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David Atkinson, Ph.D.Department Chairman,
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ResearchPLASMA LIPOPROTEINS AND APOLIPOPROTEINS:
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Research Group
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Our research focusses on molecular details of the structure, stability and dynamic properties of the plasma lipoproteins and their constituent apolipoproteins, particularly high density (HDL) and low density (LDL) lipoprotein. This information is vital to an understanding of the lipid interactions, apoprotein exchanges, lipoprotein cell surface interactions, receptor-mediated lipoprotein uptake, and lipoprotein inter-conversions that form the basis of lipid transport and metabolism. The conformational adaptability of the exchangeable apoproteins such as apoA-I, the major protein of HDL, is essential to both their structural role in lipoprotein stabilization and their functional roles as cofactors for enzymes, ligands for receptors, or mediators of reverse cholesterol transport. The precise molecular mechanism of this unique structural adaptability remain unclear, and is the focus of a major component of our research. A second focus concerns determination of the three-dimensional structure of intact LDL by cryo-electron microscopy and 3D-image reconstruction, with emphasis on the topology and the molecular conformation of the apo-B, protein component of LDL, at the lipoprotein surface . The emphasis is on the analysis of the organization of apo-B and the localization of structural and functional domains on the LDL particle, using a combination of site-specific immuno-nanogold labeling and direct visualization of the bound LDL receptor.
Our primary approaches use most of the techniques of modern molecular biophysics and structural biology. These include protein crystallography, structural electron microscopy/image processing, calorimetry/thermodymamics, circular dichroism, and molecular modelling/mechanics to probe the structure and physical properties of lipoproteins, apolipoproteins, peptide models for the apolipoproteins, and lipid/apolipoprotein reassembled model systems.
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Apolipoprotein Conformation, Structure and Stability
The sequences of the exchangeable apolipoproteins are comprised of tandem internally homologous 11/22 residue repeats that can be divided into two 11-mer sub-repeats (A and B), with N-terminal Pro located in the A-repeat (Fig. 1). The 11/22-mer repeats form amphipathic ?-helices 25-35Å long that are highly suited for an interfacial location at the lipoprotein surface. A refined consensus sequence (Fig. 2) representing an idealized version of this 11/22-mer (A/B) unit was derived in an analysis of the inter/intra-sequence homologies of apoA-I, apoE, and apo-AIV Molecular modeling showed that this 22-residue AB unit would form an idealized amphipathic ?-helix.
In addition, we have demonstrated that lipid-free apoA-I and apoA-II have a folded state in solution similar to the "molten globular" state described as an intermediate in the folding pathway of many water soluble proteins
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Our current studies concentrate on the structure and stabilizing interactions of:
Deletion and substitution mutations of apoA-1.
Point and deletion mutations in A-I are used to investigate the features responsible for conformational stability and lipid interaction. Structural and thermodynamic studies of mutant forms of apoA-I encompassing point and deletion mutants (Fig.1) probe the role of specific regions of the apoA-I molecule in its conformation and stability.
Peptides that model structural and functional units in the sequences of apoA-I.
To understand the properties of the individual segments of apoA-1, peptides are designed on the basis of both the 11/22 residue amphipathic helical segments of apoA-I (Fig.1) and on the "idealized" consensus sequence (CSP) for the fundamental 11/22-mer tandem repeat in the sequence of the apoproteins (Fig. 2). These include 44 amino acid peptides that overlap by one 22 amino acid repeat, apoA-I[99-142] (putative helices 4 and 5) and apoA-I[121-163] (putative helices 5 and 6), a peptide that models the N-terminal region (apoA-I[1-44]), and the peptide representing the C-terminus, apoA-I[198-243].
The conformation and stability of these peptides are studied using CD in solution, lipid mimicking detergents, and structure inducing solvents. Their lipid binding properties are analyzed using electron microscopy and density gradient ultracentrifugation.
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The structure of 44 amino acid consensus sequence peptides (CSP)2 that model this fundamental structural motif are studied by crystallography. Determination of the structure of 44-residue peptides with different juxtapositions of the consensus "A" and "B" repeats (e. g., BABA, BBAB), examines the role of Pro punctuation and inter-helical interactions. Comparison of the structures with those of the overlapping 44-residue "real sequence peptides" provides important information on the role of specific residues and their interactions in the structure and stability of the parent apoA-1
X-ray Crystallographic Studies of Peptide Models.
The 44-residue peptide (CSP-ABAB) has been crystallized and x-ray diffraction data collected in our crystallography laboratory under cryo-conditions extend beyond 1.9Å resolution.. Data sets, extending to 1.7Å, resolution have been recorded at the National Synchrotron Light Source (NSLS). The space group, P212121, unit cell parameters a=24.7Å, b=33.1Å, c=45.4Å. and small Matthew's volume VM=1.74 Å3/D indicate close packing of four CSP molecules in a unit cell. The diffraction pattern is dominated by strong 10-11Å reflections (inter-helical spacing) and by strong intensity on the 5.4Å layer line (helical pitch) that indicate alignment of the helices with the b-axi consistent with the predicted helical length of ~20 residues, or ~ 30Å. Comparison of the unit cell dimensions with the helical diameter (~11Å) suggests that 2 helices are packed along a and 4 helices along c, consistent with the total of 8 helices, or 4 CSP molecules, in a unit cell. The crystal-packing diagram shows that the crystal is formed of staggered four-helix bundles running along the b-axis. Molecular replacement, using an idealized 20-residue polyalanine helix as a model confirms the helical orientation along the b-axis. To advance the refinement, a Se-containing derivative peptide, Se-CSP, has been synthesized with Pro1-Met(Se) substitution. The helical contents in Se-CSP and CSP are identical, the crystallization conditions and the crystal morphology are similar and x-ray diffraction analysis of single crystals of Se-CSP indicate P212121 space group with the unit cell parameters that are very close to those of CSP. A MAD data set from a single Se-CSP crystal collected at NSLS extended to 1.8Å resolution. To further aid the refinement of the CSP structure, we have also synthesized a derivative CSP peptide containing Leu6-Met(Se) substitution.
• Chao, Yang. Conformational studies of a consensus sequence peptide (CSP) and a real sequence peptide (RSP) of apolipoproteins by circular dichroism spectroscopy and x-ray crystallography. PhD. Boston University, (2003). University Microfilms International, Publication, #AAT 3068030 ISBN: 0-493-87349-X
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Structure of Low Density Lipoprotein and the Organization of apoB
A second component of our studies is the visualization of the structure of LDL and the determination of the conformation of apoprotein B on the LDL particle by cryoelectron microscopy coupled with site specific and monoclonal antibody labeling with nanogold. The cryo-electron micrograph (right) shows a field of LDL particles imaged in vitreous ice. The figure below shows a composite image of this ovoid view of MB19-LDL with the positions of additional mAbs and Nanogold labels superimposed. In these low-contrast images, the high-density areas that represent the location of apoB appear non-uniform, and connected by areas of lower density. High-density areas are occasionally seen in the projectional interiors of the particles. Thin segments of lower density connect these areas to other dense areas on or near the particle edge. The lower figure shows two 3-D reconstructions of the LDL particle with mAb bound.
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Recent Publications
Irina N. Gorshkova, Kalliopi Liadaki, Olga Gursky, David Atkinson, and Vassilis I. Zannis. Probing the lipid-free structure and stability of apolipoprotein A-I by mutation. Biochemistry, 39(51): 15910-15919, 2000.
Irina N. Gorshkova, Tong Liu, Vassilis I. Zannis, and David Atkinson. Lipid-Free Structure and Stability of Apolipoprotein A-1: Probing the Central Region by Mutation. Biochemistry, 41(33): 10529-10539, 2002.
Fang Y, Gursky O, Atkinson D . Structural studies of N- and C-terminally truncated human apolipoprotein A-I. Biochemistry. 2003, 42(22):6881-90.
Fang Y, Gursky O, Atkinson D . Lipid-Binding Studies of Human Apolipoprotein A-I and Its Terminally Truncated Mutants. Biochemistry. 2003, 42(45), 13260-13268.
Libo Wang, David Atkinson and Donald M. Small. Interfacial Properties of an Amphipathic -Helix Consensus Peptide of Exchangeable Apolipoproteins at Air/Water and Oil/Water Interfaces. J. Biol. Chem., Vol. 278, Issue 39, 37480-37491, September 26, 2003.
Hongli L. Zhu and David Atkinson. Conformation and Lipid Binding of the N-Terminal (1-44) Domain of Human Apolipoprotein A-I Biochemistry; 2004; 43(41) pp 13156 – 13164
Libo Wang, David Atkinson and Donald M. Small. The Interfacial Properties of ApoA-I and an Amphipathic a-Helix Consensus Peptide of Exchangeable Apolipoproteins at the Triolein/Water Interface. J. Biol. Chem., Vol. 280, Issue 6, 4154-4165, 2005
Irina N. Gorshkova, Tong Liu, Horng-Yuan Kan, Angeliki Chroni, Vassilis I. Zannis, and David Atkinson. Structure and Stability of Apolipoprotein A-I in Solution and in Discoidal High-Density Lipoprotein Probed by Double Charge Ablation and Deletion Mutation. Biochemistry; 2006; 45(4) pp 1242 - 1254.
A complete list of my publications is included in my CV. Click here to download my CV.
Teaching
Foundations of Biophysics and Structural Biology (GMS BY 760 - Graduate Medical
Sciences):
This graduate level course provides a thorough grounding in the theory and major
experimental methods of Biophysics and Structural Biology. The course covers
protein thermodynamics, spectroscopy, electron microscopy, x-ray diffraction,
crystallography, and NMR. The course provides both didactic and laboratory instruction.
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Biophysics of Macromolecular Assemblies (GMS BY 771 - Graduate
Medical Sciences)
This advanced course covers the concepts of the assembly of biomacromolecules,
their structure and stabilizing forces, and biological function
as related to structure. Examples are drawn from assemblies of
proteins, lipids, lipoprotein systems, membranes and viruses.
Experimental Methods in Physiology (GMS PH 741 -Graduate Medical Sciences)
Current research methods in cellular and molecular physiology,
as applied to the study of macromolecular function, motility,
ligand binding phenomena, and membrane function. Develops problem-solving
skills and awareness of current approaches to research problems.
Biochemistry (SDM MD 512 - School Dental Medicine):
This course is designed to acquaint the student with the basic principles of
modern biochemistry. The topics to be covered include proteins, enzymes, DNA,
RNA and protein synthesis, metabolism, lipids, connective tissue, and hormones
and second messengers.
Endocrinology (GMS PH 748 -
Graduate Medical Sciences and School of Medicine):
Integrated treatment
of human endocrinology, biosynthesis of hormones, their receptor interactions,
and their
physiological effects.
Physiology/Endocrinology/Neurophysiology (SDM MD 514 - School Dental Medicine)
This course presents the physiology of cells, tissues,
organs, and integrated body functions, including the physiological
basis for the understanding of clinical conditions. An integrated
approach is taken to endocrinology and reproduction. Hormonal
aberrations and their end results in humans are presented in
clinical correlations.
Contact Us
Department of Physiology and Biophysics
Boston University School of Medicine
700 Albany Street
Boston MA 02118-2526
Phone: (617) 638-4015
e-mail: atkinson@bu.edu
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