John D. Fisk

Live‐Cell Imaging of Cellular Proteins by a Strain‐Promoted Azide–Alkyne Cycloaddition

Live and let dye: Three coumarin-cyclooctyne conjugates have been used to label proteins tagged with azidohomoalanine in Rat-1 fibroblasts. All three fluorophores labeled intracellular proteins with fluorescence enhancements ranging from eight- to 20-fold. These conjugates are powerful tools for visualizing biomolecule dynamics in living cells.

Influence of Strand Number on Antiparallel β-Sheet Stability in Designed Three- and Four-stranded β-Sheets

We describe experiments that probe whether antiparallel beta-sheet secondary structure becomes more stable as the number of strands increases. Several groups, including ours, have explored this issue with peptides designed to adopt three-stranded beta-sheet conformations, but the conclusions have not been consistent. In this study, we examine the effect on conformational stability of beta-sheet lengthening perpendicular to the strand direction via analysis of designed peptides that adopt three-stranded or four-stranded antiparallel beta-sheet conformations in aqueous solution. The findings reported here, along with the context provided by earlier studies, suggest that antiparallel beta-sheet does, in general, become more stable when the number of strands is increased from two to three. We show that this conclusion is not influenced by the rigidity of the loop segment used to link adjacent beta-strands (D-Pro-Gly versus Asn-Gly). We show that further extension, from three strands to four, leads to a further increase in antiparallel beta-sheet stability.

A BODIPY-Cyclooctyne for Protein Imaging in Live Cells

Cellular proteins that bear reactive azides can be imaged by fluorescence microscopy following strain-promoted ligation to cyclooctyne dyes. Here we describe BODIPY-cyclooctyne (BDPY), a membrane-permeant fluorophore that can be used to label intracellular proteins in live mammalian cells. Flow cytometry reveals fluorescence signals more than 25-fold above background after labeling of azide-tagged cells with BDPY.

Phage display selection of tight specific binding variants from a hyperthermostable Sso7d scaffold protein library.

Antibodies, the quintessential biological recognition molecules, are not ideal for many applications because of their large size, complex modifications, and thermal and chemical instability. Identifying alternative scaffolds that may be evolved into tight, specific binding molecules with improved physical properties is of increasing interest, particularly for biomedical applications in resource‐limited environments. Hyperthermophilic organisms, such as Sulfolobus solfataricus, are an attractive source of highly stable proteins that may serve as starting points for alternative molecular recognition scaffolds. We describe the first application of phage display to identify binding proteins based on the S. solfataricus protein Sso7d scaffold. Sso7d is a small cysteine‐free DNA‐binding protein (approximately 7 kDa, 63 amino acids), with a melting temperature of nearly 100 °C. Tight‐binding Sso7d variants were selected for a diverse set of protein targets from a 1010 member library, demonstrating the versatility of the scaffold. These Sso7d variants are able to discriminate among closely related human, bovine and rabbit serum albumins. Equilibrium dissociation constants in the nanomolar to low micromolar range were measured via competitive ELISA. Importantly, the Sso7d variants continue to bind their targets in the absence of the phage context. Furthermore, phage‐displayed Sso7d variants retain their binding affinity after exposure to temperatures up to 70 °C. Taken together, our results suggest that the Sso7d scaffold will be a complementary addition to the range of non‐antibody scaffold proteins that may be utilized in phage display. Variants of hyperthermostable binding proteins have potential applications in diagnostics and therapeutics for environments with extreme conditions of storage and deployment.

Modification of orthogonal tRNAs : unexpected consequences for sense codon reassignment

Breaking the degeneracy of the genetic code via sense codon reassignment has emerged as a way to incorporate multiple copies of multiple non-canonical amino acids into a protein of interest. Here, we report the modification of a normally orthogonal tRNA by a host enzyme and show that this adventitious modification has a direct impact on the activity of the orthogonal tRNA in translation. We observed nearly equal decoding of both histidine codons, CAU and CAC, by an engineered orthogonal M. jannaschii tRNA with an AUG anticodon: tRNAOpt. We suspected a modification of the tRNAOptAUG anticodon was responsible for the anomalous lack of codon discrimination and demonstrate that adenosine 34 of tRNAOptAUG is converted to inosine. We identified tRNAOptAUG anticodon loop variants that increase reassignment of the histidine CAU codon, decrease incorporation in response to the histidine CAC codon, and improve cell health and growth profiles. Recognizing tRNA modification as both a potential pitfall and avenue of directed alteration will be important as the field of genetic code engineering continues to infiltrate the genetic codes of diverse organisms.

Simulation of the M13 life cycle I: Assembly of a genetically-structured deterministic chemical kinetic simulation

To expand the quantitative, systems level understanding and foster the expansion of the biotechnological applications of the filamentous bacteriophage M13, we have unified the accumulated quantitative information on M13 biology into a genetically-structured, experimentally-based computational simulation of the entire phage life cycle. The deterministic chemical kinetic simulation explicitly includes the molecular details of DNA replication, mRNA transcription, protein translation and particle assembly, as well as the competing protein-protein and protein-nucleic acid interactions that control the timing and extent of phage production. The simulation reproduces the holistic behavior of M13, closely matching experimentally reported values of the intracellular levels of phage species and the timing of events in the M13 life cycle. The computational model provides a quantitative description of phage biology, highlights gaps in the present understanding of M13, and offers a framework for exploring alternative mechanisms of regulation in the context of the complete M13 life cycle.

Simulation of the M13 life cycle II: Investigation of the control mechanisms of M13 infection and establishment of the carrier state

Bacteriophage M13 is a true parasite of bacteria, able to co-opt the infected cell and control the production of progeny across many cellular generations. Here, our genetically-structured simulation of M13 is applied to quantitatively dissect the interplay between the host cellular environment and the controlling interactions governing the phage life cycle during the initial establishment of infection and across multiple cell generations. Multiple simulations suggest that phage-encoded feedback interactions constrain the utilization of host DNA polymerase, RNA polymerase and ribosomes. The simulation reveals the importance of p5 translational attenuation in controlling the production of phage double-stranded DNA and suggests an underappreciated role for p5 translational self-attenuation in resource allocation. The control elements active in a single generation are sufficient to reproduce the experimentally-observed multigenerational curing of the phage infection. Understanding the subtleties of regulation will be important for maximally exploiting M13 particles as scaffolds for nanoscale devices.

Homoisoleucine: A Translationally Active Leucine Surrogate of Expanded Hydrophobic Surface Area

Hil of a strong peptide! Homoisoleucine (Hil) serves as an effective surrogate for leucine with respect to protein translation in bacterial cells. Replacement of Leu by Hil stabilizes coiled-coil peptides, as shown by the elevation of the thermal denaturation temperature. The increase in denaturation temperature is larger than that observed previously for replacement of Leu by trifluoroleucine.

Evaluating Sense Codon Reassignment with a Simple Fluorescence Screen.

Understanding the interactions that drive the fidelity of the genetic code and the limits to which modifications can be made without breaking the translational system has practical implications for understanding the molecular mechanisms of evolution as well as expanding the set of encodable amino acids, particularly those with chemistries not provided by Nature. Because 61 sense codons encode 20 amino acids, reassigning the meaning of sense codons provides an avenue for biosynthetic modification of proteins, furthering both fundamental and applied biochemical research. We developed a simple screen that exploits the absolute requirement for fluorescence of an active site tyrosine in green fluorescent protein (GFP) to probe the pliability of the degeneracy of the genetic code. Our screen monitors the restoration of the fluorophore of GFP by incorporation of a tyrosine in response to a sense codon typically assigned another meaning in the genetic code. We evaluated sense codon reassignment at four of the 21 sense codons read through wobble interactions in Escherichia coli using the Methanocaldococcus jannaschii orthogonal tRNA/aminoacyl tRNA synthetase pair originally developed and commonly used for amber stop codon suppression. By changing only the anticodon of the orthogonal tRNA, we achieved sense codon reassignment efficiencies between 1% (Phe UUU) and 6% (Lys AAG). Each of the orthogonal tRNAs preferentially decoded the codon traditionally read via a wobble interaction in E. coli with the exception of the orthogonal tRNA with an AUG anticodon, which incorporated tyrosine in response to both the His CAU and His CAC codons with approximately equal frequencies. We applied our screen in a high-throughput manner to evaluate a 10(9)-member combined tRNA/aminoacyl tRNA synthetase library to identify improved sense codon reassigning variants for the Lys AAG codon. A single rapid screen with the ability to broadly evaluate reassignable codons will facilitate identification and improvement of the combinations of sense codons and orthogonal pairs that display efficient reassignment.

Mapping the Plasticity of the Escherichia coli Genetic Code with Orthogonal Pair-Directed Sense Codon Reassignment

The relative quantitative importance of the factors that determine the fidelity of translation is largely unknown, which makes predicting the extent to which the degeneracy of the genetic code can be broken challenging. Our strategy of using orthogonal tRNA/aminoacyl tRNA synthetase pairs to precisely direct the incorporation of a single amino acid in response to individual sense and nonsense codons provides a suite of related data with which to examine the plasticity of the code. Each directed sense codon reassignment measurement is an in vivo competition experiment between the introduced orthogonal translation machinery and the natural machinery in Escherichia coli. This report discusses 20 new, related genetic codes, in which a targeted E. coli wobble codon is reassigned to tyrosine utilizing the orthogonal tyrosine tRNA/aminoacyl tRNA synthetase pair from Methanocaldococcus jannaschii. One at a time, reassignment of each targeted sense codon to tyrosine is quantified in cells by measuring the fluorescence of GFP variants in which the essential tyrosine residue is encoded by a non-tyrosine codon. Significantly, every wobble codon analyzed may be partially reassigned with efficiencies ranging from 0.8 to 41%. The accumulation of the suite of data enables a qualitative dissection of the relative importance of the factors affecting the fidelity of translation. While some correlation was observed between sense codon reassignment and either competing endogenous tRNA abundance or changes in aminoacylation efficiency of the altered orthogonal system, no single factor appears to predominately drive translational fidelity. Evaluation of relative cellular fitness in each of the 20 quantitatively characterized proteome-wide tyrosine substitution systems suggests that at a systems level, E. coli is robust to missense mutations.