College of Liberal Arts & Sciences

Full and Partial Agonism of a Designed Enzyme Switch.

Recent Comp Bio Pubs - Tue, 12/12/2017 - 14:11
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Full and Partial Agonism of a Designed Enzyme Switch.

ACS Synth Biol. 2016 Dec 16;5(12):1475-1484

Authors: Budiardjo SJ, Licknack TJ, Cory MB, Kapros D, Roy A, Lovell S, Douglas J, Karanicolas J

Abstract
Chemical biology has long sought to build protein switches for use in molecular diagnostics, imaging, and synthetic biology. The overarching challenge for any type of engineered protein switch is the ability to respond in a selective and predictable manner that caters to the specific environments and time scales needed for the application at hand. We previously described a general method to design switchable proteins, called "chemical rescue of structure", that builds de novo allosteric control sites directly into a protein's functional domain. This approach entails first carving out a buried cavity in a protein via mutation, such that the protein's structure is disrupted and activity is lost. An exogenous ligand is subsequently added to substitute for the atoms that were removed by mutation, restoring the protein's structure and thus its activity. Here, we begin to ask what principles dictate such switches' response to different activating ligands. Using a redesigned β-glycosidase enzyme as our model system, we find that the designed effector site is quite malleable and can accommodate both larger and smaller ligands, but that optimal rescue comes only from a ligand that perfectly replaces the deleted atoms. Guided by these principles, we then altered the shape of this cavity by using different cavity-forming mutations, and predicted different ligands that would better complement these new cavities. These findings demonstrate how the protein switch's response can be tuned via small changes to the ligand with respect to the binding cavity, and ultimately enabled us to design an improved switch. We anticipate that these insights will help enable the design of future systems that tune other aspects of protein activity, whereby, like evolved protein receptors, remolding the effector site can also adjust additional outputs such as substrate selectivity and activation of downstream signaling pathways.

PMID: 27389009 [PubMed - indexed for MEDLINE]

Novel Ricin Subunit Antigens With Enhanced Capacity to Elicit Toxin-Neutralizing Antibody Responses in Mice.

Recent Comp Bio Pubs - Tue, 12/05/2017 - 11:07
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Novel Ricin Subunit Antigens With Enhanced Capacity to Elicit Toxin-Neutralizing Antibody Responses in Mice.

J Pharm Sci. 2016 05;105(5):1603-1613

Authors: Wahome N, Sully E, Singer C, Thomas JC, Hu L, Joshi SB, Volkin DB, Fang J, Karanicolas J, Jacobs DJ, Mantis NJ, Middaugh CR

Abstract
RiVax is a candidate ricin toxin subunit vaccine antigen that has proven to be safe in human phase I clinical trials. In this study, we introduced double and triple cavity-filling point mutations into the RiVax antigen with the expectation that stability-enhancing modifications would have a beneficial effect on overall immunogenicity of the recombinant proteins. We demonstrate that 2 RiVax triple mutant derivatives, RB (V81L/C171L/V204I) and RC (V81I/C171L/V204I), when adsorbed to aluminum salts adjuvant and tested in a mouse prime-boost-boost regimen were 5- to 10-fold more effective than RiVax at eliciting toxin-neutralizing serum IgG antibody titers. Increased toxin neutralizing antibody values and seroconversion rates were evident at different antigen dosages and within 7 days after the first booster. Quantitative stability/flexibility relationships analysis revealed that the RB and RC mutations affect rigidification of regions spanning residues 98-103, which constitutes a known immunodominant neutralizing B-cell epitope. A more detailed understanding of the immunogenic nature of RB and RC may provide insight into the fundamental relationship between local protein stability and antibody reactivity.

PMID: 26987947 [PubMed - indexed for MEDLINE]

Computing the Dynamic Supramolecular Structural Proteome.

Recent Comp Bio Pubs - Wed, 11/29/2017 - 08:06
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Computing the Dynamic Supramolecular Structural Proteome.

PLoS Comput Biol. 2017 01;13(1):e1005290

Authors: Nussinov R, Papin JA, Vakser I

PMID: 28103234 [PubMed - indexed for MEDLINE]

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