Structural and functional characterization of β-sheet heme binding peptides: de novo designed and naturally occurring
D'Souza, Areetha Renita
Date of Issue2018-01-22
School of Biological Sciences
Heme proteins are a specialized class of metalloproteins that play a vital role in complex biological reactions, including photosynthesis and respiration. These proteins achieve their diverse structures and functions by utilizing a large number of amino acids (~more than 100 residues). Engineering of minimalistic peptides or miniproteins that can recapitulate the features of such proteins has gained significant attention in the recent years, primarily due to their applications in creating new biocatalysts and developing model systems for a better understanding of protein structure and functional attributes. Several efforts have been made towards the design of helical heme-peptides in water and membrane environment. However, fewer studies have focussed on the design of functional β-sheet peptides due to their poorly understood folding principles and a tendency for self-aggregation. In this study, we have engineered and characterized a series of β-sheet peptides in water and membrane-like environment. First, a series of mono-heme four stranded and di-heme six stranded β-sheet peptides were designed for heme binding in a membrane-like environment. The four and six stranded peptides adopted an anti-parallel β-sheet structure and could accommodate one or two hemes respectively. The linker residues between β-strand-II/III were optimized by utilizing a β-turn (DPro-Gly) and conformationally flexible β- and w-amino acids in the four stranded peptides to modify their heme binding pocket. Designed peptides contain increasing number of methylene groups from n= 1 to 7 on the flexible linker residue to study its effect on heme binding and catalysis. An increase in binding affinity of designed peptides was observed with an increase in the length of w-amino acids. The six stranded β-sheet peptide was observed to bind two hemes cooperatively. Additionally, these peptides also serve as peroxidases and participate in electron transfer with cytochrome c in a membrane environment. The four stranded hydrophobic peptides were further modified to improve its solubility in aqueous solutions. The heme binding pocket in these water-soluble peptides was altered by changing linker residues between β-strand II/III (DPro-Gly turn, w-aminoacids, Gly-Gly-Gly residues), axial coordination of heme (His/Ser coordination), and by creating a shorter binding pocket (Val9Val11). These peptides assumed a well-defined four-stranded β-sheet topology. The optimized peptides were observed to have picomolar binding affinities that are comparable to natural hemeproteins. Additionally, the peptide/heme complex exhibited high stability towards thermal- and chemical induced denaturation. Finally, the non-native heme interaction with self-aggregating Aβ40 peptide was characterized in a membrane-like environment. Aβ40 has recently been reported to bind heme in an aqueous environment. In Alzheimer’s disease (AD), Aβ-heme complex has been found to cause a regulatory heme deficiency, initiating oxidative stress by forming a peroxidase and abrogating Aβ aggregation effectively. For understanding the coordination and binding of heme to Aβ in a membrane-bound environment, experiments were also carried out with a non-amyloidogenic segment of the peptide (Aβ-16). The unstructured Aβ-16 peptide was found to have a lower affinity compared to Aβ-40 peptide in both solution and membrane environment. Monomeric Aβ-40 has been shown to be unstructured in solution, but a bis-histidine heme coordination was observed for the α-helical Aβ peptide in DPC micelles. These results highlight the importance of the C-terminus of Aβ for maintaining heme coordination and structure of the peptide. Interestingly, heme binds to Aβ in a 2 peptide:1 heme stoichiometry and efficiently inhibits its aggregation within the membrane. It also behaves as a weak peroxidase and thereby contributes to a lesser oxidative stress on cells. These findings suggest a neuroprotective role of the Aβ/heme complex in a membrane mimetic environment.