Structure and assembly of membrane-active proteins and peptides by solid-state NMR

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Research Synopsis

Our research focuses on the structural and biophysical characterization of peptide assembly, protein-protein interactions, and protein activity at biological membranes. While it is estimated that over 80% of potential drug targets exist at biological membranes, the details of membrane protein structure and function remain elusive. To achieve a molecular-level understanding of the role played by membrane active peptides and proteins in human disease, we use a wide range of imaging and spectroscopic methods, with a focus on solid state nuclear magnetic resonance (NMR). Solid state NMR has recently emerged as a powerful tool for obtaining high-resolution structural data on biomolecules not accessible to other approaches, such as integral membrane proteins and fibrillar aggregates. Projects in the lab include investigations of viral ion channels, amyloid proteins, prions, and models of peptide assembly. In each case our goal is to define protein structure and its mechanisms of action in health and disease.

1) Viral ion channels
One primary area of investigation is the formation of ion channels by a class of small viral membrane proteins which includes the hepatitis C virus p7 protein, the human respiratory syncytial virus SH protein, the SARS coronavirus E protein and Vpu from HIV-1. These ‘viroporins' enhance the permeability of host cell membranes, a process which is essential for viral infectivity, making them excellent candidates for development of novel antiviral therapies. However, the structure and functions of viroporins have been poorly characterized. We have recently developed an experimentally derived model for the ion channel formed by HIV-1 Vpu, and are using solid state NMR to develop high resolution structures for Vpu and for other members of this family. Our goals are to define the molecular basis for channel assembly in these proteins, and to move towards structural studies of drug-channel interactions – a first step in the design of new therapeutics .

2) Role of protein-membrane interactions in prion diseases
The accumulation of misfolded proteins in a fibrillar form is characteristic of several human diseases, including type II diabetes, Alzheimer's disease and prion diseases (BSE/mad cow, CJD). It has been proposed that the degenerative nature of these conditions may result from the toxicity of protein aggregates, though an as yet unknown mechanism. Using model peptides derived from the mammalian prion protein, we are investigating the structure, pathways for assembly, and mechanisms of cytotoxicity of amyloid fibrils and non-fibrillar aggregates. Extension of this work to larger proteins, including segments of the mammalian prion protein is under way. Knowledge of the structures adopted by aggregative proteins and peptides at model membranes, and the effects of these structures on bilayer integrity, will provide insight into the physical basis for the cytotoxicity of amyloid and prion proteins.

3) Other applications of solid state NMR to peptide structure and assembly
We are involved in a number of multidisciplinary, highly collaborative projects seeking to define the molecular basis for different types of peptide assembly. Using model peptides with well-defined amino acid sequence, the goal is to understand the forces governing how peptides assemble into biopolymers, and what physical properties the resulting polymer will have. Developing a structural and dynamic understanding of these model systems will help to drive future design of biomaterials.

Walsh, P., Simonetti, K. and Sharpe, S. (2009) Core structure of amyloid fibrils formed by residues 106-126 of the human prion protein. Structure In Press.

Sharpe, S., Yau, W-M. and Tycko, R. (2006) Structure and dynamics of the HIV-1 Vpu transmembrane domain revealed by solid state NMR with magic-angle spinning. Biochemistry 45: 918-933.

Sharpe, S., Yau, W-M. and Tycko, R. (2005) Expression and purification of a peptide from the Alzheimer’s beta-amyloid protein for solid-state NMR. Protein Express. Purif. 42: 200-210.

Goodyear, D.J., Sharpe, S., Grant, C.W.M. and Morrow, M.R. (2005) Molecular dynamics simulation of transmembrane polypeptide orientational fluctuations. Biophysical Journal 88:105-117
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Sharpe, S., Kessler, N., Anglister, J.A., Yau, W-M. and Tycko, R. (2004) Solid-state NMR yields structural constraints on the V3 loop from HIV-1 gp120 bound to the 447-52D antibody Fv fragment. J. Am. Chem. Soc. 126: 4979-4990.

Sharpe, S., Barber, K.R., Grant, C.W.M., Goodyear, D. and Morrow, M.R. (2002) Organization of model helical peptides in lipid bilayers: Insight into the behaviour of single-span protein transmembrane domains. Biophysical Journal 83: 345-358.

Sharpe, S., Barber, K.R. and Grant, C.W.M. (2002) Interaction between ErbB-1 and ErbB-2 transmembrane domains in bilayer membranes. FEBS Letters 519: 103-107.

Sharpe, S., Barber, K.R. and Grant, C.W.M. (2002) Evidence of a tendency to self-association of the transmembrane domain of ErbB-2 in fluid phospholipid bilayers. Biochemistry 41: 2341-2352.

Sharpe, S., Grant, C.W.M., Barber, K.R., Giusti, J. and Morrow, M.R. (2001) Structural implications of a Val-Glu mutation in transmembrane peptides from the EGF receptor. Biophysical Journal 81: 3231 3239.

Sharpe, S. and Grant, C.W.M. (2000) A transmembrane peptide from the human EGF receptor: Behaviour of the cytoplasmic juxtamembrane domain in lipid bilayers. Biochim. Biophys. Acta. (Biomembranes) 1468: 262-272.

Sharpe, S., Barber, K.R. and Grant, C.W.M. (2000) Val659Glu mutation within the transmembrane domain of ErbB-2: Effects measured by 2H NMR in fluid phospholipid bilayers. Biochemistry 39: 6572-6580.