Engineering Heterodimeric Kinesins through Genetic Incorporation of Noncanonical Amino Acids

Kinesins are commonly homodimers with two identical heavy chains (protomers) and play indispensable roles in many intracellular processes. Engineered heterodimeric kinesins with two distinct protomers are important tools for dissecting coordination and regulation of naturally occurring kinesin homodimers. Here, we report a chemical-biology-based approach that generates kinesin heterodimers by combining genetic incorporation of reactive noncanonical amino acids and small-molecule-based cross-linking. We verified using yeast kinesin-8/Kip3 as a model system that our method yields kinesin heterodimers of desired properties without introducing unintended motility disruption. To demonstrate the utility of our method, we engineered a crippled Kip3 heterodimer that contains both a wild-type-like protomer and a catalytically inactive one, and our results revealed that the resulting heterodimer moves on the microtubule with a significant reduction in velocity but not processivity. Due to its versatility, we expect that our method can be broadly adopted to create novel heterodimers for other kinesins and will thus greatly expand the studies on kinesin mechanisms

Improved Incorporation of Noncanonical Amino Acids by an Engineered tRNA^Tyr Suppressor

The <i>Methanocaldcoccus jannaschii</i> tyrosyl-tRNA synthetase (TyrRS):tRNA^Tyr cognate pair has been used to incorporate a large number of noncanonical amino acids (ncAAs) into recombinant proteins in <i>Escherichia coli</i>. However, the structural elements of the suppressor tRNA^Tyr used in these experiments have not been examined for optimal performance. Here, we evaluate the steady-state kinetic parameters of wild-type <i>M. jannaschii</i> TyrRS and an evolved 3-nitrotyrosyl-tRNA synthetase (nitroTyrRS) toward several engineered tRNA^Tyr suppressors, and we correlate aminoacylation properties with the efficiency and fidelity of superfolder green fluorescent protein (sfGFP) synthesis <i>in vivo</i>. Optimal ncAA-sfGFP synthesis correlates with improved aminoacylation kinetics for a tRNA^Tyr amber suppressor with two substitutions in the anticodon loop (G34C/G37A), while four additional mutations in the D and variable loops, present in the tRNA^Tyr used in all directed evolution experiments to date, are deleterious to function both <i>in vivo</i> and <i>in vitro</i>. These findings extend to three of four other evolved TyrRS enzymes that incorporate distinct ncAAs. Suppressor tRNAs elicit decreases in amino acid <i>K</i>_m values for both TyrRS and nitroTyrRS, suggesting that direct anticodon recognition by TyrRS need not be an impediment to superior performance of this orthogonal system and offering insight into novel approaches for directed evolution. The G34C/G37A tRNA^Tyr may enhance future incorporation of many ncAAs by engineered TyrRS enzymes

Doping of Green Fluorescent Protein into Superfluid Helium Droplets: Size and Velocity of Doped Droplets

We report doping of green fluorescent protein from an electrospray ionization (ESI) source into superfluid helium droplets. From analyses of the time profiles of the doped droplets, we identify two distinct groups of droplets. The faster group has a smaller average size, on the order of 10⁶ helium atoms/droplet, and the slower group is much larger, by at least an order of magnitude. The relative populations of these two groups depend on the temperature of the droplet source: from 11 to 5 K, the signal intensity of the slower droplet group gradually increases, from near the detection limit to comparable to that of the faster group. We postulate that the smaller droplets are formed via condensation of gaseous helium upon expansion from the pulsed valve, while the larger droplets develop from fragmentation of ejected liquid helium. Our results on the size and velocity of the condensation peak at higher source temperatures (>7 K) agree with previous reports, but those at lower temperatures (<7 K) seem to be off. We attribute this discrepancy to the masking effect of the exceedingly large droplets from the fragmentation peak in previous measurements of droplet sizes. Within the temperature range of our investigation, although the expansion condition changes from subcritical to supercritical, there is no abrupt change in either the velocity distribution or the size distribution of the condensation peak, and the most salient effect is in the increasing intensity of the fragmentation peak. The absolute doping efficiency, as expressed by the ratio of ion-doped droplets over the total number of ions from the ESI source, is on the order of 10^–4, while only hundreds of doped ions have been detected. Further improvements in the ESI source are key to extending the technology for future experiments. On the other hand, the separation of the two groups of droplets in velocity is beneficial for size selection of only the smaller droplets for future experiments of electron diffraction

Ideal Bioorthogonal Reactions Using A Site-Specifically Encoded Tetrazine Amino Acid

Bioorthogonal reactions for labeling biomolecules in live cells have been limited by slow reaction rates or low component selectivity and stability. Ideal bioorthogonal reactions with high reaction rates, high selectivity, and high stability would allow for stoichiometric labeling of biomolecules in minutes and eliminate the need to wash out excess labeling reagent. Currently, no general method exists for controlled stoichiometric or substoichiometric labeling of proteins in live cells. To overcome this limitation, we developed a significantly improved tetrazine-containing amino acid (Tet-v2.0) and genetically encoded Tet-v2.0 with an evolved aminoacyl-tRNA synthetase/tRNA_(CUA) pair. We demonstrated <i>in cellulo</i> that protein containing Tet-v2.0 reacts selectively with cyclopropane-fused <i>trans</i>-cyclooctene (sTCO) with a bimolecular rate constant of 72,500 ± 1660 M^–1 s^–1 without reacting with other cellular components. This bioorthogonal ligation of Tet-v2.0-protein reacts <i>in cellulo</i> with substoichiometric amounts of sTCO-label fast enough to remove the labeling reagent from media in minutes, thereby eliminating the need to wash out label. This ideal bioorthogonal reaction will enable the monitoring of a larger window of cellular processes in real time

ATRP under Biologically Relevant Conditions: Grafting from a Protein

Atom transfer radical polymerization (ATRP) methods were developed in water-based media, to grow polymers from proteins under biologically relevant conditions. These conditions gave good control over the resulting polymers, while still preserving the protein’s native structure. Several reaction parameters, such as ligand structure, halide species, and initiation mode were optimized in water and PBS buffer to yield well-defined polymers grown from bovine serum albumin (BSA), functionalized with cleavable ATRP initiators (I). The CuCl complex with ligand 2,2′-bipyridyne (bpy) provides the best conditions for the polymerization of oligo­(ethylene oxide) methacrylate (OEOMA) in water at 30 °C under normal ATRP conditions (I/CuCl/CuCl₂/bpy = 1/1/9/22), while the CuBr/bpy complex gave better performance in PBS. Activators generated by electron transfer (AGET) ATRP gave well-controlled polymerization of OEOMA at 30 °C with the ligand tris­(2-pyridylmethyl)­amine (TPMA), (I/CuBr₂/TPMA = 1/10/11). The AGET ATRP reactions required slow feeding of a very small amount of ascorbic acid into the aqueous reaction medium or buffer. The reaction conditions developed were used to create a smart, thermoresponsive, protein–polymer hybrid

1,2,4-Triazines Are Versatile Bioorthogonal Reagents

A new class of bioorthogonal reagents, 1,2,4-triazines, is described. These scaffolds are stable in biological media and capable of robust reactivity with <i>trans</i>-cyclooctene (TCO). The enhanced stability of the triazine scaffold enabled its direct use in recombinant protein production. The triazine–TCO reaction can also be used in tandem with other bioorthogonal cycloaddition reactions. These features fill current voids in the bioorthogonal toolkit

Structural Basis of Improved Second-Generation 3‑Nitro-tyrosine tRNA Synthetases

Genetic code expansion has provided the ability to site-specifically incorporate a multitude of noncanonical amino acids (ncAAs) into proteins for a wide variety of applications, but low ncAA incorporation efficiency can hamper the utility of this powerful technology. When investigating proteins containing the post-translational modification 3-nitro-tyrosine (nitroTyr), we developed second-generation amino-acyl tRNA synthetases (RS) that incorporate nitroTyr at efficiencies roughly an order of magnitude greater than those previously reported and that advanced our ability to elucidate the role of elevated cellular nitroTyr levels in human disease (e.g., Franco, M. et al. Proc. Natl. Acad. Sci. U.S.A 2013, 110, E1102). Here, we explore the origins of the improvement achieved in these second-generation RSs. Crystal structures of the most efficient of these synthetases reveal the molecular basis for the enhanced efficiencies observed in the second-generation nitroTyr-RSs. Although Tyr is not detectably incorporated into proteins when expression media is supplemented with 1 mM nitroTyr, a major difference between the first- and second-generation RSs is that the second-generation RSs have an active site more compatible with Tyr binding. This feature of the second-generation nitroTyr-RSs appears to be the result of using less stringent criteria when selecting from a library of mutants. The observation that a different selection strategy performed on the same library of mutants produced nitroTyr-RSs with dramatically improved efficiencies suggests the optimization of established selection protocols could lead to notable improvements in ncAA-RS efficiencies and thus the overall utility of this technology

Increasing Enzyme Stability and Activity through Hydrogen Bond-Enhanced Halogen Bonds

The construction of more stable proteins is important in biomolecular engineering, particularly in the design of biologics-based therapeutics. We show here that replacing the tyrosine at position 18 (Y18) of T4 lysozyme with the unnatural amino acid <i>m</i>-chlorotyrosine (^<i>m</i>ClY) increases both the thermal stability (increasing the melting temperature by ∼1 °C and the melting enthalpy by 3 kcal/mol) and the enzymatic activity at elevated temperatures (15% higher than that of the parent enzyme at 40 °C) of this classic enzyme. The chlorine of ^<i>m</i>ClY forms a halogen bond (XB) to the carbonyl oxygen of the peptide bond at glycine 28 (G28) in a tight loop near the active site. In this case, the XB potential of the typically weak XB donor Cl is shown from quantum chemical calculations to be significantly enhanced by polarization via an intramolecular hydrogen bond (HB) from the adjacent hydroxyl substituent of the tyrosyl side chain, resulting in a distinctive synergistic HB-enhanced XB (or HeX-B for short) interaction. The larger halogens (bromine and iodine) are not well accommodated within this same loop and, consequently, do not exhibit the effects on protein stability or function associated with the HeX-B interaction. Thus, we have for the first time demonstrated that an XB can be engineered to stabilize and increase the activity of an enzyme, with the increased stabilizing potential of the HeX-B further extending the application of halogenated amino acids in the design of more stable protein therapeutics