The outcome of the project has been a computational pipeline for the design of symmetric protein assemblies with cubic symmetry. Based on the surface shape of a compatible building block, which could be extracted from natural proteins that form protein containers or simulated de novo, natural protein domains with similar shapes are identified. After optimization of the position of the subunit in the symmetric assembly to enable high shape-complementarity the sequence of surface residues is designed to optimize interface interactions to stabilize the assembly. The methodology results in protein capsid models with exquisite shape complimentary that form closed shells with small pores, with a variation of capsid size and pore sizes that goes far beyond the current state of the art.
The design pipeline depends on several new computational technologies developed during the project. An efficient library for 3D shape matching with Zernike descriptors was developed. A method for aligning proteins based on surface shape, ZEAL, was developed. A highly all-atom protein docking method based on differential evolution was developed, EvoDOCK. EvoDOCK is capable of docking heterodimeric complexes with high accuracy when compatible backbones are available for the binding partners and with an order of magnitude more computational efficiency. A symmetric version of EvoDOCK enables the docking of proteins with cubic symmetry, which enables de novo prediction of proteins with cubic symmetry starting from a protein sequence. This is based on a combination of EvoDOCK and Alphafold2, where Alphafold2/Alphafold3 is not able to predict structures of this kind.
To enable efficient screening an optimization of designed proteins a method for screening for expression and stability in vivo was developed. This methodology enables the relative stability of a large number of protein variants to be evaluated in one experiment by expressing a DNA library in E. Coli and screening with fluorescence-activated cell sorting. The methodology is based on the concept of monitoring the protein quality control system when a protein variant is expressed in the cell. This system also allowed us to investigate how chaperones inside cells respond to folding stress. We have also developed genetic constructs that allow us to monitor the expression of protein capsid proteins without having to fuse every subunit to a big fluorescent protein. In combination with the stability and expression screening methodology it enables rapid testing of many designs, as well as directed evolution of protein capsid designs.
The experimental and computational methodology developed so far in this project can be of great value to researchers outside this project, and find applications in the industrial setting as well.
