Folding and stability of proteins have emerged as key elements in the molecular understanding of diseases like cancer and Alzheimer?s, yet a complete understanding of these phenomena remains elusive. One highly productive approach has been the systematic redesign of proteins. A significant limitation to redesign is the characterization of the enormous number of possible variants that can be engineered. Methods for the analysis of large libraries of variants of well-studied proteins are highly desirable. An extremely well-studied model for protein folding and stability is the four-helix bundle protein Rop, which modulates ColE 1 plasmid copy number. This will serve as the basis for a screen for Rop function, wherein the production of a marker like green fluorescent protein from a ColE 1 plasmid reflects the integrity of a Rop protein variant. Such a screen will be used to identify active Rop variants from large libraries containing exhaustive mutations in loop or core residues. One interesting problem is the identification of parameters that control overall conformation. Recent experiments show that proteins can fold into highly disparate threedimensional structures despite high homology (for example, a variant of the alpha/beta B1 domain folds into the coiled-coil structure of Rop). Rop-like structures have been stabilized by metal ions through the engineering of novel metal-binding sites. We will examine the limits of conformational switching by using metal binding to stabilize a Rop-like structure from a B1 variant. We are also interested in isolating B1 variants that recapitulate Rop function. A screen for Rop function will allow us to radically expand the scope of protein redesign and refine our understanding of the parameters that control protein folding and stability.
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| Magliery, Thomas J; Regan, Lynne (2005) Sequence variation in ligand binding sites in proteins. BMC Bioinformatics 6:240 |
| Magliery, Thomas J; Wilson, Christopher G M; Pan, Weilan et al. (2005) Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism. J Am Chem Soc 127:146-57 |
| Magliery, Thomas J; Regan, Lynne (2004) A cell-based screen for function of the four-helix bundle protein Rop: a new tool for combinatorial experiments in biophysics. Protein Eng Des Sel 17:77-83 |
| Magliery, Thomas J; Regan, Lynne (2004) Combinatorial approaches to protein stability and structure. Eur J Biochem 271:1595-608 |
| Magliery, Thomas J; Regan, Lynne (2004) Beyond consensus: statistical free energies reveal hidden interactions in the design of a TPR motif. J Mol Biol 343:731-45 |
