Codon Selection Affects Recruitment of Ribosome-Associating Factors during Translation.

An intriguing aspect of protein synthesis is how cotranslational events are managed inside the cell. In this study, we developed an in vivo bimolecular fluorescence complementation assay coupled to SecM stalling (BiFC-SecM) to study how codon usage influences the interactions of ribosome-associating factors that occur cotranslationally. We profiled ribosomal associations of a number of proteins, and observed differential association of chaperone proteins TF, DnaK, GroEL, and translocation factor Ffh as a result of introducing synonymous codon substitutions that change the affinity of the translating sequence to the ribosomal anti-Shine-Dalgarno (aSD) sequence. The use of pausing sequences within proteins regulates their transit within the translating ribosome. Our results indicate that the dynamics between cellular factors and the new polypeptide chain are affected by how codon composition is designed. Furthermore, associating factors may play a role in processes including protein quality control (folding and degradation) and cellular respiration.

Slowing Translation between Protein Domains by Increasing Affinity between mRNAs and the Ribosomal Anti-Shine-Dalgarno Sequence Improves Solubility.

Recent studies have demonstrated that effective protein production requires coordination of multiple cotranslational cellular processes, which are heavily affected by translation timing. Until recently, protein engineering has focused on codon optimization to maximize protein production rates, mostly considering the effect of tRNA abundance. However, as it relates to complex multidomain proteins, it has been hypothesized that strategic translational pauses between domains and between distinct individual structural motifs can prevent interactions between nascent chain fragments that generate kinetically trapped misfolded peptides and thereby enhance protein yields. In this study, we introduce synthetic transient pauses between structural domains in a heterologous model protein based on designed patterns of affinity between the mRNA and the anti-Shine-Dalgarno (aSD) sequence on the ribosome. We demonstrate that optimizing translation attenuation at domain boundaries can predictably affect solubility patterns in bacteria. Exploration of the affinity space showed that modifying less than 1% of the nucleotides (on a small 12 amino acid linker) can vary soluble protein yields up to ∼7-fold without altering the primary sequence of the protein. In the context of longer linkers, where a larger number of distinct structural motifs can fold outside the ribosome, optimal synonymous codon variations resulted in an additional 2.1-fold increase in solubility, relative to that of nonoptimized linkers of the same length. While rational construction of 54 linkers of various affinities showed a significant correlation between protein solubility and predicted affinity, only weaker correlations were observed between tRNA abundance and protein solubility. We also demonstrate that naturally occurring high-affinity clusters are present between structural domains of ß-galactosidase, one of Escherichia coli's largest native proteins. Interdomain ribosomal affinity is an important factor that has not previously been explored in the context of protein engineering.

Influenza virus-membrane fusion triggered by proton uncaging for single particle studies of fusion kinetics.

We report a method for studying membrane fusion, focusing on influenza virus fusion to lipid bilayers, which provides high temporal resolution through the rapid and coordinated initiation of individual virus fusion events. Each fusion event proceeds through a series of steps, much like multistep chemical reaction. Fusion is initiated by a rapid decrease in pH that accompanies the "uncaging" of an effector molecule from o-nitrobenzaldehyde, a photoisomerizable compound that releases a proton to the surrounding solution within microseconds of long-wave ultraviolet irradiation. In order to quantify pH values upon UV irradiation and uncaging, we introduce a simple silica nanoparticle pH sensor, useful for reporting the pH in homogeneous nanoliter volumes under conditions where traditional organic dye-type pH probes fail. Subsequent single-virion fusion events are monitored using total internal reflection fluorescence microscopy. Statistical analysis of these stochastic events uncovers kinetic information about the fusion reaction. This approach reveals that the kinetic parameters obtained from the data are sensitive to the rate at which protons are delivered to the bound viruses. Higher resolution measurements can enhance fundamental fusion studies and aid antiviral antifusogenic drug development.