Corrole-Substituted Fluorescent Heme Proteins

Abstract

Although fluorescent proteins have been utilized for a variety of biological applications, they have several optical limitations, namely weak red and near-infrared emission and exceptionally broad (>200 nm) emission profiles. The photophysical properties of fluorescent proteins can be enhanced through the incorporation of novel cofactors with the desired properties into a stable protein scaffold. To this end, a fluorescent phosphorus corrole that is structurally similar to the native heme cofactor is incorporated into two exceptionally stable heme proteins: H-NOX from Caldanaerobacter subterraneus and heme acquisition system protein A (HasA) from Pseudomonas aeruginosa. These yellow-orange emitting protein conjugates are examined by steady-state and time-resolved optical spectroscopy. The HasA conjugate exhibits enhanced fluorescence, whereas emission from the H-NOX conjugate is quenched relative to the free corrole. Despite the low fluorescence quantum yields, these corrole-substituted proteins exhibit more intense fluorescence in a narrower spectral profile than traditional fluorescent proteins that emit in the same spectral window. This study demonstrates that fluorescent corrole complexes are readily incorporated into heme proteins and provides an inroad for the development of novel fluorescent proteins.

Corrole–protein interactions in H-NOX and HasA

“…P-1 does not bind histidine, and DFT calculations confirm that imidazole binding is destabilising. 28 The H102G variant exhibited impaired corrole incorporation (59% relative to WT H-NOX). To rescue corrole binding with a hydrogen bonding partner, H102S was prepared.…”

“…S19b, ESI † ), which likely precludes this contact from forming in the variant. It was previously demonstrated that P-1 incorporation decreases with increasing pH, 28 suggesting that protonation of this residue occurs at lower pH to enhance corrole binding. Moreover, protonation of H102 explains why the WT protein and H102 variants exhibit the same emission intensity.…”

“…Given that this corrole does not bind histidine ( i.e. , no P–N His bond) and remains a five-coordinate complex in solution, 28 it is unclear how this molecule would interact with haem proteins.…”

Corroles at work: a small macrocycle for great applications

“…100). 281 The binding of the corrole in the heme pocket of the protein was confirmed by emission and absorption spectroscopy, demonstrating the proof-of-principle and the possibility to further develop new fluorescent proteins. Interestingly, these corrole-substituted heme proteins were shown to exhibit more intense fluorescence (in a narrower spectral profile) than traditional fluorescent proteins that emit in the same spectral window.…”

“…Interestingly, these corrole-substituted heme proteins were shown to exhibit more intense fluorescence (in a narrower spectral profile) than traditional fluorescent proteins that emit in the same spectral window. 281 It was shown also that the corrole emission was enhanced in the HasA conjugate but was quenched in the H-NOX.…”