Evolutionary advances are often fueled by unanticipated innovation. Directed evolution of a computationally designed enzyme suggests that dramatic molecular changes can also drive the optimization of primitive protein active sites. The specific activity of an artificial retro-aldolase was boosted >4,400 fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes. However, structural and mechanistic studies reveal that the engineered catalytic apparatus, consisting of a reactive lysine and an ordered water molecule, was unexpectedly abandoned in favor of a new lysine residue in a substrate binding pocket created during the optimization process. Structures of the initial in silico design, a mechanistically promiscuous intermediate, and one of the most evolved variants highlight the importance of loop mobility and supporting functional groups in the emergence of the new catalytic center. Such internal competition between alternative reactive sites may have characterized the early evolution of many natural enzymes.

Enzyme catalysts of a retro-aldol reaction have been generated by computational design using a motif that combines a lysine in a non-polar environment with water-mediated stabilization of the carbinolamine hydroxyl and β-hydroxyl groups. Here we show that the design process is robust and repeatable, with 33 new active designs constructed on 13 different protein scaffold backbones. The initial activities are not high but are increased through site-directed mutagenesis and laboratory evolution. Mutational data highlight areas for improvement in design. Different designed catalysts give different borohydride-reduced reaction intermediates, suggesting a distribution of properties of the designed enzymes that may be further explored and exploited.

The fabrication of nanoscale devices requires architectural templates on which to position functional molecules in complex arrangements. Protein scaffolds are particularly promising templates for nanomaterials due to inherent molecular recognition and self-assembly capabilities combined with genetically encoded functionalities. However, difficulties in engineering protein quaternary structure into stable and well-ordered shapes have hampered progress. Here we report the development of an ultrastable biomolecular construction kit for the assembly of filamentous proteins into geometrically defined templates of controllable size and symmetry. The strategy combines redesign of protein–protein interaction specificity with the creation of tunable connector proteins that govern the assembly and projection angles of the filaments. The functionality of these nanoarchitectures is illustrated by incorporation of nanoparticles at specific locations and orientations to create hybrid materials such as conductive nanowires. These new structural components facilitate the manufacturing of nanomaterials with diverse shapes and functional properties over a wide range of processing conditions.

We report the cocrystal structures of a computationally designed and experimentally optimized retro-aldol enzyme with covalently bound substrate analogs. The structure with covalently bound substrate analog is similar but not identical to the design model, with an RMSD over the active site residues and equivalent substrate atoms of 1.4Å. As in the design model, the binding pocket orients the substrate through hydrophobic interactions with the naphthyl moiety such that the oxygen atoms analogous to the carbinolamine and β-hydroxyl oxygens are positioned near a network of bound waters. However, there are differences between the design model and the structure: the orientation of the naphthyl group and the conformation of the catalytic lysine are slightly different; the bound water network appears to be more extensive; and the bound substrate analog exhibits more conformational heterogeneity than in typical native enzyme-inhibitor complexes. Alanine scanning of the active site residues shows that both the catalytic lysine and the residues around the binding pocket for the substrate naphthyl group make critical contributions to catalysis. Mutating the set of water-coordinating residues also significantly reduces catalytic activity. The crystal structure of the enzyme with a smaller substrate analogue that lacks the naphthyl rings shows the catalytic lysine to be more flexible than in the naphthyl substrate complex; increased preorganization of the active site would likely improve catalysis. The covalently bound complex structures and mutagenesis data highlight strengths and weaknesses of the de novo enzyme design strategy.

Self-assembling protein templates have enormous potential as biomaterials for the fabrication of multifunctional nanostructures that require precise positioning of individual molecules in regular patterns over large surface areas. Furthermore, the development of protein templates that are stable under extreme conditions of heat or chemical denaturants will expand processing conditions and end-use applications for biomaterials that require exceptional stability and robustness. In the present work, we characterized the high thermal stability of a filamentous protein template, the γ-prefoldin (γPFD) from the hyperthermophile Methanocaldococcus jannaschii, and subsequently used rational design to further enhance the filament's thermal stability for application as a biotemplate in the creation of platinum nanowires. The γPFD assembles into long fibers with lengths that exceed 2 μm, which when heated to various temperatures and examined by transmission electron microscopy, revealed a T(m) of 93°C for the quaternary filament structure. Subsequently, we increased the hydrophobicity of the α-helices of the γPFD's coiled-coil, which appeared to strengthen the filamentous structure, leading to filaments of greater length at elevated temperatures. These enhanced filaments functioned as templates for the synthesis of platinum nanowires at unprecedented temperatures, and may create new opportunities for other applications of nanoscale biotemplates that require exceptional thermal stability. See accompanying commentary by Jonathan S. Dordick DOI: 10.1002/biot.201200338.