The power of the nanodisc system for controlling aggregation is illustrated
below. The nanodisc protocol for obtaining SSNMR spectra of integral membrane
proteins was tested using subunit c of ATP synthase, in a collaborative effort
between the Sligar (UIUC), McDermott and Rienstra (UIUC) laboratories. This
protein had been characterized by X-ray crystallography of the whole ATP
synthase complex[Stock, 1999 #384], and by solution NMR data of the isolated
peptide in organic solvent and aqueous micelles[Rastogi, 1999 #145]. Both data
imply that subunit c forms a hairpin of two antiparallel helices; residues 2- 40 and
47-74 (out of 79 total residues) are reported to be helical. An ongoing goal in the
McDermott laboratory has been to study the structure of ATPase subunit c as an
isolated peptide in a membrane mimetic environment, as an oligomer in a
membrane mimetic environment, and in the context of the total Fo complex,
where it is present in 12 copies. These efforts have been thwarted by the fact
that in many detergent and lipid environments, when the protein is present at
high concentrations needed for NMR, it forms an artefactual beta sheet structure,
as evidenced by diagnostic 13C shifts of all residues. Figure 6 illustrates the COCand CO-Ccorrelations in a 2D 13C homonuclear SSNMR spectrum,
measured using the DARR pulse sequence, on a 750 MHz Bruker AVANCE
system, with MAS at 14 kHz. The subunit c sample is uniformly enriched in 15N
and 13C, prepared in E. coli polar lipid vesicles vesicles at low ionic strength,
and precipitated by ultracentrifugation. The spectra were collected at –20 oC.
The Gly residues with correlations at 49,176 ppm are helical, but those at 46, 173
are sheet; the Ala residues at 55,179 are helical, but those at 51,175 are sheet.
Based on our preliminary NMR data of the whole Fo complex these sheet
structures are unexpected. Figure 6 also illustrates that comparable data (other
than slight differences in temperature and spinning frequency) collected on
nanodisc evidence no (or dramatically reduced) beta sheet formation,
presumably because protein aggregation is impeded during precipitation or
crystallization, and the membrane environment maintains its structure during
precipitation as well.
Figure 6 2D high
field MAS
based
homonuclear 13C
correlation
spectra for the c
subunit of ATP
synthase,
measured with the
DARR pulse
sequence. The
region where the
backbone CO
correlates to the
Cand C
resonances is
expanded. The key
difference between
the two
samples is the
formulation, where
the spectrum at left
was taken of a
peptide in
nanodisc and that
on the right was
taken of the
peptide in E. coli
polar lipid vesicles.
These data show
spurious beta
sheet conformers
for the case of the
vesicle but not for
the nanodisc
sample.