Computational de Novo Design and Characterization of a Protein That Selectively Binds a Highly Hyperpolarizable Abiological Chromophore

Abstract

This work reports the first example of a single-chain protein computationally designed to contain four α-helical segments and fold to form a 4-helix bundle encapsulating a supramolecular abiological chromophore that possesses exceptional nonlinear optical properties. The 109-residue protein designated SCRPZ-1, binds and disperses an insoluble hyperpolarizable chromophore, ruthenium(II) [5-(4′-ethynyl-(2,2′;6′,2″-terpyridinyl))-10,20-bis(phenyl)porphinato]zinc(II)-(2,2′;6′,2″-terpyridine)2+ (RuPZn) in aqueous buffer solution at a 1:1 stoichiometry. A 1:1 binding stoichiometry of the holoprotein is supported by electronic absorption and CD spectra, as well as equilibrium analytical ultracentrifugation and size exclusion chromatography. SCRPZ-1 readily dimerizes at μM concentrations, and an empirical redesign of the protein exterior produced a stable monomeric protein, SCRPZ-2, that also displayed a 1:1 protein:cofactor stoichiometry. For both proteins in aqueous buffer, the encapsulated cofactor displays photophysical properties resembling those exhibited by the dilute RuPZn cofactor in organic solvent: femtosecond-, nanosecond-, and microsecond-timescale pump-probe transient absorption spectroscopic data evince intensely absorbing holoprotein excited states having large spectral bandwidth that penetrate deep in the near-infrared (NIR) energy regime; the holoprotein electronically excited triplet state exhibits a microsecond timescale lifetime characteristic of the RuPZn chromophore. Hyper-Rayleigh light scattering (HRS) measurements carried out at an incident irradiation wavelength (λinc) of 1340 nm for these holoproteins demonstrate an exceptional dynamic hyperpolarizabilty (β1340 = 3100 × 10−30 esu). X-ray reflectivity measurements establish that this de novo designed hyperpolarizable protein can be covalently attached with high surface density to a silicon surface without loss of the cofactor, indicating that these assemblies provide a new approach to bio-inspired materials that have unique electro-optic functionality.

Thermophilic Ferritin 24mer Assembly and Nanoparticle Encapsulation Modulated by Interdimer Electrostatic Repulsion

“…26,27 An entropy-based, probabilistic formalism was used to calculate the probabilities of the amino acids and their rotamer conformations. 28–31 Using CHARMM19, 26 hydrogen atoms were added, and energies were calculated using the dihedral, van der Waals, and electrostatic terms, with a non-bonded cut-off of 8 Å. The probabilistic approach has an effective parameter β that is conjugate to the average energy over sequences and side chain conformations; β = 0.5 mol/kcal for these calculations.…”

Computational Redesign of Thioredoxin Is Hypersensitive toward Minor Conformational Changes in the Backbone Template

“…However, designing larger (>70 aa) globular αβ proteins with irregular contact patterns is a highly complex task and has only been achieved by employing computational methods [10–14]. In addition to this unique achievement, computational methods have been employed to full-sequence design of a variety of protein structures including early mini-proteins [15–17], tandem repeats [18,19], and ligand binders [20–21]. Despite much effort, however, the total number of full-sequence designed proteins for which an atomic resolution structure has been solved still remains low; to our knowledge, less than 10 larger globular αβ proteins have been reported in the literature [10,12–14,22].…”

Protein design with a comprehensive statistical energy function and boosted by experimental selection for foldability

“…For the other three proteins, the CD results suggest insignificant and non-cooperative loss of secondary structures at high temperatures. Such thermo-unfolding behaviours are often encountered in designed proteins [27][28][29] irrespective of energy functions or rules used during design. It could be results of the shared design strategy of stabilizing the folded structures as much as possible in all aspects according to the given energy functions or rules.…”