Biology subjects

Brunette, T.

Publications and source records attributed to Brunette, T..

6 recordsLinked to original sources

bioRxiv2024

Results of the Protein Engineering Tournament: An Open Science Benchmark for Protein Modeling and Design

The grand challenge of protein engineering is the development of computational models to characterize and generate protein sequences for arbitrary functions. Progress is limited by lack of 1) benchmarking opportunities, 2) large protein function datasets, and 3) access to experimental protein characterization. We introduce the Protein Engineering Tournament--a fully-remote competition designed to foster the development and evaluation of computational approaches in protein engineering. The tournament consists of an in silico round, predicting biophysical properties from protein sequences, followed by an in vitro round where novel protein sequences are designed, expressed and characterized using automated methods. Upon completion, all datasets, experimental protocols, and methods are made publicly available. We detail the structure and outcomes of a pilot Tournament involving seven protein design teams, powered by six multi-objective datasets, with experimental characterization by our partner, International Flavors and Fragrances. Forthcoming Protein Engineering Tournaments aim to mobilize the scientific community towards transparent evaluation of progress in the field. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/606135v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@8f9769org.highwire.dtl.DTLVardef@11da734org.highwire.dtl.DTLVardef@1cc51c0org.highwire.dtl.DTLVardef@10b3c27_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

De novo design of buttressed loops for sculpting protein functions

In natural proteins, structured loops play central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve by de novo protein design. Here we describe a solution to this problem that generates structured loops buttressed by extensive hydrogen bonding interactions with two neighboring loops and with secondary structure elements. We use this approach to design tandem repeat proteins with buttressed loops ranging from 9 to 14 residues in length. Experimental characterization shows the designs are folded and monodisperse, highly soluble, and thermally stable. Crystal structures are in close agreement with the computational design models, with the loops structured and buttressed by their neighbors as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute quite generally to efforts to design new protein functions.

biochemistry↗

bioRxiv2023

Blueprinting expandable nanomaterials with standardized protein building blocks

A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies in comparison has been much more complex, largely due to the irregular shapes of protein structures1. Here we describe extendable linear, curved, and angled protein building blocks, as well as inter-block interactions that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight "train track" assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not been previously possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank 3D canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to "back of an envelope" architectural blueprints.

biochemistry↗

bioRxiv2022

De novo design of modular peptide binding proteins by superhelical matching

General approaches for designing sequence-specific peptide binding proteins would have wide utility in proteomics and synthetic biology. Although considerable progress has been made in designing proteins which bind to other proteins, the general peptide binding problem is more challenging as most peptides do not have defined structures in isolation, and to offset the loss in solvation upon binding the protein binding interface has to provide specific hydrogen bonds that complement the majority of the buried peptides backbone polar groups (1-3). Inspired by natural repeat protein-peptide complexes, and engineering efforts to alter their specificity (4-11), we describe a general approach for de novo design of proteins made out of repeating units that bind peptides with repeating sequences such that there is a one to one correspondence between repeat units on the protein and peptide. We develop a rapid docking plus geometric hashing method to identify protein backbones and protein-peptide rigid body arrangements that are compatible with bidentate hydrogen bonds between side chains on the protein and the backbone of the peptide (12); the remainder of the protein sequence is then designed using Rosetta to incorporate additional interactions with the peptide and drive folding to the desired structure. We use this approach to design, from scratch, alpha helical repeat proteins that bind six different tripeptide repeat sequences--PLP, LRP, PEW, IYP, PRM and PKW--in near polyproline 2 helical conformations. The proteins are expressed at high levels in E. coli, are hyperstable, and bind peptides with 4-6 copies of the target tripeptide sequences with nanomolar to picomolar affinities both in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including a ladder of protein sidechain to peptide backbone hydrogen bonds. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating sequences, and for naturally occuring proteins containing disordered regions. Our approach provides a general route to designing specific binding proteins for a broad range of repeating and non-repetitive peptide sequences.

biochemistry↗

bioRxiv2022

Precisely patterned nanofibers made from extendable protein multiplexes

Molecular systems with coincident cyclic and superhelical symmetry axes have considerable advantages for materials design as they can be readily lengthened or shortened by changing the length of the constituent monomers. Among proteins, alpha helical coiled coils have such symmetric extendable architectures, but are limited by the relatively fixed geometry and flexibility of the helical protomers. Here, we describe a systematic approach to generating modular and rigid repeat protein oligomers with coincident C2 to C8 and superhelical symmetry axes that can be readily extended by repeat propagation. From these building blocks, we demonstrate that a wide range of unbounded fibers can be systematically designed by introducing hydrophilic surface patches that force staggering of the monomers; the geometry of such fibers can be precisely tuned by varying the number of repeat units in the monomer and the placement of the hydrophilic patches.

bioengineering↗

bioRxiv2020

Hierarchical design of multi-scale protein complexes by combinatorial assembly of oligomeric helical bundle and repeat protein building blocks

A goal of de novo protein design is to develop a systematic and robust approach to generating complex nanomaterials from stable building blocks. Due to their structural regularity and simplicity, a wide range of monomeric repeat proteins and oligomeric helical bundle structures have been designed and characterized. Here we describe a stepwise hierarchical approach to building up multi-component symmetric protein assemblies using these structures. We first connect designed helical repeat proteins (DHRs) to designed helical bundle proteins (HBs) to generate a large library of heterodimeric and homooligomeric building blocks; the latter have cyclic symmetries ranging from C2 to C6. All of the building blocks have repeat proteins with accessible termini, which we take advantage of in a second round of architecture guided rigid helical fusion (WORMS) to generate larger symmetric assemblies including C3 and C5 cyclic and D2 dihedral rings, a tetrahedral cage, and a 120 subunit icosahedral cage. Characterization of the structures by small angle x-ray scattering, x-ray crystallography, and cryo-electron microscopy demonstrates that the hierarchical design approach can accurately and robustly generate a wide range of macromolecular assemblies; with a diameter of 43nm, the icosahedral nanocage is the largest structurally validated designed cage to date. The computational methods and building block sets described here provide a very general route to new de novo designed symmetric protein nanomaterials.

biochemistry↗