Combinatorial and Statistical Approaches to Protein Stability, Structure and Function
Proteins adopt their conformations based on their sequence of amino acids, but we still have little more than rules of thumb relating sequence to structure or thermodynamic stability. In part, this is due to the vast number of possible variants of even small proteins. For example, one molecule of every possible 50-amino acid protein sequence would have more mass than our galaxy. Moreover, it is difficult to apply traditional biophysical techniques in a high-throughput format. We can also statistically analyze databases of natural protein variants for higher-order patterns that lead to stability, such as correlated occurrences of amino acids. We have recently constructed the consensus sequence of a large enzyme family, and found that it is active but has unusual structural properties. We are re-engineering this sequence with correlated mutations to probe their effects. Our group is also interested in developing powerful tools for in vivo detection of protein-protein interactions. We are using the recently developed power of in vivo, site-specific unnatural protein mutagenesis, and we are developing a method to rapidly scan a photoaffinity label through every position of a protein to elucidate binding partners and interaction surfaces. Separately, we are improving a split-GFP reassembly screen to trap interacting proteins and applying this to four-helix bundle interfaces in tumor suppressor proteins. We apply our technologies to improve the physical properties of proteins used for therapeutic purposes. We are part of a large NIH Center that aims to improve the activity and properties of enzymes that inactivate organophosphorous nerve agents. Our lab uses a wide variety of techniques, including DNA library construction and screening, protein expression and purification, spectroscopic analysis of proteins (CD, fluorescence, NMR and X-ray crystallography), bioinformatics and synthetic organic chemistry.
Our work is funded by the NIH, including NIGMS for our combinatorial biophysics work and NINDS's CounterACT program for our work on paraoxonase-1.
Recent Ph.D. students have gone on to postdoctoral positions at the University of Texas, Austin, and The Scripps Research Institute, Florida. Undergraduate honors thesis students have gone on to graduate school at ETH Zurich, University of Washington, Seattle, and University of Colorado, Boulder, and medical school at Penn State University, Hershey, University of Toledo and The Ohio State University.
- Muthukrishnan S, Shete VS, Sanan TT, Vyas S, Oottikkal S, Porter LM, Magliery TJ & Hadad CM. (2012) "Mechanistic insights into the hydrolysis of organophosphorus compounds by paraoxonase-1: Exploring the limits of substrate tolerance in a promiscuous enzyme," J. Phys. Org. Chem., accepted.
- Durani V, Sullivan BJ & Magliery TJ. (2012) "Simplifying protein expression with ligation-free, traceless and tag-switching plasmids," Protein Expr. Purif., accepted.
- Sullivan BJ, Nguyen T, Durani V, Mathur D, Rojas S, Thomas M, Syu T & Magliery TJ. (2012) "Stabilizing proteins from sequence statistics: The interplay of conservation and correlation in triosephosphate isomerase stability," J. Mol. Biol., 420: 384-399.
- Sarkar M, Harsch CK, Matic GT, Hoffman K, Norris JR 3RD, Otto TC, Lenz DE, Cerasoli DM & Magliery TJ. (2012) "Solubilization and humanization of paraoxonase-1," J. Lipids, 2012: Article ID 610937, 13 pages.
- Sullivan BJ, Durani V & Magliery TJ. (2011) "Triosephosphate Isomerase by Consensus Design: Dramatic Differences in Physical Properties and Activity of Related Variants," J. Mol. Biol., 413: 195-208.
- Magliery TJ, Lavinder JJ & Sullivan BJ. (2011) "Protein stability by number: high-throughput and statistical approaches to one of protein science's most difficult problems," Curr. Opin. Chem. Biol. 15: 443-451.
- Nie L, Lavinder JJ, Sarkar M, Stephany K & Magliery TJ. (2011) "Synthetic approach to stop-codon scanning mutagenesis," J. Am. Chem. Soc. 133: 6177-6186.
- Hari SB, Byeon C, Lavinder JJ & Magliery TJ. (2010) "Cysteine-free rop: A four-helix bundle core mutant has wild-type stability and structure but dramatically different unfolding kinetics," Protein Sci. 19: 670-679.
- Nadaud PS, Sarkar M, Wu B, MacPhee CE, Magliery TJ & Jaroniec CP. (2009) "Expression and purification of a recombinant amyloidogenic peptide from transthyretin for solid-state NMR spectroscopy," Protein Expr. Purif. 70: 101-108.
- Otto TC, Harsh CK, Yeung DT, Magliery TJ, Cerasoli DM & Lenz DE (2009) "Dramatic difference in organophosphorpus hydrolase activity between huam and chimeric recombinant mammalian paraoxonase-1 enzymes," Biochemistry 48: 10416-10422.