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The genetic code, elucidated in the 1960s through the work of Nirenberg, Ochoa, and Khorana, provides a set of molecular instructions for turning DNA into proteins. What if we could change the meaning of those instructions and decide for ourselves how to interpret the genetic code? Over the last decade, cells have been outfitted with modified molecular machinery that enables them to use non-standard sets of amino acids to make proteins.  These developments are leading to new approaches to macromolecular design, protein evolution, biological imaging, and proteome-wide analysis of cellular processes.

Specific Examples

S-Hydroxylation of Insulin at ProB28 Accelerates Hexamer Dissociation and Delays Fibrillation

Lieblich SA, Fang KY, Cahn JKB, Rawson J, LeBon J, Ku HT, Tirrell DA. J. Am Chem. Soc. 139(25) 8384-87 (2017)

Insulin Abstract

Daily injections of insulin provide lifesaving benefits to millions of diabetics. But currently available prandial insulins are suboptimal: The onset of action is delayed by slow dissociation of the insulin hexamer in the subcutaneous space, and insulin forms amyloid fibrils upon storage in solution. Here we show, through the use of noncanonical amino acid mutagenesis, that replacement of the proline residue at position 28 of the insulin B-chain (ProB28) by (4S)-hydroxyproline (Hzp) yields an active form of insulin that dissociates more rapidly, and fibrillates more slowly, than the wild-type protein. Crystal structures of dimeric and hexameric insulin preparations suggest that a hydrogen bond between the hydroxyl group of Hzp and a backbone amide carbonyl positioned across the dimer interface may be responsible for the altered behavior. The effects of hydroxylation are stereospecific; replacement of ProB28 by (4R)-hydroxyproline (Hyp) causes little change in the rates of fibrillation and hexamer disassociation. These results demonstrate a new approach that fuses the concepts of medicinal chemistry and protein design, and paves the way to further engineering of insulin and other therapeutic proteins.'


Cell-specific proteomic analysis in Caenorhabditis elegans.
Yuet KP, Doma MK, Ngo JT, Sweredoski MJ, Graham RLJ, Moradian A, Hess S, Schuman EM, Sternberg PW, Tirrell DA. Proc Natl Acad Sci USA. 112(9): 2705-10 (2015).

The emergence of mass spectrometry-based proteomics has revolutionized the study of proteins and their abundances, functions, interactions, and modifications. However, it is difficult to monitor dynamic changes in protein synthesis in a specific cell type within its native environment. Here we describe a method that enables the metabolic labeling, purification, and analysis of proteins in specific cell types and during defined periods in live animals. Using Caenorhabditis elegans, we show that labeling can be restricted to body wall muscles, intestinal epithelial cells, neurons, pharyngeal muscle, and cells that respond to heat shock. By coupling our methodology with isotopic labeling, we successfully identify proteins—including proteins with previously unknown expression patterns—expressed in targeted subsets of cells.

SutA is a bacterial transcription factor expressed during slow growth in Pseudomonas aeruginosa.
Babin BM, Bergkessel M, Sweredoski MJ, Moradian A, Hess S, Newman DK, Tirrell DAProc Natl Acad Sci USA. 113(5):E597-605. (2016).

Pathogens that are dormant or growing slowly play important roles in chronic infections, but studying how cells adapt to these conditions is difficult experimentally. This work demonstrates that time-selective analysis of cellular protein synthesis, using bioorthogonal noncanonical amino acid tagging (BONCAT), can provide the sensitivity needed to identify important factors in slow-growth physiology. We identified in Pseudomonas aeruginosa, a previously uncharacterized transcriptional regulator that is expressed preferentially under slow-growth conditions, binds RNA polymerase, and has widespread effects on gene expression. This factor is one of several proteins of unknown function identified in our proteomic analysis, and our results suggest that further characterization of fundamental cellular processes under these conditions will shed light on important and understudied realms of biology.

Bioorthogonal Chemoenzymatic Functionalization of Calmodulin for Bioconjugation Applications.
Kulkarni C, Lo M, Fraseur JG, Tirrell DA, Kinzer-Ursem TL. Bioconjug Chem. 26(10): 2153-60. (2015). 

Calmodulin (CaM) is a widely studied Ca2+-binding protein that is highly conserved across species and involved in many biological processes, including vesicle release, cell proliferation, and apoptosis. To facilitate biophysical studies of CaM, researchers have tagged and mutated CaM at various sites, enabling its conjugation to fluorophores, microarrays, and other reactive partners. However, previous attempts to add a reactive label to CaM for downstream studies have generally employed nonselective labeling methods or resulted in diminished CaM function. Here we report the first engineered CaM protein that undergoes site-specific and bioorthogonal labeling while retaining wild-type activity levels. By employing a chemoenzymatic labeling approach, we achieved selective and quantitative labeling of the engineered CaM protein with an N-terminal 12-azidododecanoic acid tag; notably, addition of the tag did not interfere with the ability of CaM to bind Ca2+ or a partner protein. The specificity of our chemoenzymatic labeling approach also allowed for selective conjugation of CaM to reactive partners in bacterial cell lysates, without intermediate purification of the engineered protein. Additionally, we prepared CaM-affinity resins that were highly effective in purifying a representative CaM-binding protein, demonstrating that the engineered CaM remains active even after surface capture. Beyond studies of CaM and CaM-binding proteins, the protein engineering and surface capture methods described here should be translatable to other proteins and other bioconjugation applications.

Synthesis of bioactive protein hydrogels by genetically encoded SpyTag-SpyCatcher chemistry
Sun F, Zhang W-B, Mahdavi A, Arnold FH, Tirrell DA. Proc Natl Acad Sci USA. 111: 11269-11274 (2014).


Advances in tissue engineering and regenerative medicine have created a need for new biomaterial scaffolds that facilitate cell encapsulation and transplantation. Here we present an approach to the synthesis of artificial protein scaffolds that form spontaneously under physiological conditions. These protein hydrogels may be designed to include cell-adhesion ligands, protease cleavage sites, and full-length globular proteins that carry the information needed to program the behavior of encapsulated cells. We demonstrate the approach by encapsulating mouse embryonic stem cells in a protein scaffold that includes leukemia inhibitory factor (LIF); the encapsulated cells remain pluripotent in the absence of added LIF. The results presented here illustrate a versatile strategy for the creation of information-rich biomaterials.

Programming Molecular Association and Viscoelastic Behavior in Protein Networks.
Dooling LJ, Buck ME, Zhang WB, Tirrell DA. Adv Mat. (2016)

A set of recombinant artificial proteins that can be cross-linked, by either covalent bonds or association of helical domains or both, is described. The designed proteins can be used to construct molecular networks in which the mechanism of cross-linking determines the time-dependent responses to mechanical deformation.