing, other potential structural changes at the PASp - transmembrane helix interface remain to be elucidated. It can be concluded that the C -terminal β-strand is shortened by one amino acid upon citrate binding. This nding is in agreement with observations in crystal structures of citrate-free and citrate-bound CitA PASp in K. pneumoniae (Sevvana et al., 2008). Just like in G. thermodenitricans CitA PASp, the C -terminal β-strand is one amino acid shorter in the citrate-bound state due to the contraction of the domain upon citrate binding (Sevvana et al., 2008).
4. Discussion 71
Figure 4.11: Spectral dierences between the two signalling states at the PASp-TM2-interface. A: Cα-Cβ-secondary chemical shifts indicate a secondary structure alteration at leucine 154, which switches between extended conformation in citrate-free CitApc R93A and helical conformation in citrate-bound CitApc R93A. In citrate-bound wild-type CitApc, L154 is also found in helical conformation. Upon citrate binding, the C -terminal β-strand of PASp is thus shortened by one amino acid. B: Section of PDSD spectra of CitApc R93A in both citrate-free (blue) and citrate-bound (green) states. The Cα-Cβ-crosspeak of leucine 154 is shifted upon citrate binding. Residues succeeding leucine 154 are not assigned in solid-state spectra.
Transmembrane signalling is conceivable with four principle mechanistic models: trans-lation, piston-type displacement, pivot movement parallel to the membrane or rotation perpendicular to the membrane (Hulko et al., 2006; Matthews et al., 2006). Although other mechanisms cannot easily be excluded, the shortening of the C -terminal β-strand in CitA is consistent with a piston-model for transmembrane signalling (Ottemann et al., 1999). In this model, the second transmembrane helix is pulled out of the membrane upon signal recognition. This piston-like movement could be triggered by the contraction of PASp in CitA coupled with a shortening of the C -terminal β-strand (see gure 4.12) exerting a pull on the second transmembrane helix. In addition to the piston movement, a slight helix tilt or rotation could also contribute to net helix motion.