Fibrillar superstructure formation of hemoglobin A and its conductive, photodynamic and photovoltaic effects
Introduction
The emergence of novel functions has been achieved by creating novel structures. The development of new biomaterials that serve for a functional scaffold accommodating multiple effects has been pursued with the aim of application at the bio-nanomaterial interface. Elucidation of the principles for protein assembly, therefore, is fundamental not only for assessing biological phenomena at the molecular and cellular levels but also for obtaining protein-based biomaterials. Protein suprastructure formation has been involved in various physiological and pathological conditions, including cytoskeleton formation, extracellular matrix formation and even amyloidosis, as witnessed during the cellular degeneration of various neurodegenerative disorders [1], [2], [3], [4], [5], [6], [7].
Generally, suprastructures are derived from a self-assembly process of their constitutive monomeric units. Molecular self-assembly has been the pivotal event in describing various nanostructure formations of polymers and inorganic materials. Although proteins could also be self-assembled, their assembly mechanism has not been generalized because of chemical and structural complexities intrinsic to protein molecules. Recently, we have demonstrated that amyloid fibrils of α-synuclein, a pathological component of Parkinson’s disease, were produced almost instantaneously by the treatment of either hexane or shear force [8], [9]. Structural rearrangement within the preformed oligomeric structure of α-synuclein was responsible for the dramatically facilitated fibrillar suprastructure formation. These observations led us to propose a model for protein suprastructure formation, suggesting that the structural rearrangement within the protein quaternary structure plays a crucial role in the protein self-assembly since the interactive interfaces already present between intramolecular subunits are ready to exchange their partners, leading to intermolecular assembly and subsequent protein suprastructure formation.
Hemoglobin A (HbA) has been investigated to demonstrate whether this heme-containing oxygen-transferring protein with a well-defined tetrameric quaternary structure (α2β2) converts into the fibrillar protein suprastructure observed with hemoglobin S (HbS) by maneuvering the subunit–subunit interfaces [10]. HbS is derived from HbA via a single-point gene mutation which results in the amino acid substitution of nonpolar valine for anionic glutamate as the sixth residue of the β-subunit [11]. Under low oxygen conditions, the deoxyhemoglobin undergoes structural rearrangement that exposes a hydrophobic patch located in between the E and F helices. This hydrophobic patch is then able to interact with the nonpolar valine of proximal HbS [12]. As a result, HbS exhibits a high propensity for polymerization to the fibrillar state, which causes structural distortion of red blood cells and produces the diseased state known as sickle cell anemia [13]. It is therefore presumed that normal HbA may have an innate potential to be assembled into the fibrillar suprastructure although the protein lacks the nonpolar valine of HbS that has been suggested to be critical for the polymerization [14]. In addition, intermolecular assembly of HbA is also postulated because myoglobin, which exhibits a structural similarity to the α and β-subunits of HbA, was demonstrated to form β-sheet-rich amyloid fibrils in its deoxy state [15]. Based on this circumstantial evidence, HbA has been examined to see whether the protein turns into the fibrillar protein suprastructures. Since the HbA contains the redox active irons within photoactivatable heme structures, suprastructures made of HbA could be employed in various areas, such as conductive protein wire formation, production of photovoltaic materials, photodynamic therapy and oxygen storage. This investigation therefore aims to provide the molecular principle of the protein suprastructure formation, which could allow us to develop valuable biocompatible materials that could eventually be applicable to a particular area of the bio-machine interface.
Section snippets
Materials
HbA, HbS and met-hemoglobin (met-Hb) were purchased from Sigma. The proteins were dissolved to 10 mg ml−1 in 100 mM sodium phosphate, pH 7.4, and stored at −80 °C in aliquots. For the pH effect of the CHCl3-induced HbA fibrillation, HbA was dissolved in 100 mM sodium phosphate at various pHs between 6.8 and 7.6. Most reagents, including CHCl3, ascorbic acid and terephthalic acid (TPA), were supplied from Sigma unless otherwise mentioned.
Fibrillation of HbA and disintegration of the HbA fibrils
HbA (0.1 mg ml−1 in 100 mM sodium phosphate at pH 7.4) was treated
CHCl3-induced fibrillation of HbA
The fibrillar suprastructure of HbA was formed with 0.5% CHCl3, which could alter the subunit alignment within the quaternary structure of HbA [17]. Soluble HbA was revealed in a spherical unit with an average diameter of 5.5 nm under TEM following protein adsorption onto a copper grid and negative staining with uranyl acetate (Fig. 1a). When HbA was treated with 0.5% CHCl3 at 0.1 mg ml−1 in 100 mM sodium phosphate, pH 7.4, the fibrillar structures were produced instantaneously with a brief vortex.
Conclusion
HbA became self-assembled into the fibrillar structure in the presence of 0.5% CHCl3 and the resulting fibrils were selectively disintegrated into fragments by an additional treatment of ascorbic acid. Structural rearrangement within the protein quaternary structure of HbA has been suggested to be a principle for the protein suprastructure formation. Applications of the HbA fibrils have been also pursued to show their electrical conductivity, photovoltaic effect and photodynamic effect.
Acknowledgements
This study was supported in part by the Korea Healthcare Technology R&D Project (A084217-0902-0000100) of Ministry for Health, Welfare and Family Affairs.
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