Corrole–protein interactions in H-NOX and HasA

Lemon, Christopher M.; Nissley, Amos J; Latorraca, Naomi R.; Wittenborn, E.C.; Marletta, Michael A.

doi:10.1039/d2cb00004k

Cited by 5 publications

(20 citation statements)

References 70 publications

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“…Consequently, protonated H102 can hydrogen bond to both the PO unit of the corrole and P115. This additional interaction rigidifies the heme binding pocket to enhance corrole binding, and accounts for the necessity of both residues (Figure a) …”

Section: Fluorescent Proteinssupporting

confidence: 71%

“…To elucidate the corrole− protein interactions, a series of point mutants were generated to modulate the heme binding pocket and identify critical contacts. 68 In the case of H-NOX, changes in the distal hydrogen-bonding network (W9F and Y140F) had no effect on corrole binding, indicating that the PO unit does not interact with these residues. Consequently, the distal subdomain is likely shifted away from the heme binding pocket, akin to the unliganded protein.…”

Section: ■ Fluorescent Proteinsmentioning

confidence: 99%

“…This additional interaction rigidifies the heme binding pocket to enhance corrole binding, and accounts for the necessity of both residues (Figure 8a). 68 In the case of HasA, modulation of the H32 loop had no effect on corrole binding, but variation of the Y75 loop had a profound effect. The Y75F variant decreased corrole binding by ∼50%, but no corrole binding was observed for H83 variants, indicating that this residue is essential.…”

Section: ■ Fluorescent Proteinsmentioning

confidence: 99%

“…A docking model generated from the apo HasA crystal structure and the DFT-optimized geometry of the phosphorus corrole (Figure 8c) is consistent with the FRET analysis. 68 These examples illustrate that unnatural heme analogues can bind to proteins in unexpected ways. This underscores the need to identify these interactions and ensure proper protein folding and cofactor incorporation, especially in cases of reconstitution.…”

Section: ■ Fluorescent Proteinsmentioning

confidence: 99%

“…Given that histidine residues are integral to the heme binding pocket of both Cs H-NOX and Pa HasA, it was unknown how the corrole binds to these proteins. To elucidate the corrole–protein interactions, a series of point mutants were generated to modulate the heme binding pocket and identify critical contacts . In the case of H-NOX, changes in the distal hydrogen-bonding network (W9F and Y140F) had no effect on corrole binding, indicating that the PO unit does not interact with these residues.…”

Section: Fluorescent Proteinsmentioning

confidence: 99%

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Designer Heme Proteins: Achieving Novel Function with Abiological Heme Analogues

Lemon

Marletta

2021

Acc. Chem. Res.

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Conspectus Heme proteins have proven to be a convenient platform for the development of designer proteins with novel functionalities. This is achieved by substituting the native iron porphyrin cofactor with a heme analogue that possesses the desired properties. Replacing the iron center of the porphyrin with another metal provides one inroad to novel protein function. A less explored approach is substitution of the porphyrin cofactor with an alternative tetrapyrrole macrocycle or a related ligand. In general, these ligands exhibit chemical properties and reactivity that are distinct from those of porphyrins. While these techniques have most prominently been utilized to develop artificial metalloenzymes, there are many other applications of this methodology to problems in biochemistry, health, and medicine. Incorporation of synthetic cofactors into protein environments represents a facile way to impart water solubility and biocompatibility. It circumvents the laborious synthesis of water-soluble cofactors, which often introduces substantial charge that leads to undesired bioaccumulation. To this end, the incorporation of unnatural cofactors in heme proteins has enabled the development of designer proteins as optical oxygen sensors, MRI contrast agents, spectroscopic probes, tools to interrogate protein function, antibiotics, and fluorescent proteins. Incorporation of an artificial cofactor is frequently accomplished by denaturing the holoprotein with removal of the heme; the refolded apoprotein is then reconstituted with the artificial cofactor. This process often results in substantial protein loss and does not necessarily guarantee that the refolded protein adopts the native structure. To circumvent these issues, our laboratory has pioneered the use of the RP523 strain of E. coli to incorporate artificial cofactors into heme proteins using expression-based methods. This strain lacks the ability to biosynthesize heme, and the bacterial cell wall is permeable to heme and related molecules. In this way, heme analogues supplemented in the growth medium are incorporated into heme proteins. This approach can also be leveraged for the direct expression of the apoprotein for subsequent reconstitution. These methodologies have been exploited to incorporate non-native cofactors into heme proteins that are resistant to harsh environmental conditions: the heme nitric oxide/oxygen binding protein (H-NOX) from Caldanaerobacter subterraneus (Cs) and the heme acquisition system protein A (HasA) from Pseudomonas aeruginosa (Pa). The exceptional stability of these proteins makes them ideal scaffolds for biomedical applications. Optical oxygen sensing has been accomplished using a phosphorescent ruthenium porphyrin as the artificial heme cofactor. Paramagnetic manganese and gadolinium porphyrins yield high-relaxivity, protein-based MRI contrast agents. A fluorescent phosphorus corrole serves as a heme analogue to produce fluorescent proteins. Iron complexes of nonporphyrin cofactors bound to HasA inhibit the growth of pathogenic b...

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Section: Fluorescent Proteinssupporting

confidence: 71%

Section: ■ Fluorescent Proteinsmentioning

confidence: 99%

Section: ■ Fluorescent Proteinsmentioning

confidence: 99%

Section: ■ Fluorescent Proteinsmentioning

confidence: 99%

Section: Fluorescent Proteinsmentioning

confidence: 99%

See 3 more Smart Citations

Designer Heme Proteins: Achieving Novel Function with Abiological Heme Analogues

Lemon

Marletta

2021

Acc. Chem. Res.

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Diversifying the functions of heme proteins with non-porphyrin cofactors

Lemon

2023

Journal of Inorganic Biochemistry

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Ratiometric Oxygen Sensing with H-NOX Protein Conjugates

et al. 2022

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Ratiometric sensors are self-referencing constructs that are functional in cells and tissues, and the read-out is independent of sensor concentration. One strategy for ratiometric sensing is to utilize two-color emission, where one component possesses analyte-dependent emission and the other is independent of analyte concentration, serving as an internal standard. In this way, the intensity ratio of the two components is a quantitative measure of the analyte. In this study, protein-based ratiometric oxygen sensors are prepared using the heme nitric oxide/oxygen-binding protein (H-NOX) from the thermophilic bacterium Caldanaerobacter subterraneus. The native heme cofactor is replaced with a Pd(II) or Pt(II) porphyrin as the oxygen-responsive phosphor. Mutagenesis is performed to incorporate a cysteine residue on the protein surface for thiol/maleimide coupling of the oxygen-insensitive dye, which serves as a Forster resonance energy transfer (FRET) donor for the porphyrin. While both Pd(II)-and Pt(II)-based sensors are responsive over biologically relevant ranges, the Pd sensor exhibits greater sensitivity at lower oxygen concentrations. Together, these sensors represent a new class of protein-based ratiometric oxygen sensors, and the modular platform allows the oxygen sensitivity to be tailored for a specific application. This proof-of-principle study has identified the key considerations and optimal methodologies to develop and subsequently refine protein-based ratiometric oxygen sensors.

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Corrole–protein interactions in H-NOX and HasA

Abstract: Mutagenesis was utilised to reveal corrole–protein interactions in H-NOX and HasA. The key interaction is a hydrogen bond between the PO unit of the corrole and a protonated histidine residue.

Cited by 5 publications

References 70 publications

Designer Heme Proteins: Achieving Novel Function with Abiological Heme Analogues

Designer Heme Proteins: Achieving Novel Function with Abiological Heme Analogues

Diversifying the functions of heme proteins with non-porphyrin cofactors

Ratiometric Oxygen Sensing with H-NOX Protein Conjugates

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