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PROTEIN FOLDING AND BIOMOLECULAR NMR SPECTROSCOPY
Research Interests Protein Folding and Biomolecular NMR Spectroscopy: Amyloidoses are diseases involving abnormal protein aggregation and deposition in tissues by normally soluble proteins, and are well documented in more than twenty clinically defined cases1. Protein folding research – the study of how the information contained within a given amino acid sequence leads to a functional, three dimensional structure - has evolved to seek how and why such certain amino acid sequences misfold or aggregate to the detriment of the host. Despite very significant advances (e.g. the method of phi-value analysis for studying folding transition states2, the concept of energy landscapes3, the generic nature of amyloid fibrils4, and proof confirming the self-replication of infectious scrapie prions5), conclusive and concrete answers that can provide predictive and therapeutic powers in ameliorating these diseases have only just begun to show promise. One serendipitous finding is that partially – or alternatively - folded molecules such as HAMLET6 may serve to act as agents in selectively killing tumorigenic cells. Hence along with our understanding that misfolding may incur harm, it is becoming evident that misfolding may also yield benefit to its host, further underscoring the need for a detailed, biophysical investigation. In this context, we will attempt to address the “protein folding-misfolding problem” using the following three-pronged approach: 1. Development of new NMR spectroscopy-based methodologies to probe protein folding and misfolding. In obtaining atomic-level resolution of the protein folding-misfolding process, NMR spectroscopy has continuously played a significant role7. For the past several years, we have been involved in developing novel methodologies in laser-polarized photo-CIDNP8 (Chemically- Induced Dynamic Nuclear Polarization) and NMR spectroscopy, with the aim of characterizing ill-defined partially-folded intermediates and denatured states previously intractable with other methods9. Such partially folded or denatured molecules have been shown to form protein aggregates, some with morphologies that are strikingly similar to amyloid fibrils found in the tissues of neurodegenerative disease patients1. To this end, we have designed and constructed an in situ injection device that permits rapid homogenous mixing of solutions in the NMR magnet within 50 ms, permitting the characterization of transient, kinetic intermediate species present in real-time protein folding experiments10. In addition, by CIDNP pulse-labelling the denatured state of the 20-residue Trp-cage (TC5b)11 and transferring its magnetization to the well-defined native state, we have been able to obtain direct NOE-based distances of side chains involved in the residual structure of the ill-characterized denatured state12. 2. Investigating the structural basis of the physiological activity of alternatively folded proteins. HAMLET6,13, a potentially new biomolecular assembly that selectively induces apoptosis in tumor cells, is being explored from a biophysical, structure-and-dynamics-based perspective. HAMLET, BAMLET (the bovine analogue), and other analogues represent a new type of tumoricidal molecular complex, exhibiting broad activities against tumors from > 40 different lymphomas and carcinomas, yet leaving healthy, differentiated cells unharmed14. Using a wide range of biophysical techniques (e.g. NMR, EM, AFM, SAXS, etc), the ultrastructural and physico-chemical properties of this complex are being determined and the common structural assembly rules deduced. In addition, the molecular basis of cytotoxic activity – in comparison with those of amyloid pre-fibrils – will be investigated. 3. Inhibition of amyloid fibril growth and/or control of fibril morphology by incorporating synthetic enantiomeric peptides. We will attempt to build upon previous experience of implementing an enantiomeric peptide strategy15 - albeit now in the context of protein misfolding – to use as means for potential drug design for fibillogenesis inhibition. The first test cases will be using a human transthyretin peptide, which has shown promise. Experiments on this topic will additionally serveas anintriguing starting point for bio-nanotechnology applications. References Research PersonnelVisiting Research Fellow Visiting Graduate Student Academic Collaborators:Prof PJ Hore, Department of Chemistry, University of Oxford, UK. Selected Publications by the Protein Folding and Biomolecular NMR Spectroscopy Group |
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Contact: bbutler@tcd.ie. |
Last updated: Nov 08 2011.
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