A computational method for the design of nested proteins by loop-directed domain insertion.

The computational design of novel nested proteins-in which the primary structure of one protein domain (insert) is flanked by the primary structure segments of another (parent)-would enable the generation of multifunctional proteins. Here we present a new algorithm, called Loop-Directed Domain Insertion (LooDo), implemented within the Rosetta software suite, for the purpose of designing nested protein domain combinations connected by flexible linker regions. Conformational space for the insert domain is sampled using large libraries of linker fragments for linker-to-parent domain superimposition followed by insert-to-linker superimposition. The relative positioning of the two domains (treated as rigid bodies) is sampled efficiently by a grid-based, mutual placement compatibility search. The conformations of the loop residues, and the identities of loop as well as interface residues, are simultaneously optimized using a generalized kinematic loop closure algorithm and Rosetta EnzymeDesign, respectively, to minimize interface energy. The algorithm was found to consistently sample near-native conformations and interface sequences for a benchmark set of structurally similar but functionally divergent domain-inserted enzymes from the α/ß hydrolase superfamily, and discriminates well between native and nonnative conformations and sequences, although loop conformations tended to deviate from the native conformations. Furthermore, in cross-domain placement tests, native insert-parent domain combinations were ranked as the best-scoring structures compared to nonnative domain combinations. This algorithm should be broadly applicable to the design of multi-domain protein complexes with any combination of inserted or tandem domain connections.

Computational Design of a Photocontrolled Cytosine Deaminase.

There is growing interest in designing spatiotemporal control over enzyme activities using noninvasive stimuli such as light. Here, we describe a structure-based, computation-guided predictive method for reversibly controlling enzyme activity using covalently attached photoresponsive azobenzene groups. Applying the method to the therapeutically useful enzyme yeast cytosine deaminase, we obtained a ∼3-fold change in enzyme activity by the photocontrolled modulation of the enzyme's active site lid structure, while fully maintaining thermostability. Multiple cycles of switching, controllable in real time, are possible. The predictiveness of the method is demonstrated by the construction of a variant that does not photoswitch as expected from computational modeling. Our design approach opens new avenues for optically controlling enzyme function. The designed photocontrolled cytosine deaminases may also aid in improving chemotherapy approaches that utilize this enzyme.

Mathematical Models of Protease-Based Enzymatic Biosensors.

An important goal of synthetic biology is to build biosensors and circuits with well-defined input-output relationships that operate at speeds found in natural biological systems. However, for molecular computation, most commonly used genetic circuit elements typically involve several steps from input detection to output signal production: transcription, translation, and post-translational modifications. These multiple steps together require up to several hours to respond to a single stimulus, and this limits the overall speed and complexity of genetic circuits. To address this gap, molecular frameworks that rely exclusively on post-translational steps to realize reaction networks that can process inputs at a time scale of seconds to minutes have been proposed. Here, we build mathematical models of fast biosensors capable of producing Boolean logic functionality. We employ protease-based chemical and light-induced switches, investigate their operation, and provide selection guidelines for their use as on-off switches. As a proof of concept, we implement a rapamycin-induced switch in vitro and demonstrate that its response qualitatively agrees with the predictions from our models. We then use these switches as elementary blocks, developing models for biosensors that can perform OR and XOR Boolean logic computation while using reaction conditions as tuning parameters. We use sensitivity analysis to determine the time-dependent sensitivity of the output to proteolytic and protein-protein binding reaction parameters. These fast protease-based biosensors can be used to implement complex molecular circuits with a capability of processing multiple inputs controllably and algorithmically. Our framework for evaluating and optimizing circuit performance can be applied to other molecular logic circuits.

Photoswitchable affinity reagents: Computational design and efficient red-light switching.

Photo-controlled affinity reagents seek to provide modular spatiotemporal control of bioactivity by conferring photo-switchability of function on an affinity reagent scaffold. Here we used Rosetta-based computational methods to screen for sites on the Fynomer affinity reagent structure for attachment of photoswitchable cross-linkers. Both established UV-based cross-linkers (azobenzene-iodoacetamide (IAC)) and an azonium-based efficient red light switchable cross-linker, piperazino-tetra-ortho-methoxy azobenzene (PIP), were then tested experimentally. Several sites compatible with Fynomer function were identified, including sites showing rapid (<10s) red light (633 nm) modulation of function. While a range of overall target binding affinities were observed, the degree of photo-switchability of Fynomer function was generally small (<2-fold). Computational models suggest that local flexibility limits the degree of switching seen in these designs.

Computational assessment of thioether isosteres.

Replacement of the sulfur atom in biologically active diaryl and heteroaryl thioethers (Ar-S-Ar', HAr-S-Ar, and HAr-S-HAr') with any of several one-atom or two-atom linkers can be expected to reduce the susceptibility of the analogue to metabolic oxidation, a well-documented problem for thioethers intended for medicinal chemistry applications. Ab initio calculations indicate how well various proposed thioether isosteric groups, including some new and unusual ones, may perform structurally and electronically in replacing the bridging sulfur atom. Four of these are calculationally evaluated as proposed substructures in Axitinib analogues. The predicted binding behavior of the latter within two different previously crystallographically characterized protein-Axitinib binding sites (VEGFR2 kinase and ABL1 T315I gatekeeper mutant kinase), and an assessment of their suitability and anticipated shortcomings, are presented.