An evolution-based model for designing chorismate mutase enzymes.

The rational design of enzymes is an important goal for both fundamental and practical reasons. Here, we describe a process to learn the constraints for specifying proteins purely from evolutionary sequence data, design and build libraries of synthetic genes, and test them for activity in vivo using a quantitative complementation assay. For chorismate mutase, a key enzyme in the biosynthesis of aromatic amino acids, we demonstrate the design of natural-like catalytic function with substantial sequence diversity. Further optimization focuses the generative model toward function in a specific genomic context. The data show that sequence-based statistical models suffice to specify proteins and provide access to an enormous space of functional sequences. This result provides a foundation for a general process for evolution-based design of artificial proteins.

Learning the pattern of epistasis linking genotype and phenotype in a protein.

Understanding the pattern of epistasis-the non-independence of mutations-is critical for relating genotype and phenotype. However, the combinatorial complexity of potential epistatic interactions has severely limited the analysis of this problem. Using new mutational approaches, we report a comprehensive experimental study of all 213 mutants that link two phenotypically distinct variants of the Entacmaea quadricolor fluorescent protein-an opportunity to examine epistasis up to the 13th order. The data show the existence of many high-order epistatic interactions between mutations, but also reveal extraordinary sparsity, enabling novel experimental and computational strategies for learning the relevant epistasis. We demonstrate that such information, in turn, can be used to accurately predict phenotypes in practical situations where the number of measurements is limited. Finally, we show how the observed epistasis shapes the solution space of single-mutation trajectories between the parental fluorescent proteins, informative about the protein's evolutionary potential. This work provides conceptual and experimental strategies to profoundly characterize epistasis in a protein, relevant to both natural and laboratory evolution.

Electric-field-stimulated protein mechanics.

The internal mechanics of proteins-the coordinated motions of amino acids and the pattern of forces constraining these motions-connects protein structure to function. Here we describe a new method combining the application of strong electric field pulses to protein crystals with time-resolved X-ray crystallography to observe conformational changes in spatial and temporal detail. Using a human PDZ domain (LNX2PDZ2) as a model system, we show that protein crystals tolerate electric field pulses strong enough to drive concerted motions on the sub-microsecond timescale. The induced motions are subtle, involve diverse physical mechanisms, and occur throughout the protein structure. The global pattern of electric-field-induced motions is consistent with both local and allosteric conformational changes naturally induced by ligand binding, including at conserved functional sites in the PDZ domain family. This work lays the foundation for comprehensive experimental study of the mechanical basis of protein function.

Evolution-based design of proteins.

Statistical analysis of protein sequences indicates an architecture for natural proteins in which amino acids are engaged in a sparse, hierarchical pattern of interactions in the tertiary structure. This architecture might be a key and distinguishing feature of evolved proteins-a design principle providing not only for foldability and high-performance function but also for robustness to perturbation and the capacity for rapid adaptation to new selection pressures. Here, we describe an approach for systematically testing this design principle for natural-like proteins by (1) computational design of synthetic sequences that gradually add or remove constraints along the hierarchy of interacting residues and (2) experimental testing of the designed sequences for folding and biochemical function. By this process, we hope to understand how the constraints on fold, function, and other aspects of fitness are organized within natural proteins, a first step in understanding the process of "design" by evolution.

Surface sites for engineering allosteric control in proteins.

Statistical analyses of protein families reveal networks of coevolving amino acids that functionally link distantly positioned functional surfaces. Such linkages suggest a concept for engineering allosteric control into proteins: The intramolecular networks of two proteins could be joined across their surface sites such that the activity of one protein might control the activity of the other. We tested this idea by creating PAS-DHFR, a designed chimeric protein that connects a light-sensing signaling domain from a plant member of the Per/Arnt/Sim (PAS) family of proteins with Escherichia coli dihydrofolate reductase (DHFR). With no optimization, PAS-DHFR exhibited light-dependent catalytic activity that depended on the site of connection and on known signaling mechanisms in both proteins. PAS-DHFR serves as a proof of concept for engineering regulatory activities into proteins through interface design at conserved allosteric sites.

Dynamic scaffolding in a G protein-coupled signaling system.

The INAD scaffold organizes a multiprotein complex that is essential for proper visual signaling in Drosophila photoreceptor cells. Here we show that one of the INAD PDZ domains (PDZ5) exists in a redox-dependent equilibrium between two conformations--a reduced form that is similar to the structure of other PDZ domains, and an oxidized form in which the ligand-binding site is distorted through formation of a strong intramolecular disulfide bond. We demonstrate transient light-dependent formation of this disulfide bond in vivo and find that transgenic flies expressing a mutant INAD in which PDZ5 is locked in the reduced state display severe defects in termination of visual responses and visually mediated reflex behavior. These studies demonstrate a conformational switch mechanism for PDZ domain function and suggest that INAD behaves more like a dynamic machine rather than a passive scaffold, regulating signal transduction at the millisecond timescale through cycles of conformational change.

Evolutionary information for specifying a protein fold.

Classical studies show that for many proteins, the information required for specifying the tertiary structure is contained in the amino acid sequence. Here, we attempt to define the sequence rules for specifying a protein fold by computationally creating artificial protein sequences using only statistical information encoded in a multiple sequence alignment and no tertiary structure information. Experimental testing of libraries of artificial WW domain sequences shows that a simple statistical energy function capturing coevolution between amino acid residues is necessary and sufficient to specify sequences that fold into native structures. The artificial proteins show thermodynamic stabilities similar to natural WW domains, and structure determination of one artificial protein shows excellent agreement with the WW fold at atomic resolution. The relative simplicity of the information used for creating sequences suggests a marked reduction to the potential complexity of the protein-folding problem.