A Simple Method to Dual Site-Specifically Label a Protein Using Tryptophan Auxotrophic Escherichia coli

Site-specifically labeling proteins with multiple dyes or molecular moieties is an important yet non-trivial task for many research, such as when using Föster resonance energy transfer (FRET) to study dynamics of protein conformational change. Many strategies have been devised, but usually done on a case-by-case basis. Expanded genetic code provided a general platform to incorporate non-canonical amino acids (ncAA), which can also enable multiple site-specific labeling, but it’s technically complicated and not suitable for some applications. Here we present a streamlined method that could enable dual site-specific protein labeling by using a tryptophan auxotroph of Escherichia coli to incorporate a naturally found tryptophan analog, 5-hydroxytryptophan into a recombinant protein. As a demonstration, we incorporated 5-hydroxytryptophan into E. coli release factor 1 (RF1), a protein known to possess two different conformations, and site-specifically attached two different fluorophores, one on 5-hydroxytryptophan and another on a cysteine residue. This method is simple, generally applicable, efficient, and can serve as an alternative way for researchers who want to install an additional labeling site in their proteins.

Measuring the tolerance of the genetic code to altered codon size

SummaryProtein translation using four-base codons occurs in both natural and synthetic systems. What constraints contributed to the universal adoption of a triplet-codon, rather than quadruplet-codon, genetic code? Here, we investigate the tolerance of the E. coli genetic code to tRNA mutations that increase codon size. We found that tRNAs from all twenty canonical isoacceptor classes can be converted to functional quadruplet tRNAs (qtRNAs), many of which selectively incorporate a single amino acid in response to a specified four-base codon. However, efficient quadruplet codon translation often requires multiple tRNA mutations, potentially constraining evolution. Moreover, while tRNAs were largely amenable to quadruplet conversion, only nine of the twenty aminoacyl tRNA synthetases tolerate quadruplet anticodons. These constitute a functional and mutually orthogonal set, but one that sharply limits the chemical alphabet available to a nascent all-quadruplet code. Our results illuminate factors that led to selection and maintenance of triplet codons in primordial Earth and provide a blueprint for synthetic biologists to deliberately engineer an all-quadruplet expanded genetic code.

In situ EPR spectroscopy of a bacterial membrane transporter using an expanded genetic code

The membrane transporter BtuB is site-directedly spin labelled on the surface of living bacteria via Diels–Alder click chemistry.

Controlling Phosphate Removal with Light: The Development of Optochemical Tools to Probe Protein Phosphatase Function

Protein phosphatases play an essential role in cell signaling; however, they remain understudied compared with protein kinases, in part due to a lack of appropriate tools. In order to provide conditional control over phosphatase function, we developed two different approaches for rendering MKP3 (a dual-specific phosphatase, also termed DUSP6) activatable by light. Specifically, we expressed the protein with strategically placed light-removable protecting groups in cells with an expanded genetic code. This allowed for the acute perturbation of the Ras/MAPK signaling pathway upon photoactivation in live cells. In doing so, we confirmed that MKP3 does not act as a thresholding gate for growth factor stimulation of the extracellular signal-regulated kinase (ESRK) pathway.

Aminoacyl-tRNA Synthetases and tRNAs for an Expanded Genetic Code: What Makes them Orthogonal?

In the past two decades, tRNA molecules and their corresponding aminoacyl-tRNA synthetases (aaRS) have been extensively used in synthetic biology to genetically encode post-translationally modified and unnatural amino acids. In this review, we briefly examine one fundamental requirement for the successful application of tRNA/aaRS pairs for expanding the genetic code. This requirement is known as “orthogonality”—the ability of a tRNA and its corresponding aaRS to interact exclusively with each other and avoid cross-reactions with additional types of tRNAs and aaRSs in a given organism.