Functionalization of Bacterial Microcompartment Shell Proteins With Covalently Attached Heme

Abstract

Heme is a versatile redox cofactor that has considerable potential for synthetic biology and bioelectronic applications. The capacity to functionalize non-heme-binding proteins with covalently bound heme moieties in vivo could expand the variety of bioelectronic materials, particularly if hemes could be attached at defined locations so as to facilitate position-sensitive processes like electron transfer. In this study, we utilized the cytochrome maturation system I to develop a simple approach that enables incorporation of hemes into the backbone of target proteins in vivo. We tested our methodology by targeting the self-assembling bacterial microcompartment shell proteins, and inserting functional hemes at multiple locations in the protein backbone. We found substitution of three amino acids on the target proteins promoted heme attachment with high occupancy. Spectroscopic measurements suggested these modified proteins covalently bind low-spin hemes, with relative low redox midpoint potentials (about −210 mV vs. SHE). Heme-modified shell proteins partially retained their self-assembly properties, including the capacity to hexamerize, and form inter-hexamer attachments. Heme-bound shell proteins demonstrated the capacity to integrate into higher-order shell assemblies, however, the structural features of these macromolecular complexes was sometimes altered. Altogether, we report a versatile strategy for generating electron-conductive cytochromes from structurally-defined proteins, and provide design considerations on how heme incorporation may interface with native assembly properties in engineered proteins.

Mechanisms of Scaffold-Mediated Microcompartment Assembly and Size Control

“…The spontaneous curvature of microcompartment shell proteins is not known. Hexamers may have zero spontaneous curvature, since systems of only hexamer proteins can form tubular structures or rolled sheets (rosettes) under some conditions, ,− and assemble flat sheets on surfaces or interfaces. , However, in the latter systems interfacial tension exerts a strong driving force favoring flat sheets. Moreover, recombinant expression of hexamers, pseudo-hexamers, and pentamers can result in small, icosahedral, empty shells, ,, implying that there is a net spontaneous curvature for at least some stoichiometries of microcompartment proteins.…”

“…In particular, recent experiments on recombinant systems exhibit assembly of small (∼20–30 nm) empty microcompartment shells, suggesting a small shell spontaneous curvature radius. Yet, other experiments observe extensive flat sheets of hexamers, ,− suggesting the possibility of a low or zero spontaneous curvature. In this regard, both the simulations and theoretical model predict that shells with a small spontaneous curvature radius exhibit only narrow fluctuations in size when presented with scaffolds in molecules of different lengths, whereas shells with a large spontaneous curvature radius can assemble over a wide range of sizes to accommodate scaffolds with different lengths.…”

Mesoscopic to Macroscopic Electron Transfer by Hopping in a Crystal Network of Cytochromes

“…2 There are currently intensive bioengineering efforts to divert electron flow into high-energy or high-value products. For example, synthetic proteins toward the formation of nanowires have been constructed, 3,4 providing proof-ofprinciple for the concept of controlled and targeted longrange ET.…”

MCPdb: The bacterial microcompartment database

“…Access to validated biological structures as well as structural and biophysical annotations is necessary for well-informed scientific investigation surrounding MCPs. As a relatively new field, the structural biology of MCPs is an area of growing scientific and bioengineering interest [53][54][55][56][57][58][59][60][61][62][63][64]. Not only a tool for experts in the field, the MCPdb provides novices and young…”