Dehydration effects on D96N bacteriorhodopsin films

ELSEVIER

Thin Solid Films 283 (1996) I--4

Letter

Dehydration effects on D96N bacteriorhodopsin films Tatyana V. Dyukova 1,,, Evgeny P. Lukashev 2 National Institute of Standards and Technology, A353/222, Gaithersburg, MD 20899, USA Received 8 Match 1995; accepted 15 December 1995

Abstract Dehydration effects on gelatin films of the D96N mutant bacteriorhodopsin (BR) and wild-type BR were studied. Unlike the wild-type BR films, wherein dehydration to 12% humidity results in an approximate 200-fold increase in the lifetime of the M state, D96N BR films dehydrated to the same extent have been shown to exhibit only a 17- to 20-fold increase in the lifetime of the M state. Chemically-enhanced D96N BR films possess a total bleaching efficiency of the initial-to-M-statetransition that is close to theoretical maximum (1.0) over a wide range of relative humidity (35 to 85%). This provides an additional benefit to the D96N BR films as a material for storage and retrieval of optical information. Keywords: Bacteriorhodopsin; D96N mutant; Gelatin films; Biomateriais; Multilayers; Optical spectroscopy; Water

1. Introduction

2. Experimental section

Bacteriorhodopsin (BR) is a light-sensitive transmembrahe protein from the purple membranes (PMs) of the halophilic bacterium Halobacterium salinarium. Upon light excitation, BR goes through a cyclic sequence of spectraUy distinct intermediates (I, J, K, L, M, N and O) and protonation-deprotonation steps, resulting in a proton transiocation from the cytoplasm to the extracellular medium [ ! ]. Since the mid-1980s, there have been numerous reports of possible applications of both wild-type (WT) BR and BR genetic mutants. One feasible application for this biological material is optical information storage and processing [2-7]. The reversibility of all photoprocesses in the BR molecule make various types of optical recording possible with BR films. The possibility of controlling the lifetime of the M state by various chemical, physical or genetic means, combined with excellent photochemical and thermal stability of the protein, makes the BR film a promising material for this technical application. We report here, a study of the humidity dependence of the M-state decay time constants and the bleachiag efficiency of D96N BR films. This work was carried out to better understand the utilization limits of this material.

Arcaine sulfate, N,N,N',N'-tetramethylethylenediamine, sodium azide, dimethyldichlorosilane and the 300 Bloom Type A gelatin (from porcine skin) were purchased from Sigma Chemical Company (St. Louis, MO) and the 1,2diaminopropane was purchased from Aldrich Chemical Company (Milwaukee, WI). All chemicals were reagent grade and were used without further purification. PM fi'agments were isolated from the cells ofl~alobacterium salinarium WT ET 1000 and the D96N mutant BR (wherein Asp-96 is substituted with non-ionizable Asn) according to a published procedure [ 8]. The purified PMs were sonicated in an ice bath for 4 min at 20 kHz. Next, a preparation of 8% aqueous gelatin was added to each of the samples, followed by stirring 10 min at 38 °C. The final gelatin concentration in the mixture was 5%. Chemically-enhanced BR films contained additives that increased the lifetime of the M state [9]. The following additives: arcaine sulfate, N,N,N',N'-tetramethylethylenediamine and 1,2-diaminopropane at final concentrations of 2.5 × 10 -4 mol I- ', 5.9 × 10 -4 mol I- ~ and 1.35 × 10- l mol i- t respectively, were successively added to the mixture. Chemically-unmodified BR films contained no additives. Sodium azide, an antibacterial agent, was added to the stock gelatin solution for selected samples at a final concentration of 4 × 10-3 mol 1-~. As a reference, some of the samples contained no sodium azide. "lhe final pH of the chemically-enhanced BR mixtures was 10.2. Each mixture was then introduced between two 50 × 50 mm 2glass supports

* Corresponding author. Tel.: ( + 1) (301)975-2085; fax: ( + 1 ) (301) 330-3447; e-mail: [email protected]. Permanent address: Institute of Theorefical and Experimental Biophysics, Russian Academy of Sciences, Pashchino, Moscow 142292, Russia. 2 Permanent address: Moscow State University, Biology Faculty, Department of Biophysics, Moscow 119899, Russia. 0040-6090/96/$15.00 © 1996 Elsevier Science S.A. All fights reserved SSD10040-6090 ( 9 5 ) 08528-9

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T.V. Dyukova. E.P. Lukashev/Thin Solid Films 283 (1996) 1-4

separated by 800 p,m spacers and was allowed to gel at 9 °C. The upper glass support had been pre-treated with dimethyldichlorosilane to produce a non-sticking surface. After 1 h, the upper plate was removed. The samples were allowed to dry at 9 °C at 10 to 15% relative humidity (RH). The thickness of the dried samples was approximately 50 ~m. Sample cuts (25 X 12 × I mm 3) of the films were mounted at 45 ° in a 4 5 × 1 0 × 1 0 nun 3 plastic cuvette. The desired humidity values (85, 68, 52, 43, 32, 12 and 3% RH) for the films were produced by equilibration of the films for at least 24h in the vapors of saturated salt solutions [10] at the bottom of sealed cuvettes. Spectra, kinetic measurements, and photoinduced absorbance changes of the films at the peak of the M-state absorption were obtained using a Hewlett Packard 8452A Diode Array spect-ophotometer. In the course of the measurements, all samples were enclosed in sealed cuvettes. A Kodak 300 W, 120 V bulb Ektagraphic projector, model B-2 was used as an excitation light source in combination with a yellow long-pass filter which cuts off irradiation of less than 530 nm wavelengths. The maximum power density of the incident light on the sample measured with an Aerotech Laser Radiometer, Model 71 was approximately 30 mW cm- z. All films were previously light-adapted for 3 min, after which they were exposed to blue light for 15 s followed by total darkness for 3 to 5 min to allow complete recovery of the initial state and to obtain equivalent starting conditions. The parameter of bleaching efficiency was used to compare different samples with respect to their ability to transit from the initial state to the M state. Bleaching efficiency was defined as the ratio of photoinduced absorbance change at 412 nm to the maximum absorbance of the initial state, AA4t2/A,~ m~. In the event that complete bleaching of the initial state was observed, indicating that all BR molecules were in the M state, this ratio was considered to be equal to i.0.

3, Results and discussion Absorbance changes in D96N BR films at different RH were compared with that of the WT BR films. Fig. I shows the dehydration-induced spectral changes in chemicallyunmodified BR films of the WT (A) and the D96N mutant (B). Since all the films tested showed similar changes, indicating that both mutant and WT BR molecules apparently experienced analogous transformations upon dehydration, only these two sets of spectra are presented. As previously reported for the WT BR [ l 1-13], a blue shift in the absorbance maximum for the D96N BR film along with diminished absorption were observed with decreasing RH. Dramatic changes in the observed absorbance maxima and amplitudes at RH values between 12 and 3% are probably due to the dissociation of water molecules specifically-bound in the vicinity of the retinal chromophore that induces deprotonation of the Schiff base [ 11 ], a key element of the attached retinal chromophore.

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Fig. I. Dehydration-inducedspectralchangesin chemically-unmodifiedBR fihns of the WT (A) and D96N mutant (B). Absorbancevalues were normaliz~zdto be equal at 526 nm. which is the isosbestic point for the dehydrationinducedabsorbancechanges [ 12]. This correlationwas made assumingthat the absorbancevaluesat 700 nm wereequal to zeroand that all specu'apassedthroughthe isosbesticpointat 526 nm. Relativehumidities a~, iu orderof decreasingabsorbancevalues: (A) 85, 68, 43, 32, 12 and 3°ARH; (B) 85, 68, 52, 43, 32, 12 and 3% RH. The M-state decay curves for cbemically-unmodifiod D96N BR film at different RH were fit, in a least squares fashion, to an equation of the forra: Y ( t ) = A [ e x p ( - t / ~'t)] + B [ e x p ( - t / r 2 ) ] +C. We assume that A + B + C = 1 and A and B represent the relative amplitudes of the time constants "r~ and ~'2 respectively. Results of mono- and biexponential fitting of the curves are summarized in Table 1. As shown in Table 1, M-state decay time constants did not vary more than 17- to 20-fold upon dehydration ofD96N BR films from 85 to 12% RH. A similar degree of dehydration in airdried PM films of WT BR is known to result in a decrease in the M-state decay rate by at least three orders of magnitude [ 14]. Analogous RH dependence has been observed in polyvinyl alcohol films and air-driod PM films of D96N BR, indicating that the observed changes reflected the behavior of the protein rather than the gelatin binder. Fig. 2 shows the RH dependence on the bleaching efficiency of the unmodified and chemically-enhanced D96N BR

T, V. Dyukova, E.P. Lukashev / Thin Solid Films 283 (1996) 1-4 Table I The M-state decay time constants ~ ( r s ) in D96N BR and WT BR films at different relative humidities Sample

RH (%)

WT, additives ~'

85

68

43

32

12

3

0.2+0.03(0.75) 0.8+0,1 (0.25)

45:0.2(0.48) 21 + 1.7 (0.52) -

15+1.1 (0.45) 124+7.4 (0.55) -

10+0.6(0.47) 30+6.5 (0.44) + (0.09) c

115:0.7(0.40) 141 +8.9(0.50) + (0.10)

95:1.2(0.39) 1605: 12(0.50) + (0.11 )

0.5 &0.04 (0.53) 45:0.28 (0.47) -

0.9+0.05 (0.48) 9+0.43 (0.44) + (0.08)

1 5:0.2 (0.46) 12+0.9 (0.47) + (0.07)

2+0.3 (0.48) 175:1.0 (0.40) + (0.12)

3+0.5 (0.30) 485=3.4 (0.50~ + (0.20)

-

45:0.3(0.70) 15 5=0.8 (0.30) -

22+1.1 (0.31) 159+8.1 (0.60) + (0.09)

155:0.8(0.34) 156+7.8 (0.55) + (0. I I )

95=0.6(0.38) lSI 5:9 (0.45) + (0.17)

135=0.9(0.32) 192 5=9.8 (0.48) + (0.20)

9+0.72(I.00) -

12-.k0.7(037) 285= 1.9 (0.63) -

235:1.4(0.34) 1725=8.6 (0.66) -

13+0.7(0.38) 1605:8.3 (0.48) + (0.14)

175:0.6(0.33) 190+ I1. (0.51) + (0.16)

15+0.9(0.29) 190:t:17 (0.51) + (0.20)

-

D96N, no additives

0.3+0.04 (0.50) I 5:0.15 (0.50) -

D96N, additives and NaN3

0.95:0.14(1.00) -

D96N, additives, no NaN 3

a In cases where kinetic curves were fit to two or more components, the components are shown as a grouping with the first component r, followed by successive components, ~-~_and ~'3.The relative amplitudes are given in brackets next to the corresponding time constants, ~', and r2. b Chemical additives: arcaine sulfate, N,N,N',N'-tetramethylethylenediamine and 1,2-diaminopropane were used to increase :he lifetime of the M state. c The third, slowest kinetic component of the M-state decay is approximately several minutes. It has been defined only as a fraction of a total amplitude.

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Relative Humidity (%) 1.0-

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films compared with the chemically-enhanced WT BR film. When the two curves, (A I ) and (B), for the two chemicallyenhanced D96N BR and WT BR films are compared, one can see that the bleaching efficiency of the D96N BR film is essentially independent of RH over a wide range from 35 to 85%, unlike the WT BR film, wherein the RH-independent region is only from 35 to 55% RH. This gives a specific, additional advantage to D96N mutant BR fihn as a photochromic material. The humidity-dependence curves for chemically-enhanced D96N BR films with and without sodium azide (Fig. 2, (A 1) and (A2)) are similar except for two extreme humidities (68 and 85% RH). This is the result of an acceleration of the M-state decay in the D96N BR in the presence of sodium azide. The M-state decay in D96N BR is known to be significantly slowed since Asp-96, an interior proton donor within the protein, is absent and the Schiff base is directly reprotonated from the medium [ 15]. The azide molecule serves as a mobile proton donor for the Schiffbase resulting in a partial 'recovery' of the Ash-96 lost protonation function [ 16].

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4. Conclusions

0.2'

0.0.

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Fig. 2. Bleaching efficiency vs. relative ,air humidity for D96N BR (A) and WT BR (B) films. (A): ( I ) with additives (arcaine sulfate, N,N,N',N'tetramethylethylenediamine and 1,2-diaminopropane) and no NAN,; (2) with additives and NaNj; (3) with no additives and no NaN.~. (B) WT BR film with additives and NAN3.

Dramatic differences in the dehydration-induced changes in the kinetic parameters of WT and D96N BR films, result from the different pathways of the Schiff base reprotonation in these two proteins. The major reason that the M-stale lifetimes in D96N BR fihns are largely unaffected by humidity, is the lack of the proton translocation step from Asp-96 to the Schiff base. This particular step was shown to be the

4

T.V. Dyukova, E.P. Lukashev/Thin Solid Films 283 (1996) 1.-4

most humidity-dependent in the WT BR [ 17]. The data presented herein demonstrate that D96N BR films, earlier reported as a suitable material for use in storage and retrieval of optical information, possess an additional, specific advantage. Chemically-enhanced D96N BR films exhibit bleaching efficiencies that are close to 1.0 and independent of humidity over the range of 35 to 85% RH. Acknowledgements The authors would like to thank Dr. R. Ashton, Dr. P. Chen and Dr. H. Weetall for helpful discussion. This work is partially supported by a grant 70 NAN B5 HO114 from NIST.

References [ 1] J. Lanyi, Biochim. Biopttys. Acta, 1183 (1993) 241. [2] G. lvanitsky (ed.), Collected articles: Photosensitive Biological Complexes and Optical Information Recording, Biol. Sci. Research Center, Pushchino, Russia, 1985, p. 98 tin Russian).

[3] N. Vsevolodov and T. Dyukova, Trends Biotechnol., 12 (3) (1994) 81. [4] Q.W. Song, C. Zhang, R. Gross and R. Birge, Optics Lett., 18 (10) (1993) 775. [5] N. Hampp, R. Thoma, D. Zeisel, C. Brauchle and D. Oesterhelt, Adv. Chem. Ser.: MoL Biomol. Electron., 240 (1994) 51 l. [6] D. Haronian and A. Lewis, Appl. Phys. Len., 61 (18) (1992) 2237. [7] D. Oesterhelt, C. Bmuchl¢ and N. Hampp, Quart. Rev. Biophys., 24 ( 1991 ) 425. [8] B. Becher and J.Y. Cassim, Prep. Biochem., 5 (1975) 161. [9] T. Dyukova and N. Vsevolodov, Photochromic compositions and materials containing bacteriorhodopsin, US patent pending, 1994. [ 10] R.C. Weast (ed.), Handbook of Chemistry and Physics, CRC Press, 62nd edn., 1981-1982, p. E-44. [ 1! ] P. Hildebrandt and M. 3tockburger, Biochemistry. 23 (1984) 5539. [ 12] R. Renthal and R. Regalado, Photochem. Photobiol., 54 (1991) 931. [ 13] Yu. ~ v and E. Terpugov, Biochim. Biophys. Acta, 590 (1980) 324. [ 14] R. Korenstein and B. Hess, Nature, 270 (1977) 184. [ 15] A. Miller and D. Oesterhelt, Biochim. Biophys. Acta, 1020 (1990) 57. [ 16] J. Tittor, M. Wahl, U. Schweiger and D. Oeslerhelt, Biochim. Biophys. Acta, !187 (1994) 191. [17] Y. Cao, G. Varo, M. Chang, B. Ni, R. Needleman and I.K. Lanyi, Biochemistry, 30 (1991) 10972.