News and Publications

Bioorganic, Protein, and Medicinal Chemistry

J.W. Kelly, H. Bekele, E. Koepf, H. Lashuel, A. Lentz, V. Oza, M. Petrassi, E. Powers, H. Purkey, P. Raman, G. Ratnaswamy, T. Walkup, L. Woo, Y. Xie

The central themes of our research are to understand the chemistry and biology of peptides and proteins and to develop new approaches for manipulating these properties with purposefully designed small molecules. We use spectroscopic and biophysical methods in combination with chemical synthesis and recombinant DNA technology to accomplish these aims. Specific research projects include the following.

CONFORMATIONAL CHANGES AND AMYLOID DISEASE

An invariant feature of the 16 human amyloid diseases (e.g., Alzheimer's disease) is the presence of amyloid fibrils close to dead or dying neurons. We are studying 3 amyloidogenic proteins: transthyretin, gelsolin, and amyloid-ß peptide. For each protein, conversion from a well-defined tertiary structure into an alternative conformation and ultimately a quaternary structure rich in ß-sheets appears to be the causative event in the associated disease (Fig. 1).

We use analytical ultracentrifugation and mass spectrometry to study the quaternary structural changes and circular dichroism, fluorescence, and nuclear magnetic resonance spectroscopic methods to study the tertiary structural changes required for formation of amyloid fibrils. Electron and atomic force microscopy are used to study the mechanism of assembly of the amyloidogenic intermediate, and hydrogen/deuterium exchange with electrospray or matrix-assisted laser desorption/ionization mass spectrometry is used to examine the dynamic characteristics of the amyloidogenic intermediate.

We now know that marked changes in tertiary structure are required for the conversion of these proteins into amyloid. The challenge remaining is to understand the details of these changes, the dynamic properties of the intermediates, and the self-assembly mechanism that yields amyloid.

Neurotoxic effects, a common feature of all amyloid diseases, are poorly understood. Most likely the toxic effects are due to multiple mechanisms. Using our expertise in the preparation of amyloid and its soluble precursors in combination with the cell and neurobiology expertise at TSRI, we have embarked on a project to understand why amyloid fibrils kill nerve cells.

We have introduced a new therapeutic strategy in which a high-affinity ligand is used to prevent the quaternary and tertiary structural changes required for formation of amyloid fibrils. In this project, we use x-ray crystallography or nuclear magnetic resonance in combination with synthetic chemistry to discover high-affinity inhibitors. Recent cocrystal structures of the normally folded form of transthyretin with second-generation inhibitors suggest new molecules to synthesize and test. Our philosophy on synthesis is to develop simple parallel syntheses that can be used to prepare libraries to quickly sort out relationships between structure and activity.

ß-SHEET STRUCTURAL PRINCIPLES

Peptides shorter than 40 amino acids typically do not adopt well-defined structures in aqueous solution, a situation that makes it difficult to study ß-sheet tertiary structure. In an effort to better understand ß-sheet structure in aqueous solution, we have designed and synthesized amino acids that act as the nucleus for formation of ß-sheets when incorporated into a surprising range of sequences composed of 6 or more natural amino acids. The challenge is to convert these dynamic ß-sheet structures into well-defined ß-sheets that are proteinlike.

In parallel we are studying the WW domain (57 residues), an important and recently discovered signal transduction domain that adopts an isolated 3-stranded ß-sheet structure in aqueous solution, as discerned from nuclear magnetic resonance and x-ray crystallographic studies. This protein is excellent for determining the contributions of hydrogen bonding and hydrophobic interactions to ß-sheet structure. We are particularly interested in using a kinetic approach to study the structural nature of transition states in protein folding. This goal will be accomplished by using by solid-phase peptide synthesis to incorporate unnatural amino acids into the WW sequence and then testing the physical and spectroscopic properties of the resulting analogs, with emphasis on the rate of folding.

MINERALIZED SURFACES IN CHEMISTRY AND BIOLOGY

The use of small peptidomimetic ß-sheet structures in combination with electrostatic intermolecular interactions leads to ß-sheet building blocks that self-assemble to form a monolayer coating at an air-water interface or on a hydrophobic surface. Controlled self-assembly allows a periodic presentation of functional groups on a surface that can act as a nucleus for mineral crystallization if the lattice match is good. Taking clues from biology, we have accomplished calcite mineralization. The challenge and potential of this approach are the formation of unnatural inorganic surfaces that can be used for a variety of applications such as catalysis.

USE OF PEPTIDES FOR SYNTHESIS OF COMPLEX NATURAL AND DESIGNED SMALL MOLECULES

Peptides have been underused as starting materials for the formation of complex conformational constrained organic molecules that lack amide bonds. A major effort is under way to develop these methods to create high-affinity ligands for both proteins and RNA. Using a variety of chemical reactions, some of which are newly discovered, we are producing a range of natural products and their analogs in just a few steps.

PUBLICATIONS

Bekele, H., Nesloney, C.L., McWilliams, K.W., Zacharius, N.M., Chitnumsub, P., Kelly, J.W. Improved synthesis of the Boc and Fmoc derivatives of 4-(2´-aminoethyl)-6-dibenzofuranpropionic acid: An unnatural amino acid that nucleates beta-sheet folding. J. Org. Chem. 62:2259, 1997.

Chen, H.I., Einbond, A., Kwak, S.-J., Linn, H., Koepf, E., Peterson, S., Kelly, J.W., Sudol, M. Characterization of the WW domain of the human Yes-associated protein and its polyproline-containing ligands. J. Biol. Chem. 272:17070, 1997.

Colon, W., Lai, Z., Lashuel, H.A., McCulloch, J., McCutchen, S.L., Miroy, G.J., Peterson, S.A., Kelly, J.W. Transthyretin quaternary and tertiary structural changes facilitate misassembly into amyloid: A new therapeutic strategy based on preventing the amyloidogenic conformational changes. Adv. Protein Chem. 50:161, 1997.

Dikler, S., Kelly, J.W., Russell, D.H. Improving mass spectrometric sequencing of arginine-containing peptides by derivatization with acetylacetone. J. Mass. Spectrom. 32:1337, 1997.

Kelly, J.W. The alternative conformations of amyloidogenic proteins and their multi-step assembly pathways. Curr. Opin. Struct. Biol. 8:101, 1998.

Kelly, J.W. Amyloid fibril formation and protein misassembly: A structural quest for insights into amyloid and prion diseases. Structure 5:595, 1997.

Kelly, J.W. The environmental dependency of protein folding best explains prion and amyloid diseases. Proc. Natl. Acad. Sci. U.S.A. 95:930, 1997.

Lai, Z., McCulloch, J., Kelly, J.W. GdnHCl-Induced denaturation and refolding of transthyretin exhibits a marked hysteresis: Equilibria with high kinetic barriers. Biochemistry 36:10230, 1997.

Nettleton, E.J., Sunde, M., Lai, Z., Kelly, J.W., Dobson, C.M., Robinson, C.V. rotein subunit interactions and structural integrity of amyloidogenic transthyretins: Evidence from electrospray mass spectrometry. J. Mol. Biol., in press.