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Influence of circular permutations on the structure and stability of a six-fold circular symmetric designer protein.

ABSTRACT: The β-propeller fold is adopted by a sequentially diverse family of repeat proteins with apparent rotational symmetry. While the structure is mostly stabilized by hydrophobic interactions, an additional stabilization is provided by hydrogen bonds between the N-and C-termini, which are almost invariably part of the same β-sheet. This feature is often referred to as the "Velcro" closure. The positioning of the termini within a blade is variable and depends on the protein family. In order to investigate the influence of this location on protein structure, folding and stability, we created different circular permutants, and a circularized version, of the designer propeller protein named Pizza. This protein is perfectly symmetrical, possessing six identical repeats. While all mutants adopt the same structure, the proteins lacking the "Velcro" closure were found to be significantly less resistant to thermal and chemical denaturation. This could explain why such proteins are rarely observed in nature. Interestingly the most common "Velcro" configuration for this protein family was not the most stable among the Pizza variants tested. The circularized version shows dramatically improved stability, which could have implications for future applications.

SUBMITTER: Mylemans B

PROVIDER: S-EPMC7679965 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Circular permutation (CP) refers to situations in which the termini of a protein are relocated to other positions in the structure. CP occurs naturally and has been artificially created to study protein function, stability and folding. Recently CP is increasingly applied to engineer enzyme structure and function, and to create bifunctional fusion proteins unachievable by tandem fusion. CP is a complicated and expensive technique. An intrinsic difficulty in its application lies in the fact that not every position in a protein is amenable for creating a viable permutant. To examine the preferences of CP and develop CP viability prediction methods, we carried out comprehensive analyses of the sequence, structural, and dynamical properties of known CP sites using a variety of statistics and simulation methods, such as the bootstrap aggregating, permutation test and molecular dynamics simulations. CP particularly favors Gly, Pro, Asp and Asn. Positions preferred by CP lie within coils, loops, turns, and at residues that are exposed to solvent, weakly hydrogen-bonded, environmentally unpacked, or flexible. Disfavored positions include Cys, bulky hydrophobic residues, and residues located within helices or near the protein's core. These results fostered the development of an effective viable CP site prediction system, which combined four machine learning methods, e.g., artificial neural networks, the support vector machine, a random forest, and a hierarchical feature integration procedure developed in this work. As assessed by using the hydrofolate reductase dataset as the independent evaluation dataset, this prediction system achieved an AUC of 0.9. Large-scale predictions have been performed for nine thousand representative protein structures; several new potential applications of CP were thus identified. Many unreported preferences of CP are revealed in this study. The developed system is the best CP viability prediction method currently available. This work will facilitate the application of CP in research and biotechnology.

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Influence of circular permutations on the structure and stability of a six-fold circular symmetric designer protein.

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