Water, Solute, and Ion Transport in De Novo-Designed Membrane Protein Channels.

Biological organisms engineer peptide sequences to fold into membrane pore proteins capable of performing a wide variety of transport functions. Synthetic de novo-designed membrane pores can mimic this approach to achieve a potentially even larger set of functions. Here we explore water, solute, and ion transport in three de novo designed β-barrel membrane channels in the 5-10 Å pore size range.

An evolved artificial radical cyclase enables the construction of bicyclic terpenoid scaffolds via an H-atom transfer pathway.

While natural terpenoid cyclases generate complex terpenoid structures via cationic mechanisms, alternative radical cyclization pathways are underexplored. The metal-catalysed H-atom transfer reaction (M-HAT) offers an attractive means for hydrofunctionalizing olefins, providing access to terpenoid-like structures. Artificial metalloenzymes offer a promising strategy for introducing M-HAT reactivity into a protein scaffold.

Sculpting conducting nanopore size and shape through de novo protein design.

Transmembrane β-barrels have considerable potential for a broad range of sensing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels, which provide suboptimal starting points. By contrast, de novo protein design can in principle create an unlimited number of new nanopores with any desired properties.

Validation of de novo designed water-soluble and transmembrane β-barrels by in silico folding and melting.

In silico validation of de novo designed proteins with deep learning (DL)-based structure prediction algorithms has become mainstream. However, formal evidence of the relationship between a high-quality predicted model and the chance of experimental success is lacking. We used experimentally characterized de novo water-soluble and transmembrane β-barrel designs to show that AlphaFold2 and ESMFold excel at different tasks.

Rationale in Custom Design of Transmembrane β-Barrel Pores.

Biological nanopores incorporated into synthetic membranes are widely used for single-molecule analytical applications such as DNA sequencing. The ability to engineer custom membrane proteins with a pore would allow the generation of a multitude of nanopores for the sensing/sequencing of small molecules and (bio)polymers. The de novo design of transmembrane β-barrel pores has recently enabled the generation of nanopores with custom size, shape, and properties.

Sculpting conducting nanopore size and shape through protein design.

Transmembrane β-barrels (TMBs) are widely used for single molecule DNA and RNA sequencing and have considerable potential for a broad range of sensing and sequencing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels such as CsgG, which have evolved to carry out functions very different from sensing, and hence provide sub-optimal starting points. In contrast, protein design can in principle create an unlimited number of new nanopores with any desired properties.

Design and optimization of enzymatic activity in a de novo β-barrel scaffold.

While native scaffolds offer a large diversity of shapes and topologies for enzyme engineering, their often unpredictable behavior in response to sequence modification makes de novo generated scaffolds an exciting alternative. Here we explore the customization of the backbone and sequence of a de novo designed eight stranded β-barrel protein to create catalysts for a retro-aldolase model reaction. We show that active and specific catalysts can be designed in this fold and use directed evolution to further optimize activity and stereoselectivity.

Principles and Methods in Computational Membrane Protein Design.

After decades of progress in computational protein design, the design of proteins folding and functioning in lipid membranes appears today as the next frontier. Some notable successes in the de novo design of simplified model membrane protein systems have helped articulate fundamental principles of protein folding, architecture and interaction in the hydrophobic lipid environment. These principles are reviewed here, together with the computational methods and approaches that were used to identify them.

De novo design of transmembrane β barrels.

Transmembrane β-barrel proteins (TMBs) are of great interest for single-molecule analytical technologies because they can spontaneously fold and insert into membranes and form stable pores, but the range of pore properties that can be achieved by repurposing natural TMBs is limited. We leverage the power of de novo computational design coupled with a "hypothesis, design, and test" approach to determine TMB design principles, notably, the importance of negative design to slow β-sheet assembly. We design new eight-stranded TMBs, with no homology to known TMBs, that insert and fold reversibly into synthetic lipid membranes and have nuclear magnetic resonance and x-ray crystal structures very similar to the computational models.

Incorporation of sensing modalities into de novo designed fluorescence-activating proteins.

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 Ca for real-time fluorescence reporting.

De novo design of a fluorescence-activating β-barrel.

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.

Escherichia coli D-malate dehydrogenase, a generalist enzyme active in the leucine biosynthesis pathway.

The enzymes of the β-decarboxylating dehydrogenase superfamily catalyze the oxidative decarboxylation of D-malate-based substrates with various specificities. Here, we show that, in addition to its natural function affording bacterial growth on D-malate as a carbon source, the D-malate dehydrogenase of Escherichia coli (EcDmlA) naturally expressed from its chromosomal gene is capable of complementing leucine auxotrophy in a leuB(-) strain lacking the paralogous isopropylmalate dehydrogenase enzyme. To our knowledge, this is the first example of an enzyme that contributes with a physiologically relevant level of activity to two distinct pathways of the core metabolism while expressed from its chromosomal locus.