Thomas J. Magliery

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Identification of protein binding partners is one of the key challenges of proteomics. We recently introduced a screen for detecting protein-protein interactions based on reassembly of dissected fragments of green fluorescent protein fused to interacting peptides. Here, we present a set of comaintained Escherichia coli plasmids for the facile subcloning of(More)
Human serum paraoxonase-1 (HuPON1) has the capacity to hydrolyze aryl esters, lactones, oxidized phospholipids, and organophosphorus (OP) compounds. HuPON1 and bacterially expressed chimeric recombinant PON1s (G2E6 and G3C9) differ by multiple amino acids, none of which are in the putative enzyme active site. To address the importance of these amino acid(More)
Consensus design methods have been used successfully to engineer proteins with a particular fold, and moreover to engineer thermostable exemplars of particular folds. Here, we consider how a statistical free energy approach can expand upon current methods of phylogenetic design. As an example, we have analyzed the tetratricopeptide repeat (TPR) motif, using(More)
Naturally occurring tRNA mutants are known that suppress +1 frameshift mutations by means of an extended anticodon loop, and a few have been used in protein mutagenesis. In an effort to expand the number of possible ways to uniquely and efficiently encode unnatural amino acids, we have devised a general strategy to select tRNAs with the ability to suppress(More)
Consensus design, the selection of mutations based on the most common amino acid in each position of a multiple sequence alignment, has proven to be an efficient way to engineer stabilized mutants and even to design entire proteins. However, its application has been limited to small motifs or small families of highly related proteins. Also, we have little(More)
The low stability of natural proteins often limits their use in therapeutic, industrial, and research applications. The scale and throughput of methods such as circular dichroism, fluorescence spectroscopy, and calorimetry severely limit the number of variants that can be examined. Here we demonstrate a high-throughput thermal scanning (HTTS) method for(More)
BACKGROUND In an effort to expand further our ability to manipulate protein structure, we have completed the first step towards a general method that allows the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the construction of an 'orthogonal' suppressor tRNA that is uniquely acylated in vivo, by an(More)
Understanding the determinants of protein stability remains one of protein science's greatest challenges. There are still no computational solutions that calculate the stability effects of even point mutations with sufficient reliability for practical use. Amino acid substitutions rarely increase the stability of native proteins; hence, large libraries and(More)
Cysteine residues can complicate the folding and storage of proteins due to improper formation of disulfide bonds or oxidation of residues that are natively reduced. Wild-type Rop is a homodimeric four-helix bundle protein and an important model for protein design in the understanding of protein stability, structure and folding kinetics. In the native(More)