{"title":"De novo design of four-helix bundle proteins to bind metalloporphyrin cofactors.","authors":"Karen R Coronado, Yixuan Zhu, Samuel I Mann","doi":"10.1016/bs.mie.2025.06.038","DOIUrl":null,"url":null,"abstract":"<p><p>The versatility of heme proteins in nature stems from the intricate control exerted by their protein scaffolds. De novo protein design offers a powerful means to dissect and recreate these structure-function relationships, enabling construction of novel metalloproteins with tailored functionalities. Here, we describe the computational design and characterization MPP1, a four-helix bundle protein designed to bind an abiological Mn-diphenylporphyrin (MnDPP) cofactor. Using parameterized coiled-coil backbones, flexible backbone sequence design in Rosetta, and structure-guided loop building, MPP1 was designed to accommodate the cofactor with precise positioning of axial ligands and second-shell interactions, as well as purposeful accessibility for oxidants and substrates. The resulting protein was the first crystallographically characterized de novo designed porphyrin-binding protein. MPP1 demonstrated the ability to stabilize a high-valent Mn(V)-oxo species and mediate thioether oxidation. This chapter details the computational strategies, cofactor incorporation, and solution characterization necessary to design and evaluate four-helix bundle proteins capable of binding porphyrin and porphyrin-like cofactors with atomic-level precision. Keywords: de novo design, protein design, bioinorganic chemistry, metalloporphyrins, heme proteins.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"720 ","pages":"1-22"},"PeriodicalIF":0.0000,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12497995/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods in enzymology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1016/bs.mie.2025.06.038","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/7/14 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 0
Abstract
The versatility of heme proteins in nature stems from the intricate control exerted by their protein scaffolds. De novo protein design offers a powerful means to dissect and recreate these structure-function relationships, enabling construction of novel metalloproteins with tailored functionalities. Here, we describe the computational design and characterization MPP1, a four-helix bundle protein designed to bind an abiological Mn-diphenylporphyrin (MnDPP) cofactor. Using parameterized coiled-coil backbones, flexible backbone sequence design in Rosetta, and structure-guided loop building, MPP1 was designed to accommodate the cofactor with precise positioning of axial ligands and second-shell interactions, as well as purposeful accessibility for oxidants and substrates. The resulting protein was the first crystallographically characterized de novo designed porphyrin-binding protein. MPP1 demonstrated the ability to stabilize a high-valent Mn(V)-oxo species and mediate thioether oxidation. This chapter details the computational strategies, cofactor incorporation, and solution characterization necessary to design and evaluate four-helix bundle proteins capable of binding porphyrin and porphyrin-like cofactors with atomic-level precision. Keywords: de novo design, protein design, bioinorganic chemistry, metalloporphyrins, heme proteins.
期刊介绍:
The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.