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Journal of Photochemistry & Photobiology A: Chemistry

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Short note

Effect of macromolecular crowding on the conformational behaviour of aporphyrin rotor

Shuto Kozakaa, Rie Wakabayashia, Onofrio Annunziatab, Milan Balazc,⁎, Masahiro Gotoa,Noriho Kamiyaa,⁎, Sergei V. Dzyubab,⁎

a Department of Applied Chemistry and Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 812-8581, JapanbDepartment of Chemistry and Biochemistry, Texas Christian University, Fort Worth, TX 76129, USAcUnderwood International College, Integrated Science and Engineering Division, Yonsei University, Seoul, 03722, Republic of Korea

A R T I C L E I N F O

Keywords:FluorescenceFluorescence probesPorphyrinoidsSolvent effectsBiosensorsMacromolecular crowding

A B S T R A C T

Macromolecular crowding modulates the conformational preference of a small molecular rotor, which is used asa molecular viscometer. The shift towards the planar conformation of the porphyrin rotor was observed in thepresence of increasing concentrations and sizes of polyethylene glycols. This observation highlights the differ-ences between the behaviour of this molecular viscometer in the crowding and non-crowding types of media.

Numerous bio(macro)molecular and biochemical events are influ-enced by the structural and physical constraints of the environment.Proteins, lipids, carbohydrates, and nucleic acids occupy substantialvolumes in cells, thus creating a highly crowded, heterogeneous en-vironment. This so-called macromolecular crowding effect has beenextensively studied in past decades, in order to relate the processesobserved in vivo or in cellulo to the results observed in vitro or in spec-troscopic investigations [1–5]. Both biological and synthetic polymers,known as crowders, have been used to mimic cellular environments.Numerous studies have shown that steric repulsion among biomacro-molecules, imposed by crowders, favours compact conformations ofproteins as well as biomolecular association processes [1–5]. The effectof macromolecular crowding has been extensively explored in regard toprotein stability, structure, and function, as well as protein-protein in-teractions [1–6]. Yet, the effect of crowding on the conformationalbehaviour of small molecules, i.e., molecules that are smaller thanmacromolecular crowders (yet larger than the molecules of the sol-vent), has not been addressed. This might have significant implications,especially in the field of environment-sensitive probes, including mo-lecular rotors [7]. The molecular rotors whose conformational beha-viour and photophysical properties are sensitive to changes in the en-vironment have garnered considerable interest in recent years due tothe ease of accessibility and structural and functional tunability forvarious applications [7,8]. Among various molecular rotors, smallmolecule-based viscometers have received a lot of attention, as they

allow assessing media’s viscosity without imposing significant pertur-bations to the media [8]. On the other hand, exploring the effects of themedia on the conformation of small molecule solutes also represents aninteresting paradigm. Here, we report on the conformational behaviourof the zinc porphyrin dimer (PD, Fig. 1) in a macromolecular crowdingenvironment, using various concentrations of polyethylene glycols(PEGs) of different sizes. PEGs are water-soluble, chemically inert,biocompatible polymers, which are commonly used as macromolecularcrowding agents to mimic intracellular crowding density [1–6].

PD (Fig. 1) has been extensively used as an environmental probe forvarious types of environments, including molecular and ionic liquids,reverse micelles, cellular milieu, and polymer film [9–15]. On thestructural level, rotation around the diyne moiety produces a set ofrotamers, with the twisted (λem at ca. 700–720 nm) and planar (λem atca. 780–800 nm) rotamers representing the extremes (Fig. 1A).

The triethylene glycol moieties on the porphyrin core were in-troduced to minimize the aggregation of PD; thus, the effects of themedia on the conformational behaviour of PD could be easily assessedusing the emission spectra of PD [9]. Importantly, it was established,that PD acts a molecular viscometer, as the variations of media’s po-larity, pH, etc had no significant impact on the distribution of the ro-tamers [9], and over a range of concentrations in some molecular sol-vents, no significant conformational changes were noted (Fig. S1). Ingeneral, the amount of twisted PD appeared to increase with increasingof the media’s viscosity (Fig. 1B and C), due to viscosity depended

https://doi.org/10.1016/j.jphotochem.2018.10.006Received 31 August 2018; Received in revised form 28 September 2018; Accepted 2 October 2018

⁎ Corresponding authors.E-mail addresses: mbalaz@yonsei.ac.kr (M. Balaz), kamiya.noriho.367@m.kyushu-u.ac.jp (N. Kamiya), s.dzyuba@tcu.edu (S.V. Dzyuba).

Journal of Photochemistry & Photobiology A: Chemistry 369 (2019) 115–118

Available online 06 October 20181010-6030/ © 2018 Elsevier B.V. All rights reserved.

T

twisted to planar rotamer conversion in the excited state [9,10,15,16].In order to evaluate the effect of macromolecular crowding on the

conformational preference of PD, various concentrations of PEG-2 kDa,PEG-20 kDa, and PEG-40 kDa were evaluated (Fig. 2). Consistent with

the literature account [17], PD was not emissive in water, but uponaddition of various amounts of PEG, the emission was restored (Figs.S2–S4). Importantly, the amount of the twisted conformation of PDdecreased as the amount of PEG increased (Fig. 2, bottom). The shift

Fig. 1. Structure of PD (A) and its conformational preference as a function of viscosity: molecular solvents (B) and ionic liquids (C); modified from Ref. [13].

Fig. 2. Top: normalized emission spectra of PD (normalization done at 740 nm) in the presence of various amounts of crowding agents in water: PEG-2 kDa – A, PEG-20 kDa – B, and PEG-40 kDa – C. Bottom: amounts of twisted PD as a function of PEG concentration (left) and media viscosity (right). Conditions: [PD]=5 μM;[DMSO]=0.5% (v/v); λex= 475 nm; amount of PEG in % (weight PEG/volume of water; wv).

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from the twisted to planar rotamer was observed with increasing con-centrations of PEG as well as with increasing viscosity of the solution(Table S1), regardless of the molecular weight of PEG (Figs. 2, S2–S4).

The effect of PEG-20 kDa on the conformational bias of PD wasdistinct from that imposed by both PEG-2 kDa and PEG-40 kDa. Inprinciple, this is not surprising, considering the complexity of crowding,and the fact that different sized PEGs are unlikely to provide similartypes of crowding environments [18–21].

Arguably, this initial observation indicated that PD could be used asa small molecule probe for evaluating macromolecular crowding. Thesetypes of probes are not currently available, and only several spectro-scopic probes, based on protein-scaffolds conjugated to fluorophores,have been developed to test and monitor the effect of macromolecularcrowding [22–26].

It was previously established that by using different excitation wa-velengths (λex) around the Soret band absorption maximum of por-phyrin rotors, one could elucidate the effect of the media on the dis-tribution of porphyrin rotamers [14–16]. Specifically, if the planar/twisted ratio could be altered under different λex, then both con-formations could be assumed to be present in a given media. On theother hand, if the planar/twisted ratio would remain unchanged withvarying λex, then the existence of a single conformation could be as-sumed. It should be noted that λex below 475 nm should preferentiallyexcite the twisted rotamer, whereas λex above 475 nm should pre-ferentially excite the planar rotamer. Importantly, at λex of 475 nm,both rotamers should be equally excited. Here, we examined theplanar/twisted ratio (i.e., the amount of twisted or planar PD) as afunction of amount and identity of PEG over a range of λex (Figs.S5–S9). Not surprisingly, in all cases the planar/twisted ratio changedin favour of the planar conformation as λex was changed from 460 to510 nm. However, the amount of twisted PD decreased with increasingconcentrations of PEG as well (Fig. S10). This observation indicatedthat an increase of macromolecular crowding favoured the more com-pact, i.e., planar, conformation of PD. All of the available emission data(Figs. 2, S5–S10) confirmed the conformational preference of planar PDin macromolecular crowding environments, which was also consistentwith the excluded-volume model (see Appendix S1).

Although UV–vis spectroscopy has rarely been used for assessingconformational preference of PD, we found that changes in the UV–visspectra were also consistent with the decrease of the twisted con-formation of PD as a function of increasing amounts of PEG (Figs.S11–S13). Specifically, the longer-wavelength Q-band became moreprominent, while the relative intensity of the shorter-wavelength Q-band decreased (Figs. S11–S13). Thus, the changes were found to be inagreement the increased amount of the planar conformation [27].

In order to decouple the effect of the crowding effect of PEG fromthe viscosity effect of PEG solutions on conformation of PD, PEG-2 kDawas dissolved in ethanol (EtOH) and emission and UV–vis spectra of PDwere acquired (Fig. S14).1 The amount of twisted PD appeared to in-crease with increasing viscosity of PEG-2 kDa/EtOH solutions, as evi-dent by the positive slope values (Fig. S14, Table S2). Considering thatthe solubility and solvation of PEG in alcohols is inferior to those ob-served in water, PEG was likely to be in a collapsed/condensed con-formation, and as a result was not acting as a crowding agent in EtOH(see Appendix S1). Thus, PEG in EtOH appeared to behave similar toEtOH solutions that contain viscous polyols, such as ethylene glycol(EG) or glycerol (Fig. 1B).

In addition, we examined the conformational behaviour of PD insolutions with similar viscosities, i.e., PEG-2 kDa/40% wv (17.0 mPa s),PEG-40 kDa/10% wv (19.8 mPas), and EG/neat2 (17.1 mPa·s) (Fig. 3

and S15, Table S1). It appeared that the amount of twisted PD did notdepend on the viscosity of the media, but rather on the identity of themedia. This experiment provided further confirmation that thecrowding effect of PEG was modulating the conformation of PD.

Importantly, since the conformational behaviour of PD appeared tobe sensitive to the crowding environment, its ability to shed light onchanges in the viscosity of the media with crowding agents should betaken with caution. Specifically, due to the fact that glycerol/alcohol(or water) mixtures (i.e., media that are routinely used for constructingcalibration curves for viscosity measurements) could be consideredhomogenous media that are void of nanoscopic heterogeneities,whereas structural heterogeneity and compartmentalization are thestaples of cellular environments.

Thus, it could be argued that glycerol/alcohol mixtures might notaccurately mimic cellular environments, and that the use of media thatcontain crowding agents would be a more appropriate option for gen-erating calibration curves.

Overall, we have demonstrated for the first time that macro-molecular crowding modulates conformational behaviour of a smallmolecule, by favouring the more compact, planar conformation of PD.This contrasts with the conformational preference towards the twistedconformation that is observed in molecular solvents. Arguably, PDmight be used as a probe for monitoring and quantifying macro-molecular crowding, and we are currently investigating the effects ofcrowding using macromolecular and molecular crowders on the con-formational behaviour of various molecular rotors.

Conflicts of interest

There are no conflicts to declare.

Acknowledgement

This work was supported in part by JSPS KAKENHI Grant NumberJP16H06369 from the Ministry of Education, Cultures, Sports, Scienceand Technology (MEXT) of Japan.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the

Fig. 3. Effect of media (EG/neat (blue), PEG-2 kDa/40% wv in H2O (red), PEG-40 kDa/10% wv in H2O (green)) with similar viscosities on the conformation ofPD. Conditions: [PD]= 5 μM, [DMSO]= 0.5% (v/v); λex= 475 nm; [PEG-2 kDa]=40% (wv); [PEG-40 kDa]= 10% wv; EG – neat. Emission spectrawere normalized at 740 nm; inset shows the amount of PD in EG/neat (blue),PEG-2 kDa/40% wv (red), and PEG-40 kDa/10% wv (green).

1 PEG-20kDa and PEG-40kDa were not soluble in EtOH. Also, PEG 2kDa wasfound to precipitate from EtOH when it was allowed to stand at room tem-perature overnight.2 EG is not a crowding agent, and it was used only as a solvent of a certain

(footnote continued)viscosity for comparison.

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online version, at doi:https://doi.org/10.1016/j.jphotochem.2018.10.006.

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