X-ray observations of single bio-supramolecular photochirogenesis
Graphical abstract
Introduction
Human serum albumin (HSA) is the most abundant protein in human plasma and functions as a transporter/reservoir of various endogenous and exogenous hydrophobic molecules such as fatty acids, bilirubins, hormones, and many pharmaceuticals in its ligand-specific binding sites [[1], [2], [3], [4], [5], [6], [7], [8], [9], [10]]. These hydrophobic binding sites are three-dimensional, well organized, and inherently chiral. We have employed these binding pockets as a chiral environment for affecting the enantiodifferentiating photocyclodimerization of 2-anthracenecarboxylate (AC) to obtain a mixture of four stereoisomeric cyclodimers 1–4 (Fig. 1), two of which are chiral and obtained in higher enantiomeric excesses of up to 90% upon irradiation with HSA than other photo-catalysts [[11], [12], [13], [14]]. We have elucidated the AC binding sites and stoichiometries as well as their photochirogenic performance in a series of spectroscopic and photochemical studies [[15], [16], [17], [18]]. Nevertheless, we still know little about the events occurring inside HSA upon AC complexation and the supramolecular consequences of AC-HSA complex photoirradiation, which hinders our molecular-level understanding of the dynamic complexation and photochirogenesis processes.
In this study, we visualize the binding and photochirogenic behaviour of HSA with and without AC molecules bound to its first to fourth binding sites at a single-molecule level using the real-time monitoring of the Brownian motion of AC-HSA complexes (AC/HSA = 0, 1, 5, and 10) with a diffracted X-ray tracking (DXT) method (Fig. 2) [[19], [20], [21], [22], [23], [24], [25], [26], [27]]. DXT is a state-of-the-art technique invented by one of the present authors for tracking the Brownian motion of a single target molecule with high angular accuracy (< 0.05°), while avoiding any significant physical or chemical perturbations, which is enabled by chasing the X-ray spots diffracted from a gold nanocrystal (<100 nm) attached to the target (< 10 nm) of a comparable or larger size. Although it shall be assigned using the real size effect of this HSA, the target host molecule in DXT is known, as the size of gold nanocrystal has little effect on the dynamics of the target molecules. For a molecular-level understanding of HSA-mediated bio-supramolecular photochirogenesis, DXT is expected to allow us to directly observe the dynamic behaviour of a gold nanocrystal-tagged single HSA molecule before and after the binding of different equivalents of AC as well as the effects of photoirradiating the AC-HSA complex track on in-situ Brownian motion in real time. Combining the dynamic physical inspection via the static (photo)chemical investigation, we can draw a more precise dynamic picture of this highly efficient, but dynamically less elucidated bio-supramolecular photochirogenesis. (See Fig. 3.)
Section snippets
DXT of HSA and AC-HSA complexes
Conventionally, gold nanocrystals are tethered to a single biomolecule anchored to an amorphous gold substrate with a long-chain N-succinimidyl 3- (2-pyridyldithio)propionate (LC-SPDP) [28–0]. For HSA, we chose sulfosuccinimidyl 6-(3′-(2- pyridyldithio) propionamido) hexanoate (sulfo- LC-SPDP) as the tether to link a gold nanocrystal to a Lys residue on the HSA surface [[28], [29], [30]]. The binding sites of gold to the Lys residues (59 residues) were random; thus, it was assessed that the
DXT under photoirradiation
We further performed real-time DXT monitoring of 10 AC irradiated in-situ at 355 nm with a Nd:YAG laser in the midst of monitoring and compared its MSD behaviour before and after the irradiation (for the detailed protocol, see Fig. 4a and Scheme S4 in the Supporting Information). As shown inFig. 4b and 4c, the MSD growth curve unequivocally declined immediately after laser irradiation. This real-time, in-situ DXT observation revealed that the release of the photoproduct from the
Conclusion
We have demonstrated that the state-of-the-art DXT method is a powerful and unique tool for real-time tracking and accordingly visualizing supramolecular events occurring in HSA at a single-molecule level, which is not achievable by other conventional techniques. The methodology developed and the findings obtained in this study should not be restricted to HSA or related proteins, but be expandable to a vast variety of supramolecular, polymer, and biomolecular systems and provide us with
Acknowledgments
We are grateful to prof. Tadashi Mori of The Osaka University, Graduate School of Engineering and Prof. Haruo Kozono of Tokyo University of Science, Graduate School of Biological Science for crucial logistical help. This research was supported by a Grant-in-Aid for Scientific Research (A), (Grant Nos. 22241032 and 21245011) from the Ministry of Education, Culture, Sports, Science and Technology, Japan in 2010–2012 and was performed with the approval of the SPring-8 (Proposal Nos. 2011B1299,
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