Expanding heme-protein folding space using designed multi-heme β-sheet mini-proteins

Abstract

Nature has primarily exploited helical proteins, over β-sheets, for heme/multi-heme coordination. Understating of heme-protein structures has motivated the design of heme proteins utilizing coiled-coil helical structure. By contrast, de novo designed β-sheet proteins are less successful. However, designing proteins with discretely folded β-sheet structures encoding specific functions would have great potential for the development of new synthetic molecules e.g. enzymes, inhibitors. Here we report the design and characterization of multi-heme binding four-, six-, eight-, and twelve-stranded β-sheet mini-proteins (<40 amino acids) and proteins. Atomic-resolution structures demonstrate an expected β-sheet structural topology. The designed β-sheet mini-proteins pack or latch multiple hemes with high affnity in versatile orientations either by stacking or sideways, mimicking naturally occuring multi-heme protein conduits. The designed multi-stranded β-sheet heme proteins could serve as a platform for the generation of novel synthetic β-sheet protein mimics.

Mimochrome, a metalloporphyrin‐based catalytic Swiss knife†

“…Poor peroxidase activity was detected, suggesting a strong binding affinities between the iron ion and two axial His ligands. Remarkably, some analogues were soluble and monomeric in solution, allowing the structural characterization by NMR of the apo forms [217]. Unfortunately, the lack of high-resolution structures of the holo states did not allow a clear correlation between peptide sequence/coordination geometry and heme-binding affinity.…”

Pushing and Pulling: A Dual pH Trigger Controlled by Varying the Alkyl Tail Length in Heme Coordinating Peptide Amphiphiles

“…The split Soret is a demonstrated phenomenon of two chromophores being electronically coupled due to their proximity and ordering . This feature is not common in synthetic peptides binding to heme with bis-histidine coordination. ,,− Further analysis of the binding affinity (equation S1) is consistent with an earlier report, where the ideal ratio of peptide:heme is ∼10:1 . Analyzing the binding suggests that shorter alkyl chains yield lower affinity binding sites than the peptides with longer alkyl chains ( K D values: c8-Pep = 49 μM, c12-Pep = 0.9 μM, c16-Pep = 0.56 μM, c18-Pep = 0.13 μM).…”

“…Therefore, a peptide that yields a greater β-sheet content will yield a higher binding affinity due to the fact that more structured binding sites are available for heme binding. This assertion is supported by a series of heme and multi-heme coordinating peptides by Bhattacharjya and co-workers. − In early iterations, β-hairpin peptide structures in DPC (dodecylphosphocholine) detergent micelles yielded binding affinities ranging from K D = 0.5–9 μM. The peptides were designed to bind either 1 or 2 heme molecules per peptide.…”

De Novo-Designed β-Sheet Heme Proteins

“…To achieve this, we designed and characterized a series of β-sheet miniproteins that can coordinate two or four heme molecules with tight binding affinities. 83 The six-stranded β-sheet peptide (peptide 7) that binds two hemes in membranes was optimized by replacing some hydrophobic residues with polar residues to help improve its water solubility (Figure 8). Miniprotein 1 was designed to coordinate two heme molecules in a sidewise orientation (Figure 8A), and a longer four-stranded β-sheet structure, miniprotein 2, was designed to create a binding pocket between strands 2 and 3 by placing four histidines to bind two heme molecules in a stacking orientation (Figure 8A).…”

“…The water-soluble β-sheet miniproteins were further redesigned to partially mimic a multiple-heme arrangement similar to that of MtrF protein (Figure C). To achieve this, we designed and characterized a series of β-sheet miniproteins that can coordinate two or four heme molecules with tight binding affinities . The six-stranded β-sheet peptide (peptide 7) that binds two hemes in membranes was optimized by replacing some hydrophobic residues with polar residues to help improve its water solubility (Figure ).…”