Photoresponsive behavior of self-assembling systems by amphiphilic α-helix with azobenzene unit

ELSEVIER

Photoresponsive behavior of self-assembling systems by amphiphilic a-helix with azobenzene unit

Abstract A photoresponsive amphiphilic polypeptide compo& of two amphiphilic cu.helical polypeptides, polyl (y-methyl L-glutamate)-co( 12-glutamic acid) 1, joined with an azohenzene moiety. was prepared via the monolayer reaction method. We investigated the photoinduced changes in the aggregate structure of the polypeptide in aqueous solution. The polypeptides were soluble in aqueous solution and associated with each other to form a globular aggregate = 10 nm in diameter in the dark. The globular aggregate grew into e twisted ribl)oll-like structure which could be observed with an optical microscope. The trans.to-ci.y isomerization of- the azs.>benzenc umt 111 the main chain induced a hending of the polypeptide rod, which resulted in a dissociation of the aggregate. In this procr\s, the degree of dixaggregation was not linear to the irradiated light intensity, i.e., the photoinduccd cffcct appeared over a certain value of the light intensity. Such on-offdeformatiolr4 ol the supramolecular order may be due to a native character of self-organiLinu b Yystems supported by weak hut cooperative inter-molecular

interactions.

0 1998 Elsevier Science S.A.

Kc~\~~o,-fl.~: PhotoresponGve

amphiphilic

polypeptidea:

Self-asemhly:

Supramolcu~lcs:

1. Introduction It is welt known that structure formation assembling is essential for unique functions systems.

For

cxamplc.

Tobacco

mosaic

virus,

by molecular of biological microtubu-

lines, and ion channels are typical examples of biological supramolecutar systems. Among them, the acetylchotine receptor [ I ] consists of a ring of five similar subunits, and forms a channel pore in the center [ 21. Moreover. the pore is formed by a tubular assembly of tive amphiphitic oc-helical segments, M2. in each subunit [ 31. Recently. the self-assembly and morphology of biomimetic artiticial systems, such as amphiphites [ 4-8 ] and potypeptides [9-l 21, have been studied. These molecular units have been employed to build up functional structures [ 13-t 61. Ihara et al. [ t7,18 ] reported that

the

amphiphiles,

double-chain

alkyl

compounds

with

hydrophilic polypeptide-head groups, formed twisted ribbonlike aggregates by self-assembling in aqueous solution, with drastic morphological changes (globule -+ globule with tail + untwisted filament ---) double-helix + twisted ribbon). It was estimated that the twisted ribbon-like aggregates are constructed from single-walled bilayer membranes. Ghadiri

IO1 I-1 Wl/9X/Sl9 P//SI0I1~1~11(98)0006h

n(l (i:i

IYYX El. SEVICT Science 9

S.A.

All

rishts

reserved

Photoisomeriration:

Morphological

changea

et at. ] I Y-24 ] reported the construction of open-ended hollow tubular objects, peptide nanotubes, haacd on the aetfassembly of flat, ring-shaped cyclic peptide subunits made up of alternating D- and L-amino acid residues. The peptide nanotuhes acted as transmembrane ion channels and pores in a lipid

hilayer

membrane.

On the other hand. the introduction of photosensitive units into such supramolecular systems may be important for the understanding of complex photobiological processes and also for their practical applications. A numberofexcetlent reviews [ 25-271 on photoresponsive systems have recently been published.

In previous studies. we reported I 28.29 ] on the preparation of a photoresponsivc amphiphilic polypeptide and its porcforming activity in a lipid bitayer membrane. The potypeptide consisted

of two

amphiphilic

a-helical

r-ads

that

are

hydro-

philic on one fact (which consists of t>-gtutamic acid side chain) and hydrophobic on the opposite face (which comprises

y-methyl

L-glutamate)

joined

by

an

azobenzene

moiety. The polypeptides were incorporated into the hilayer membrane to form a transmemhrane hundle in the dark. The transmemhrane bundle acted a\ an ion-permeable pore through the membrane. Photoisomcrization of the aLobenzene moiety mduced reversible changes in the high-order

structure of the ionic pore in the membrane owing to the denaturation of the amphiphilic character of the polypeptide. As a result, the ion-transport properties through the membrane could be regulated by photoirradiation. Furthermore. the polypeptide formed an aggregate in aqueous solution and hydrophobic compounds were incorporated into the interior of the aggregate in the dark. Photoinduced high-order structural changes of the aggregate resulted in a release of the hydrophobic compounds to the external aqueous phase. Here, we report on the photo- and thermo-induced change\ in the high-order structure of the aggregate composed of the photoresponsive amphiphilic polypeptides. The polypeptides formed twisted ribbon-like aggregates by self-assembling in the aqueous solution in the dark. accompanied by drastic morphological changes ( globule + fibril -+ twisted ribbon). Photoirradiation of the globular particle resulted in the destruction of the aggregate structure (globule + disordered small particle and precipitation ) owing to the photoinduced denaturation of the amphiphilic character of the polypeptide. Furthermore, the relation hetwcen the intensity of the photo-stimulation and the degree of the induced destruction was also examined to clarify the photoresponsive behavior of the supramolecular system in more detail.

2. Experimental

A poly( y-methyl L-glutamate) with an azobenzene moiety in the main chain ( MAzoM) was obtained by the polymerization of N-carboxy anhydride of L-glutamic acid y-methyl ester [ 301 with p,p’-diaminoazobenzene as the initiator in dimethylformamide solution. The molar ratio of the anhydride to initiator was 60. An average molecular weight of I I 000 was estimated from the molar ratio of the azobenzene moiety to the y-methyl [>-glutamate residues of MAzoM. The ratio was determined from the absorbance at 375 nm on the basis of the molar extinction coefficient of the azobenzene in MAzoM dimethylformamide solulion. A photoresponsive amphiphilic polypeptide (am.MAzoM) was prepared by selective saponification of MAzoM side chains according to the monolayer reaction method, which has been described in detail elsewhere [ 3 I 1. The L-glutamic acid content of the resulting polypeptide wa\ estimated to be 34 mol% from H’-NMK analysis. Furthermore, the sequence of the polypeptide components. determined by gel filtration chromatographic analysis of the oligopeptide fraction resulting from theenzymatic hydrolysis of am.-MAzoM with Stuphy1ococcu.s LILIWW. Strain VX protease [ 32 I. could be written as (G,G,M,,M,,G,M,,M,,M,,G,M,M,,G,M,;M,M,,G,M,;M,; ),, where M,; and G,, denote y-methyl L-glutamate and I.-glutamic acid residues, respectively. The circular dichroism spectrum of am.-MAzoM in trimethyl phosphate (TMP) solution exhibited the two negative bands at 208 and 222 nm typical of ;I

stable right-handed a-helix. The helical content was estimated to be X0% from the value of the molar ellipticity at 222 nm. Therefore. am.-MAzoM formed an amphiphilic a-helix that is hydrophilic on one face (which consists of L-glutamic acids) and hydrophobic on the opposite face (which consists of y-methyl [,-glutamate side chains). IUV light irradiation induced a t,-llrl.v-to-cis photoisomerization of the azobenzene moiety of am-MAzoM. The main absorption band at 375 nm assigned to the truns v-r* transition was decreased by U\’ light irradiation for5 min in trifluoroethanol solution. It was estimated that 60% of the trmr form was converted to the cis isomer from the changes in the absorbance at 375 nm (Jasco, V-530 spectrophotometer ).

3. Methods

Am.-MAzoM was dissolved in N.N-dimethylformamide and the solution was poured into a glass flask. A thin film was formed on the inner surface of the flask by evaporating the solvent. After adding on aqueous solution containing 0. I M KCI at pH 6.9 to this flask, it was sonicated in an ice-water bath under a stream of nitrogen by using an ultrasonic processor ( Branson Sonifier model 250) for IO min. The concentration ot am.-MAzoM was I .92 X IO-” M. After sonication the solution was allowed to stand for three months to form superstructures by self-assembling of the polypeptides.

Nitrobenzofurazane-modified am.-MAzoM (NBD-am.MAzoM) was obtained as follows. Am.-MAzoM was dispersed in an aqueous solution containing 0. I M KCI. pH 6.9 ( IO ml) by ?,onication with a Branson Sonitier model 250 under a nitrogen atmosphere at 0°C in the dark. The concentration of am.-MAzoM in the aqueous solution was I .92 X IO ’ M. ‘The am-MAzoM aqueous solution was incubated with IO ~1 of 4-fiuoro-7-nitrohenzofurazane ( NBD) /ethanol solution (0. I M ) for I min. at 60°C [ 33 1. After the reaction. this solution was rapidly quenched to 0°C.

The anisotropy, y= (I, - I, ) /(I,, + 2I L ). of NBD-am.MAzoM in the aqueous solution was determined with a fluorescence polarization spectrophotometer (Otsuka Elcctronics. FS-SO I 11at 25°C. I., and I, are the fluorescence intensities polarized parallel and perpendicular to the direction of the polarized excitation beam. The concentration of NBDam.-MAzoM was I .92 X IO ’ M. The excitation wa\jelength of NBD-am.-MAzoM wa< 375 nm and rhe fluorescence was observed at 533 nm. Circular dichroism cpectra of am.-MAzoM in TMP and in 0.1 M KCI aqueous solution were measured with a Jaaco

J-600 spectropolarimeter. The concentration of am.-MAzoM was fixed at 1.92X lo-’ M. The secondary structure was estimated from the molar ellipticity. 3.4. Dynumic light-scattering

measurements

The size and distribution of the aggregates of am.-MAzoM in 0.1 M KC1 aqueous solution at pH 6.9 were measured at 25°C with a dynamic light-scattering spectrophotometer ( Otsuka Electronics, DLS-700). 3.5. Microscopic

TND glass filters. The relative intensity is indicated in percent, where 100% corresponds to the intensity in the absence of the TND filter.

measurements

Optical microscopic observation of the aggregates of am.MAzoM was carried out with a differential interference microscope (Olympus Optical. BXSO-%FLADI. >( 1000). The shape of the aggregates was observed with an atomic force microscope (Digital Instruments, Nanoxcope Il1a1 using the tapping mode. An aliquot ol‘the am.-MAzoM aqueous solution containing 0. I M KC1 at pH 6.9 was placed on freshly cleaved mica. allowing the polypeptide to be adsorbed on its surface for 5 min at room temperalure, rinsed with Milli-Q treated and doubly distilled water to remove excess salt. :tnd dried under ambient conditions. Atomic force microscopy ( AFM) images were done in ‘height’ mode using silicon cantilevers (125 mm. tip radius S-10 nm). A IO mm X IO mm scanner was used for imaging.

The solubility of am.-MAzoM in aqueous solution was followed by quantitation of the terminal amino group ofamMAzoM by a fluorescence technique using NBD as a probe. It is well known that NBD, which itself is nonfluorescent. rapidly reacts with primary amino groups yielding a uscfui fluorescent probe [ 331. As the am.-MAzoM had no primary amino groups in the side chains, NBD could be introduced at the amino-terminal end only. An am-MAzoM aqueous solution containing 0.1 M KCI, pH 6.9. was filtered using a membrane filter (Millipore. 0. I pm). The permeate solution (5 ml ) was incubated at 60°C with 5 p.1of an ethanol solution of NBD (0.1 M) for 1 min. After the reaction. it was quenched rapidly to 0°C. The initial concentration of am.MAzoM in the aqueous solution was fixed at I .92 X IO- ’ M. Strong fluorescence could be observed in the resulting solution containing am.-MAzoM carrying NBD. The excitation wavelength of NBD was 475 nm and the fluorescence was observed at 541 nm with a spectrofiuorophotometer ( Jasco. F-777).

4. Results and discussion

The conformation of the photoresponsive amphiphilic polypeptide, am.-MAzoM, was characterized using circular dichroism (CD). The CD spectra of am.-MAzoM in TMP and in an aqueous solution containing 0. I M KC1 at pH 6.9 in the dark are shown in Fig. I. The CD spectrum of the TMP solution of am.-MAzoM exhibited two negative bands at 208 and 222 nm typical of a right-handed a-helix. On the other hand. the CD spectrum of am.-MAzoM in the aqueous sohtion showed red shifting of the 222 nm band toward 227 nm and a decrease in the molar ellipticity at 208 nm. This distortion of the spectrum implied that the o-helical rods of am.MAzoM formed aggregates [ 34-361. Furthermore, wte investigated the stability of the am -MAzoM aggregate with respect to pH in aqueous solution in the dark. The pH dependence of the molar el:lipticity at 227 nm, [ O]Z1,r of am.MAzoM in aqueous solution is shown in Fig. 2. The value of [ H]227 reveals a negative maximum in the neutral pH region. This result arises from the increase in the solubility of am.MAzoM in this region compared IO that in acidic and alkaline solutions. In acidic solution. the protonation of t.-glutamic acid side chains of am.-MAzoM resulted in precipitation. On the other hand, the repulsion between ionized t.-glutamic acid side chains of am.-MAzoM in the alkaline solution disturbed the a-helical conformation of the polypeptide. The conformational changes induced the denaturation of the amphiphilic

*

b x c-2 E

3.7. lrrcrdiution The light source used for UV irradiation ( 250 nm
0 -2.0 -4.0

hfnm Fig. 1. CD yxxtra

containing

of am-MAzoM in TMP I -~ 0. I M KC1 at pH 6.9 (-.-) BI 2S”c’.

) and ill aqueous

solution

7

6

Fig. 2. pH dependence am.-MAzoM

in aqueous

of the minimum solution

containing

8

PH ellipticity.

9

1 H]21i.

0.1 M KCI

of dxk~adapted

at 25’C.

character of am.-MAzoM and resulted in the decrease in solubility. It is also noted here that a random copolypeptide containing the same amount of L-glutamic acid and y-methyl t--glutamate residues was insoluble in aqueous solution O\;C’I the pH range 5 to 9. These results suggest that am.-MA/oM has an o-helical conformation in aqueous solution in the neutral pH region, and can form stable aggregates by scliassociation of several amphiphilic o-helical rods. Further more, we have already reported 1291 that the am.-MAzoM aggregate in aqueous solution has a micellar structure in which hydrophobic compounds could bc incorporated.

The am.-MAzoMs were dispersed in aqueous solution by sonication and were allowed to stand for several months. The structure of the am.-MAzoM aggregates in aqueous solution was observed with optical microscopy. The differential interference micrograph of am.-MAr.oM aggregates in aqueous solution containing 0. I M KCI at pH 6.9, which was allowed to stand for three months in the dark. is shown in Fig. 3. The twisted ribbon-like aggregates are clearly seen in the micrograph. However, no aggregates could be observed immediately after sonication. We investigated the process of aggregate formation with dynamic light-scattering measurcments. The changes in the size ofthc am-MAzoM aggregates by aging in the dark at 25°C are shown in Fig. 4. The initial size of the aggregates was about IO nm in diameter. The development of the process of attaining the stable superstructure consists of two stages (before and after 20 h) To elucidate the two-stage growth in further detail, the morphological changes of the aggregates were directly observed by atomic force microscopy. A typical AFM image of am.-MAzoM in aqueous solution. which was allowed to stand for IO h in the dark at 25°C. is shown in Fig. 5(a) Globular particles with diameters of 3ONO nm wcrc clearly observed. On the other hand. the fibrous aggregates could be seen after aging for six days ( Fig. S(b) ). The fibrous aggrcgates and the amphiphilic character of am.-MAzoM allow us

Fig. 3. Differential aqueous solution stand

for three

interference containing months

I000

micrograph of am.-MAzoM 0.1 M KC1 at pH 6.9 which

in the dark

(II 25°C

after

aggregates was allowed

in to

sonication.

1

,,p”c

to postulate a cylindrical bilayer structure [ 37 \ (Fig. 6) as a primary aggregate. The postulated structure may be energetically favorable in aqueous solution in order to avoid polarapolar interaction between water and hydrophobic side chains of am.-MAzoM. Moreover. it can be seen that the fibrous aggregate developed from the globular particles ( rnarked by the arrow in FIN. S(b) ). The development of the tail-like microtibrils composed of cylindrical bilayers from globular particles may provide the firs\ stage in the morphological changes. The formation of the rihhon-like aggregate (Fig. 3). fibril association, can be ascribed to hydrogen bonds between the L-glutamic acid faces of the hbrils. The tibril association may be the second stage in the growing processes (Fig. 6).

Azobenzene is known to change the overall geometry from planar to nonplanar [ 38.39 ] by trLln.s-cI.sphotoisomerization. We have already reported 140 ] that the geometry of the am.MAzoM is controllable by the photoisomerization of the a/;o-

147

Fig. 5. AFM images of am-MAzoM 3°C lor IO h ( a I and six days (b).

aggregates

in aqueous solution

containing

benzene unit. We investigated here the photoinduced deformation behavior of the aggregates, i.e.. the relationship between the stability of the aggregates and the irradiated UV light intensity. The photoinduced changes in the anisotropy, y, of NBDam.-MAzoM in an aqueous solution of pH 6.9 containing 0. I M KC1 at 25°C are shown in Fig. 7. The y value of NBD-am.-MAzoM reflected the fluidity of the aggregates composed of am.-MAzoMs. In addition. the yvalue of NBDlabeled poly( I.-glutamic acid) (NBD-PGA. Mn z=9200)

0. I M KC1 at pH 6.9: typical

images when the solution

was aged in the dark at

which has a-helical (pH 4.2) and random coil (pH 7.5) conformation is also plotted in this Figure. On UV light irradiation for 5 min, the y value was decreased. The equilibrium y value of NBD-am.-MAzoM is nearly equal to that of CLhelical NBD-PGA, which is almost dispersed in a molecular level. Keeping the sample in the dark after UV light irradiation did not induce any changes in the y value. This implied that the UV light irradiation induced the partial irreversible dissociation of the aggregates. A part of the aggregates was precipitated, which was confirmed by visual inspection.

twisted ribbon (associated fibrils)

ib) first stage I

UV irra. * 4

Fig. 8. Histogram <,f the particle si/.e of the watrr-wlublc aggrepatex conposed of am-MA/oM in aqueous solulinn contuming 0. I M KCI. ptl 6.9. at 75‘C immediately afler wnication: (a) hcio~-r and (I,) after IW light irradiation.

somcation in the dark adap.

bilayer in globular particles (primary aggregate) IOnm Fig. 6. Dimensions ofthe structural

disordered

cylmdncal

to photo-

IO’

d/rim

responses

ofthe

aggregate and precqxtate IOnrn . am.-MAzoM

aggregate

80

and thermo-stimul;ltions.

Oo

0

60 i

-. dark ad

PG.4 (pH 4.2, u hcl,xl 0.1

PFA (pH 7.5, random cool)

0 Irradiated Fig. 9. (a) Relaticmrhlp hctween NBD-am-MAtoM wstcr-soluble Fig. 7. Photoinduced aqueous solution

changes in the anisotropy. y, of NBD-an.-MA/oM containing 0.1 M KCI. pH 6 Y. at 25°C.

111

Photoinduced changes in the sizes ofthe aggregates were also measured by dynamic light scattering (Fig. 8). The sample solutions were prepared by filtration using a membrane tilter ( Millipore, 0. I pm) to remove the precipitate. UV light irradiation reduced the size of the aggregate from 10.6 to 3. I nm. These results suggest that the photoisomerization of the azobenzene moiety induced a loss of the amphiphilic character of am.-MAzoM. which resulted in the destruction of the aggregate into a smaller one consisting of a few am.-MAzoM molecules accompanied by precipitation.

light intcnyity. soluble agfregaw

(hi

Relationship and the irradiated

20

40 UV

60 light

80 intensity

100 / %

the relative Iluorsscenw Intensity apfrqate produced and the irradiated hetwcen the par~lcle UV light intcn\tty.

sue

of the

ol the UV hater-

The influence of the intensity of the applied UV light on the solubility and size of am.-MAzoM aggregates in the ayueous solution was studied. UV light irradiation was applied for 5 min at 25°C. The relationship between the relative fluorescence intensity of the NHD-am.-M4zoM and the irradiated UV light intensity is shown in Fig. 9. In this case, the decrease in the colubility of the polypeptide resulted in a decrease in the iluorescence intensity. The fluorescence intensity significantly decreased above 70% light intensity. In addition. the particle size of 1he water-soluble aggregate also

drastically decreased when the UV light intensity attained 70% (Fig. 9(b)). both indicating an on-off manner of the aggregate destruction. This may be related to the cooperative phenomena of well-organized systems. The am.-MAzoM molecules self-organize their aggregates based on a cylindrical bilayer structure, so that the macroscopic destruction over a certain degree of this well-defined structure may be required for the molecular deformation ( photoisomerization) of indi-Cdual am.-MAzoM polypeptide\.

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