1. Structures of Human Apolipoprotein Fragments.
   

   Using distance geometry calculations of NOE-derived distance restraints I have successfully solved the structures of fragments of apoA-I, apoA-II and apoE in lipid mimetic environments. These structures confirmed that amphipathic helices are structural motifs in lipoproteins. The average structure of apoA-I(142-187) is a V-shaped helix-hinge-helix motif in which the hydrophobic residues of the two helices are found on the inner face in either SDS or DPC. This result, together with intermolecular NOE data, allows us to propose a simplified model for the complex, wherein the V-shaped structure straddles the micelle.

   Using what I have called 'peptide-aided signal assignment' (PASA) of segments from apoA-I ranging between 20 to 66 residues, I was able to assign some resonances for the intact apoA-I spectra (243 aa).

2. Peptide/lipid Interaction.
   

   The main interaction between apolipoprotein fragments and lipids is hydrophobic. The structural difference between apoE(263-286) and apoE(267-289) led us to propose that aromatic and paired hydrophobic side chains are excellent lipid anchors. The Protein Data Bank structures are PDB number 1OEF for apoE(263-286) and PDB number 1OEG for apoE(267-286). The enhancement of anionic lipid binding by cationic side chains has been substantiated using intermolecular NOE and other observations. Finally, we believe that interfacial, acidic side chains are responsible for lipid selectivity.

   Supported by the Natural Sciences and Engineering Council of Canada V5A 1S6 (NSERC).

Complete Solution Structure of Apolipoprotein C-I.


Human apolipoprotein C-I is a 57 residue exchangeable apolipoprotein distributed mainly in HDL and VLDL. ApoC-I is a secondary activator of the enzyme LCAT and thought to be important for people suffering from Tangier disease and familial apoA-I/apoC-III deficiency. ApoC-I also inhibits binding of VLDL to the low density lipoprotein receptor-related protein (LRP), probably by displacing apoE from the VLDL surface.

The structure of apoC-I in the presence of SDS was calculated from 522 distance restraints based on the homonuclear NOESY spectrum collected at 100 ms. ApoC-I contains two helices, L8-K30 and R39-E53, linked by an extended hinge region. Both helices are amphipathic with nonpolar residues forming the hydrophobic face and polar residues forming the hydrophilic face. The distribution of charged side chains fits the description of a class A2 amphipathic helix, with basic side chains at the polar-nonpolar interface and acidic side chains (with the exception of E19) at the center of the polar face. The Protein Data Bank structures of apoC-I in SDS may be viewed PDB number 1IOJ. Poretions of ApoC-I in SDS may be viewed: apoC-I(7-24), PDB number 1ALE; apoC-I(35-53), PDB number 1ALF; apoC-I(1-38), PDB number 1OPP

Supported by a Student Traineeship from the British Columbia Heart Foundation, 1994-1996.

1. A Multidimensional NMR Approach to the Study of the Nicotinic Acetylcholine Receptor and its Ligands.

   The Nicotinic Acetylcholine Receptor, nAChR, is a ligand-gated ion channel and thus mediates cholinergic neurotransmission, particularly at the neuromuscular junction. The architecture of nAChR is that of a pseudosymmetric lipid bilayer spanning pentamer with a central pore. Four homologous subunits with stoichiometry a2bdg assemble to yield the quaternary structure of the nAChR at the neuromuscular junction. The b subunit resides between the two a subunits each of which expose an acetylcholine binding site of differing affinities. Each subunit has a large extracellular N-terminal domain, four membrane spanning domains M1,M2,M3,M4 of no less than four hundred amino acids each, and an intracellar domain linking M3 and M4. Affinity binding and site directed mutagenesis studies have disclosed a four fold model of the acetylcholine binding sites with one fold associated with neighboring subunits. Cysteines 192,193 form an unusual vicinal disulfide which has been the central point of most NMR binding studies employing peptides and fusion proteins.

   I have used NMR to study a 28 amino acid peptide fragment centered on tyrosine 93 which has been shown to be a cationic subsite of the agonist binding site. This fragment is only four units downstream from the Main Immmunogenic Region, the epitope antigenic to the autoimmunity antibodies causing the human neuromuscular disease Myasthenia Gravis. Using the peptide and interesting ligands I have done some revealing 3D NMR work.

2. Structure of Apolipoprotein A-I.
   

   I have participated in acquiring 3D NOESY-HMQC and TOCSY-HMQC NMR data sets for a 243 residue human lipid transport protein ApoA-I. Theory and practise indicates the necessity to find an alternative to discrete Fourier Transform in the case of 3D data which by nature is incomplete and noisy. I am doing theoretical and consultative work on Maximum Entropy Reconstruction and Wavelet spectroscopy. MaxEnt provides in principle a framework for artifact free reconstruction of 3D NMR data. Although it may be only a desired goal computationally, it does provide a standard for the evaluation of new emerging NMR data processing techniques.

   Supported by NSERC.

Structure of Apolipoprotein C-II.


NMR spectra of apoC-II(44-79) have been recorded in the presence of SDS and DPC and completely assigned. This fragment activates LPL as well as the intact protein. Preliminary structure calculations indicate that apoC-II(44-79) is mainly a-helical in lipid-mimetic solution. CD analysis gives 35% helix in SDS; 41% in DPC.

2D NMR spectra of mature apoC-II (74 residues) have been recorded in the presence of SDS (140:1 molar ratio) at pH=5.0 and both 310K and 320K. Active, mature apoC-II is formed by cleavage at the QQDE site, releasing the six N-terminal residues (TQQPQQ). The NMR specta show extensive overlap, hence 3D and 4D NMR techniques will be required for structure determination. Our preliminary results indicate a structure of apoC-II be may be possible with 15N label only.

Supported by the British Columbia Heart Foundation.

Interactions of apoA-I with Lecithin:cholesterol Acyltransferase.

In collaboration with prof. Andy Lacko, N. Texas Univ., I have just commenced an NMR study on the interaction of apoA-I(122-187) with LCAT in the presence of perdeuterated dodecylphosphocholine. This peptide, supplied by our collaborator, prof. Jim Sparrow, Baylor College of Medicine, has 43% the LCAT-activating ability of intact apoA-I. LCAT (EC 2.3.1.43) is an enzyme which transesterifies the sn-2 fatty acid from phosphatidylcholine to cholesterol to yield cholesteryl esters.

Supported by NSERC.

Dr. Mark Okon (mokon@sfu.ca)

Structure of Apolipoprotein A-I


I have just begun a heteronuclear multidimensional NMR study of [N15]-labelled apoA-I (243 amino acids) in SDS. The labelled protein was prepared by our collaborator, Dr. Yves Marcel, University of Ottawa Heart Institute.

Supported by the British Columbia Heart Foundation and NSERC.

NMR/ front page/ terminology/introduction/references

Created by Kathy Cushley
last modified: February 29,2000

URL: http://www.sfu.ca/~cushley