De novo design of a fluorescence-activating β-barrel

Abstract

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.

Comparison of Rosetta flexible‐backbone computational protein design methods on binding interactions

“…These steps are iterated through cycles of simulated annealing, during which the weight of the repulsive component of the Lennard‐Jones potential is increased stepwise to enable amino acid changes that may introduce unfavorable clashes but can be subsequently relaxed in the minimization step. FastDesign has been used in a variety of application to design new functions …”

“…Highly stable, idealized folds can be designed de novo, but design of proteins with new functions remains challenging. In most cases where new functions have been designed computationally, the designed protein is modeled on natural “scaffold” proteins with minimal changes in backbone conformation, although there are recent notable exceptions of functions designed into de novo proteins . Moreover, attaining sufficiently high activities typically requires optimization of the desired function by directed evolution .…”

“…In most cases where new functions have been designed computationally, the designed protein is modeled on natural "scaffold" proteins with minimal changes in backbone conformation, [11][12][13][14][15] although there are recent notable exceptions of functions designed into de novo proteins. 16,17 Moreover, attaining sufficiently high activities typically requires optimization of the desired function by directed evolution. 14,15,18 Function often depends on hydrogen bonds, which require precise backbone and side chain geometry, which remains difficult to design 19 especially when a novel function requires "reshaping" of an existing protein conformation.…”

Fluorogen activating protein toolset for protein trafficking measurements

“…While there have been considerable improvements with FP modification, engineering brighter and photostable FPs is a difficult challenge due to their buried chromophores that are not amenable to chemical modification, and their environment sensitivity, that is, fluorescence quenching at low pH . In a breakthrough advancement in de novo design of beta‐barrel protein folds, Dou et al, were able to design beta‐barrel backbone models with cavity shapes that accommodated the fluorogenic compound, DFHBI . The generated mini‐fluorescence‐activating proteins (mFAPs), demonstrated a 3‐step approach to creating a de novo design of ligand‐binding proteins.…”

“…1 In a breakthrough advancement in de novo design of beta-barrel protein folds, Dou et al, were able to design betabarrel backbone models with cavity shapes that accommodated the fluorogenic compound, DFHBI. 98 The generated mini-fluorescenceactivating proteins (mFAPs), demonstrated a 3-step approach to creat-…”