Supramolecular dimerization of a hexameric hemoprotein via multiple pyrene-pyrene interactions

Abstract

Protein assemblies are being investigated as a new-class of biomaterials. A supramolecular assembly of a mutant hexameric tyrosine coordinated hemoprotein (HTHP) modified with a pyrene derivative is described. Cysteine was first introduced as a site-specific reaction point at position V44 which is located at the bottom surface of the cylindrical structure of HTHP. [Formula: see text]-(1-pyrenyl)maleimide was then reacted with the mutant. The modification was confirmed by MALDI-TOF mass spectrometry and UV-vis absorption spectroscopy, indicating that approximately 90% cysteine residues are attached via the pyrene derivative. Size exclusion chromatography (SEC) measurements for pyrene-attached HTHP include a single peak which elutes earlier than the unmodified HTHP. Further investigation by SEC and dynamic light scattering (DLS) measurements indicate the desired size corresponding to the dimer of the hemoprotein hexamers. The multivalent effect of pyrene–pyrene interactions including hydrophobic and [Formula: see text]–[Formula: see text] stacking interactions appears to be responsible for including formation of the stable dimer of the hexamers. Interestingly, the assembly dissociates to the hexamer by removal of heme. In the case of the apo-form of pyrene-attached HTHP, the pyrene moiety appears to be incorporated into the heme pocket because the modification point is located at the adjacent residue of the Tyr45 coordinating to heme in the holo-form of HTHP. Subsequent addition of heme into the apo-form of pyrene-attached HTHP regenerates the dimer of the hexamers. The present study demonstrates a unique heme-dependent system in which HTHP is assembled to form a dimer of hexamers in the presence of heme and disassembled by removal of heme.

A Supramolecular Assembly of Hemoproteins Formed in a Star-Shaped Structure via Heme–Heme Pocket Interactions

“…The size of the assembly in the crude mixture generated by mixing equimolar (1-Cyt b 562 N80C ) n and apoHTHP with respect to the amount of heme binding site at 45 • C was evaluated by size exclusion chromatography (SEC) analysis. The elution volume of a major SEC peak of this assembly is 14.4 mL (Figure 5A), whereas the elution volumes of Cyt b 562 and apoHTHP are 17.2 mL [29] and 16.0 mL, respectively [21]. This result indicates the existence of a structure which is larger relative to Cyt b 562 and apoHTHP.…”

“…In contrast, HTHP is thermally stable at well over 45 • C [25] with the T m value of around 130 • C. Then, it is likely that a competitive binding affinity of the thermally stable apoHTHP towards the exposed artificial heme on the surface of Cyt b 562 N80C at the ends of the linear (1-Cyt b 562 N80C ) n is favorable, allowing the binding of the artificial heme to HTHP. Thus, apoHTHP prepared from the wild-type protein indicates absence of absorbance in the visible region in the UV-Vis spectrum (Figure 3A) [21]. Then, unmodified Cyt b 562 containing a prosthetic heme was mixed with apoHTHP under an equimolar condition in the same amount as that of the heme-binding site, as shown in Figure 3B.…”

“…It is considered as an interesting building block for artificial protein assemblies due to its symmetric structure and thermal stability with a denaturation midpoint, T m , above 130 • C. Chemical modifications of HTHP, via an engineered cysteine residue, enable construction of various assemblies such as a stacked dimer, a two-dimensional sheet, and a micelle-like structure [19][20][21]. In addition to these features, HTHP allows the replacement of heme with artificial cofactors [21][22][23]. Thus, HTHP is one of the useful building blocks for the generation of supramolecular assemblies.…”

Dynamic Protease Activation on a Multimeric Synthetic Protein Scaffold via Adaptable DNA‐Based Recruitment Domains

“…The V44 residue was selected as the mutation site, being located on the top face of the hexamer with an approximately 3 nm distance between adjacent V44 residues. The HTHP cysteine mutant (HTHP V44C ) was expressed in E. coli and purified by anion exchange and size exclusion chromatography (SEC) [24] . Because of the strong intermolecular interactions between HTHP monomers, the protein is always obtained in its stable hexameric form [22] .…”

Dynamic Protease Activation on a Multimeric Synthetic Protein Scaffold via Adaptable DNA‐Based Recruitment Domains

“…The HTHP cysteine mutant (HTHP V44C ) was expressed in E. coli and purified by anion exchange and size exclusion chromatography (SEC). [24] Because of the strong intermolecular interactions between HTHP monomers, the protein is always obtained in its stable hexameric form. [22] After reaction, the HTHP-DNA bioconjugates were purified using SEC, and characterized by SDS-PAGE and analytical SEC (Figure S1 and S3).…”