Complex I
3-D reconstructions of Complex I from N. crassa (wire frame) and E.
Coli (surface rendered) showing that
the two complexes have similar structure despite their large difference
in molecular weight. The cytoplasmic
domain for E. Coli is coloured in gold and the membrane domain in
blue. The surfaces of the membrane
are shown schematically in red.
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Studies of mitochondrial NADH-ubiquinone Oxidoreductase (Complex
I) from Neurospora crassa and E. coli are being made in collaboration
with Hanns Weiss and Thorsten Friedrich (University of Düsseldorf).
This work has mainly been carried out by an EMBL predoctoral student
(Vincent Guénebaut). He first made a 3-D image reconstruction of
negatively stained single particles of the whole complex from N. crassa
(Mw approx 1.1 MDda) using the conical tilt method. Complex I is particularly
well suited to this approach because of its large size, and although an
asymmetric structure, it has a very characteristic shape. The 25Å
resolution 3-D model confirms the general "L"-shape of the molecule, with
arms of equal length and corroborates the hypothesis of a subdivision of
the whole complex into three functional domains. The membrane part of Complex
I , which in the reconstruction is masked by bound detergent, constitutes
the first domain, containing all the mitochondrially encoded subunits.
The second and third domains, forming the lower and upper halves of the
matrix (cytoplasmic) arm, are composed exclusively of nuclear encoded subunits,
amongst them all the subunits binding a detectable redox group. Immuno-labelling
with Fab to the 49 kDa subunit which is known to be in the cytoplasmic
part of the complex permitted its localisation on the matrix arm and confirmed
the previous division of the complex into membrane and matrix arms. He
then extended this study to the smaller redox enzyme complex from E. coli
(Mw 530kDa). Although less stable than the N. crassa complex, it was
possible to obtain enough single particle images to make a 3-D reconstruction
by the same method. Surprisingly, although the E. coli complex is about
half the molecular weight, it has the same overall size and shape as the
mitochondrial enzyme. The additional protein mass of the mitochondrial
complex is distributed along both arms but especially around the junction
between the two arms and around the membrane arm. It appears that the basic
structural framework found in prokaryotic complex I is stabilised by this
additional mass.
Colicin-N
A study of the E.coli toxin colicin-N bound to its receptor ,
the outer membrane porin OmpF, is being carried out in collaboration with
Franc Pattus and Marek Cyrklaff (CNRS Strasbourg)and J. Lakey (Newcastle).
In the presence of the receptor binding domain (but not in its absence)
it is possible to make tubular membrane crystals of OmpF with colicin-N
forming a regular layer on the inside of the tubes. We are currently carrying
out computer analysis of cryo-EM images of these tubes, which have helical
symmetry. A model for the density envelope of colicin-N and the receptor
should allow the X-ray structures for these molecules to be docked.
Lipid Monolayer crystallisation
We have been using the Kornberg lipid monolayer technique to obtain
ordered arrays of soluble proteins bound to lipid monolayers at the air-water
interface. A pre-doctoral student, Thomas Wendt has used the method to
obtain aligned 2-D layers of insect tropomyosin-troponin complex. We
have also recently been investigating the use of nickel-chelating lipids
to bind specifically his-tagged expressed proteins which are readily available.
References
Three Dimensional Structure of NADH dehydroge nase from Neurospora crassa
by Electron Microscopy and Conical Tilt Reconstruc tion.
Guenebaut, V., Vincentelli, R., Mills, D. Weiss, H. and Leonard K.
J.Mol Biol. (1997) 265, 409-418
Consistent Structure Between Bacterial and Mitochondrial NADH:Ubiquinone
Oxidoreductase. (Complex I)
V. Guénebaut,, A. Schlitt, H. Weiss, K. Leonard and T. Friedrich.
J. Mol. Biol. (1998) 276, 105-12
Structure of the Lethocerus troponin-tropomyosin complex as determined
by electron microscopy.
T. Wendt, V. Guenebaut and K.R.Leonard
J. Struct. Biol. (1997), 118, 1-8