An Evolution-Based Approach to De Novo Protein Design and Case Study on Mycobacterium tuberculosis

Computational protein design is a reverse procedure of protein folding and structure prediction, where constructing structures from evolutionarily related proteins has been demonstrated to be the most reliable method for protein 3-dimensional structure prediction. Following this spirit, we developed a novel method to design new protein sequences based on evolutionarily related protein families. For a given target structure, a set of proteins having similar fold are identified from the PDB library by structural alignments. A structural profile is then constructed from the protein templates and used to guide the conformational search of amino acid sequence space, where physicochemical packing is accommodated by single-sequence based solvation, torsion angle, and secondary structure predictions. The method was tested on a computational folding experiment based on a large set of 87 protein structures covering different fold classes, which showed that the evolution-based design significantly enhances the foldability and biological functionality of the designed sequences compared to the traditional physics-based force field methods. Without using homologous proteins, the designed sequences can be folded with an average root-mean-square-deviation of 2.1 Å to the target. As a case study, the method is extended to redesign all 243 structurally resolved proteins in the pathogenic bacteria Mycobacterium tuberculosis, which is the second leading cause of death from infectious disease. On a smaller scale, five sequences were randomly selected from the design pool and subjected to experimental validation. The results showed that all the designed proteins are soluble with distinct secondary structure and three have well ordered tertiary structure, as demonstrated by circular dichroism and NMR spectroscopy. Together, these results demonstrate a new avenue in computational protein design that uses knowledge of evolutionary conservation from protein structural families to engineer new protein molecules of improved fold stability and biological functionality.Author Summary: The goal of computational protein design is to create new protein sequences of desirable structure and biological function. Most protein design methods are developed to search for sequences with the lowest free-energy based on physics-based force fields following Anfinsen's thermodynamic hypothesis. A major obstacle of such approaches is the inaccuracy of the force-field design, which cannot accurately describe atomic interactions or correctly recognize protein folds. We propose a novel method which uses evolutionary information, in the form of sequence profiles from structure families, to guide the sequence design. Since sequence profiles are generally more accurate than physics-based potentials in protein fold recognition, a unique advantage lies on that it targets the design procedure to a family of protein sequence profiles to enhance the robustness of designed sequences. The method was tested on 87 proteins and the designed sequences can be folded by I-TASSER to models with an average RMSD 2.1 Å. As a case study of large-scale application, the method is extended to redesign all structurally resolved proteins in the human pathogenic bacteria, Mycobacterium tuberculosis. Five sequences varying in fold and sizes were characterized by circular dichroism and NMR spectroscopy experiments and three were shown to have ordered tertiary structure.