We are studying two main topics,     
1)  Electron microscopy of integral membrane proteins and soluble proteins bound to lipids.    
2)  The study of muscle proteins, in particular those of the "muscle cytoskeleton" and those involved in the unsolved problem of regulation of muscle contraction.    

1) Membrane Proteins    

Complex I    
 Studies of mitochondrial  Complex I (NADH-ubiquinone Oxidoreductase) from Neurospora crassa  and E. coli are being carried out in collaboration with Hanns Weiss and Thorsten Friedrich (University of Düsseldorf).    

 A 3-D image reconstruction of negatively stained single particles of  the whole complex from N. crassa (Mw approx 1.1 MDda) has been carried out 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.    

  We have now  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.  The E. coli complex is about half the molecular weight but 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  (Fig. 1). It appears that the basic structural framework found in  prokaryotic complex I is stabilised by this additional mass in the eukaryotic enzyme.    
     
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.  This has been  used 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.     

2) Muscle proteins    

Invertebrate muscle proteins    
 In carrying out muscle studies, we are using two systems - for genetics, mutational studies and most sequencing work we are using Drosophila and for the biochemistry and some electron microscopy where large amounts of protein are required we are using the giant water bug, Lethocerus.    

Kettin     
 Kettin is a high molecular weight (700KDa) modular protein found only in insect muscle. The Drosophila genomic DNA sequence has now been completed in collaboration with Bernhard Kolmerer (Labeit group) using, in part, p-element mutants mapped to the kettin locus. The protein consists almost entirely of immunoglobulin C2 (IG2)   domains, but instead of being contiguous as in other proteins of the titin type, the domains are linked by 35 residue peptide spacers. Immuno-electron microscopy of  kettin in both Lethocerus and Drosophila flight muscle shows that the N-terminus is near the centre and the C-terminus just outside the periphery of the  Z-disc, the molecule running parallel to the actin filaments. This is supported by our finding  that purified whole kettin and an expressed kettin domain-linker sequence bind to actin with high affinity, unlike other modular muscle proteins such as titin and twitchin which are not actin-binding. The role of kettin may be to stabilise the anti-parallel actin-actin interactions which are present in the Z-disc. Kettin is a specific target for calpain, an endogenous calcium-activated protease which breaks down the Z-disc.      

Thin Filament Proteins    
 Insect flight muscle thin filaments do not fully activate myosin in the presence of calcium which suggests there is additional regulation. The regulatory complex in Drosophila has tropomyosin, TnC, TnI and TnT similar to the components in vertebrate striated muscle and TnH which is a fusion protein of tropomyosin and a hydrophobic proline-alanine rich C-terminal half. Lethocerus troponin has TnC, TnT and a TnH which includes TnI sequence and acts as the inhibitory component.    
     
 In Drosophila, we have found that an isoform of glutathione-S-transferase (GST-2) is associated with the hydrophobic extension of TnH. When the C-terminal half of TnH is selectively removed, the tropomyosin half stays bound to the thin filament and GST-2 is released.  Unlike other GSTs, the molecule has a 50 amino acid hydrophobic extension at the N-terminus which is similar in sequence to the C-terminus of TnH and which binds specifically to it.    
  In collaboration with Pavlos Agianian in the group of Paul Tucker, crystals of expressed Drosophila GST-2 have been obtained which diffract X-rays to 3Å. We are hoping that solution of the crystal structure will reveal the conformation of the N-terminal extension and help to understand its interaction with TnH. A number of Drosophila muscle proteins have such proline-alanine rich extensions (TnH, TnI, myosin regulatory light chain, GST-2) -  their function is unknown.    

 Tropomyosin-troponin from Lethocerus has has been isolated as a stable complex. When this complex is adsorbed to  lipid monolayers by the tropomyosin rod, the troponin head region of the complex can rotate freely around an axis in the plane of the  monolayer giving many different projected views.  We have now  carried out a single particle tomographic 3D reconstruction of the troponin part of the complex to 33Å resolution.  The model has also been fitted to EM projection images of troponin on isolated Lethocerus thin filaments and docked to a 3-D reconstruction of  frog thin filaments by aligning the troponin with  the tropomyosin density (Fig. 2).     
     

Figure legends    

Fig.1    

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 is coloured in Acapulco gold and the membrane domain in Pacific blue. The surfaces of the membrane are shown schematically in red.    

Fig 2.    
Two models of insect troponin complex (green and gold) docked on  the frog muscle thin filament (white) - the latter kindly provided by Dr. W. Lehman, Boston University. The arrow indicates the direction of the tropomyosin filament