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De novo synthetic biliprotein design, assembly and excitation energy transfer.

ABSTRACT: Bilins are linear tetrapyrrole chromophores with a wide range of visible and near-visible light absorption and emission properties. These properties are tuned upon binding to natural proteins and exploited in photosynthetic light-harvesting and non-photosynthetic light-sensitive signalling. These pigmented proteins are now being manipulated to develop fluorescent experimental tools. To engineer the optical properties of bound bilins for specific applications more flexibly, we have used first principles of protein folding to design novel, stable and highly adaptable bilin-binding four-?-helix bundle protein frames, called maquettes, and explored the minimal requirements underlying covalent bilin ligation and conformational restriction responsible for the strong and variable absorption, fluorescence and excitation energy transfer of these proteins. Biliverdin, phycocyanobilin and phycoerythrobilin bind covalently to maquette Cys in vitro A blue-shifted tripyrrole formed from maquette-bound phycocyanobilin displays a quantum yield of 26%. Although unrelated in fold and sequence to natural phycobiliproteins, bilin lyases nevertheless interact with maquettes during co-expression in Escherichia coli to improve the efficiency of bilin binding and influence bilin structure. Bilins bind in vitro and in vivo to Cys residues placed in loops, towards the amino end or in the middle of helices but bind poorly at the carboxyl end of helices. Bilin-binding efficiency and fluorescence yield are improved by Arg and Asp residues adjacent to the ligating Cys on the same helix and by His residues on adjacent helices.

SUBMITTER: Mancini JA

PROVIDER: S-EPMC5938588 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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De novo synthetic biliprotein design, assembly and excitation energy transfer.

Mancini Joshua A JA Sheehan Molly M Kodali Goutham G Chow Brian Y BY Bryant Donald A DA Dutton P Leslie PL Moser Christopher C CC

Journal of the Royal Society, Interface 20180401 141

Bilins are linear tetrapyrrole chromophores with a wide range of visible and near-visible light absorption and emission properties. These properties are tuned upon binding to natural proteins and exploited in photosynthetic light-harvesting and non-photosynthetic light-sensitive signalling. These pigmented proteins are now being manipulated to develop fluorescent experimental tools. To engineer the optical properties of bound bilins for specific applications more flexibly, we have used first pri ...[more]

PMID: 29618529

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Project description:The major advances in molecular and structural biology and automated peptide and DNA synthesis of the 1970s and 1980s generated fertile conditions in the 1990s for the exploration of designed proteins as a new approach for inorganic chemists to generate biomolecular mimics of metalloproteins. This Account follows the development of the TRI peptide family of three-stranded coiled coils (3SCC) and α3D family of three-helix bundles (3HB) as scaffolds for the preparation of metal binding sites within de novo designed constructs. The 3SCC were developed using the concept of a heptad repeat (abcdefg) putting hydrophobes in the a and d positions. The TRI peptides contain four heptads with capping glycines. Via substitution of leucine hydrophobes, metal ligands can be introduced into the a or d sites in order to bind metals. First, the ability to use cysteine-substituted 3SCC aggregates to impose higher or lower coordination numbers on Hg(II) and Cd(II) or matching the coordination preferences of As(III) and Pb(II) is discussed. Then, methods to develop dual site peptides capable of discriminating metals based on their type (e.g., Cd(II) vs Pb(II)), their preference for a vs d sites, and then their coordination number is described. Once these principles of metal site differentiation are described, we shift to building dual site peptides using both cysteine and histidine metal binding sites. This approach provides a construct with both a Hg(II) structural and a Zn(II) hydrolytic center, the latter of which is capable of hydrating CO2. With these Zn(II) proteins, we consider the relative importance of the location of the catalytic center along the primary sequence of the peptide and show that only minor perturbations in catalytic efficiencies are observed based on metal location. We then assess the feasibility of preparing enzymes competent to reduce nitrite with copper centers in a histidine-rich environment. As part of this discussion, we examine the influence of surface residues on catalyst reduction potentials and catalytic efficiencies. We end describing approaches to prepare asymmetric proteins that can incorporate acid-base catalysts or water channels. In this respect, we highlight modifications of a helix-turn-helix-turn-helix motif called α3D and show how this 3HB can be modified to bind heavy metals or to make Zn(II) centers, which are active hydrolytic catalysts. A comparison is made to the comparable parallel 3SCC.

Dataset Information

De novo synthetic biliprotein design, assembly and excitation energy transfer.

Publications

De novo synthetic biliprotein design, assembly and excitation energy transfer.

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