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-α-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 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 to improve the efficiency of bilin binding and influence bilin structure. Bilins bind and 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.

A Colorful Pallet of B-Phycoerythrin Proteoforms Exposed by a Multimodal Mass Spectrometry Approach

“…2 Furthermore, the different N-terminal regions of the distinct g subunits that participate in linker-linker contacts might influence the absorbance and emission properties of the involved prosthetic groups because the protein microenvironment influences the fluorophore-mediated light transmission. 36 Additionally, we show the most abundant g subunits (g 2059. 29 and g 2100.…”

Rational construction of compact de novo-designed biliverdin-binding proteins

“…The cofactor B-, C-, and D-rings were further immobilized by positioning histidine residues (L75H and F120H) to pi-stack to the pyrrole rings and provide hydrophobic core bulk to restrict protein movement and core water access (Build Step 4). Such introduction of aromatic core bulk has previously stabilized similar maquettes that bind tetrapyrroles, as evident by increased melting temperatures and reduced helical movement 7,9 . Rosetta modeling of the final product suggests that S5 may hydrogen bond to the oxygen of the A-ring, and that H71 also hydrogen bonds to S5 to further stabilize the A-ring through increased steric constraint (Figure 3a-b).…”

“…De novo-designed proteins are powerful tools for exploring the principles and expanding the boundaries of protein folding, protein-protein interaction, and protein biochemical functions that build on structure-function and protein sequence diversity landscapes that are entirely distinct from those of natural protein scaffolds [1][2][3][4] . Self-assembling tetrahelical bundles [5][6][7][8][9][10][11][12][13][14][15] , which are created by simple binary patterning of hydrophobic and hydrophilic residues with high -helical propensity 16 , comprise the best established class of de novo-designed scaffolds. These scaffolds can provide stable single-chain frames into which biological cofactors can bind, as designed protein maquettes 6-10 that are useful for rationally engineering artificial holoproteins because the cofactor-interacting function of an individual residue within the core is largely isolated from the functions of the others (Figure 1a).…”

“…Previously reported maquette holoproteins have typically incorporated rigid and planar cofactors, such as hemes, chlorins, porphyrins, and flavins. Recently, we reported that maquettes can also bind highly flexible bilins or linear tetrapyrroles, and identified structure-function determinants for effective autocatalytic ligation of phycocyanobilin (PCB), namely the presence of a free cysteine and the stabilization of the bilin propionates 9 .…”

Rational Construction of Compact de Novo-Designed Biliverdin-Binding Proteins

“…Recently, we reported that they also bind flexible bilins or linear tetrapyrroles and identified determinants for autocatalytic ligation of phycocyanobilin (PCB), namely, a free cysteine and the stabilization of the bilin propionates. 6 …”