Custom-designed ligand-binding proteins represent a promising class of macromolecules with exciting applications toward the design of new enzymes or the engineering of antibodies and small-molecule recruited proteins for therapeutic interventions. However, several challenges remain in designing a protein sequence such that the binding site organization results in high affinity interaction with a bound ligand. Here, we study the dynamics of explicitly solvated designed proteins through all-atom molecular dynamics (MD) simulations to gain insight into the causes that lead to the low affinity or instability of most of these designs, despite the prediction of their success by the computational design methodology. Simulations ranging from 500 to 1000 ns per replicate were conducted on 37 designed protein variants encompassing two distinct folds and a range of ligand affinities, resulting in more than 180 μs of combined sampling. The simulations provide retrospective insights into the properties affecting ligand affinity that can prove useful in guiding further steps of design optimization. Features indicate that entropic components are particularly important for affinity, which are not easily incorporated in the empirical models often used in design protocols. Additionally, we demonstrate that the application of machine learning approaches built upon the output from the simulations can help discriminate between successful and failed binders, such that MD could act as a screening step in protein design, resulting in a more efficient process.

Through the efforts of many groups, a wide range of fluorescent protein reporters and sensors based on green fluorescent protein and its relatives have been engineered in recent years. Here we explore the incorporation of sensing modalities into de novo designed fluorescence-activating proteins, called mini-fluorescence-activating proteins (mFAPs), that bind and stabilize the fluorescent cis-planar state of the fluorogenic compound DFHBI. We show through further design that the fluorescence intensity and specificity of mFAPs for different chromophores can be tuned, and the fluorescence made sensitive to pH and Ca²⁺ for real-time fluorescence reporting. Bipartite split mFAPs enable real-time monitoring of protein-protein association and (unlike widely used split GFP reporter systems) are fully reversible, allowing direct readout of association and dissociation events. The relative ease with which sensing modalities can be incorporated and advantages in smaller size and photostability make de novo designed fluorescence-activating proteins attractive candidates for optical sensor engineering.

The regular arrangements of β-strands around a central axis in β-barrels and of α-helices in coiled coils contrast with the irregular tertiary structures of most globular proteins, and have fascinated structural biologists since they were first discovered. Simple parametric models have been used to design a wide range of α-helical coiled-coil structures, but to date there has been no success with β-barrels. Here we show that accurate de novo design of β-barrels requires considerable symmetry-breaking to achieve continuous hydrogen-bond connectivity and eliminate backbone strain. We then build ensembles of β-barrel backbone models with cavity shapes that match the fluorogenic compound DFHBI, and use a hierarchical grid-based search method to simultaneously optimize the rigid-body placement of DFHBI in these cavities and the identities of the surrounding amino acids to achieve high shape and chemical complementarity. The designs have high structural accuracy and bind and fluorescently activate DFHBI in vitro and in Escherichia coli, yeast and mammalian cells. This de novo design of small-molecule binding activity, using backbones custom-built to bind the ligand, should enable the design of increasingly sophisticated ligand-binding proteins, sensors and catalysts that are not limited by the backbone geometries available in known protein structures.