Fluorescence Anisotropy Data

Home , Anisotropy

Proc. Nati. Acad. Sci. USA Vol. 76, No. 12, pp. 6361-6365, December 1979 Biophysics Structural order of lipids and proteins in membranes: Evaluation of fluorescence anisotropy data (order parameter/lipid bilayers/lipid-protein interaction/protein dynamics) FRITZ JAHNIG Max-Planck-Institut fur Biologie, Corrensstrasse 38, D-74 Tubingen, West Germany Communicated by Manfred Eigen, October 1, 1979

ABSTRACT The limiting long-time value of fluorescence anisotropy in membranes is correlated with the orientational r,, order parameter, which characterizes the structural anisotropy of membranes. Existing experimental results for diphenylhexatriene in lipid bilayers are evaluated for the order parameter of lipid order. Steady-state measurements of fluorescence an- rt isotropy can provide the order parameter in good approximation. Proteins in a fluid lipid phase increase the lipid order parameter so determined. Upon comparison with the order parameter from deuterium magnetic resonance, it is concluded that proteins increase the order of the surrounding lipids in off-normal directions. Order parameters of protein order obtained from the limiting value of protein fluorescence anisotropy are discussed with respect to the influence of lipid order FIG. 1. Time dependence of FA. The dotted line applies to Per-. on protein order. nfI'slaw. Fluorescence depolarization is an extensively used technique of x7 was consonant with the general concepts of membrane in membrane research. A short pulse of polarized light falls on fluidity. a suspension of membranes containing fluorescent molecules, The derivation of Eq. 4 proceeds from the following concept: either extrinsic probes or intrinsically fluorescent proteins. The The polarized light excites dipole moments of a certain orien- intensity I of the fluorescence is recorded as a function of time tation that emit light over their lifetime r. Initially the emitted t for the two polarizations parallel and perpendicular to the light is polarized parallel, and rt is large. Due to rotational initial polarization. The fluorescence anisotropy (FA) is ob- diffusion the orientation of the emitting dipoles becomes in- tained as creasingly disordered, and rt decreases. Assuming the envi- ronment of the dipoles to be isotropic, their final distribution rt -III] will also be isotropic, and rt decreases to zero (Fig. 1). The larger X and therefore 0, the longer the initially created anisotropy Often measurements are performed under constant illumina- is preserved and consequently detected in steady-state mea- tion, yielding the steady-state FA surements yielding a large rs. III - I1L Recent time-resolved FA measurements with pure lipid rs = [2] membranes (2-4), lipid membranes containing cholesterol (5, + 6), and cell membranes (7, 8), as well as earlier measurements* in which IIl and IV are the time integrals of III and I-. The with excitable membranes (9), have shown rt not to decrease steady-state FA can be expressed by the time-resolved FA as to zero but to reach a finite level r. (Fig. 1). Thus the final distribution of emitting dipoles must be anisotropic. For mol- s= J dtrtIt/J dtIt, [3] ecules in membranes this is conceivable. So the final FA value furnishes information on the structural order in membranes, in which the total fluorescence intensity It = I II + 2Ij has been while the relaxation time provides information on kinetic introduced. properties such as microviscosity. Because both factors enter Since the work of Shinitzky (for a review see ref. 1) the the steady-state FA, the evaluation of rs has to be improved. steady-state FA has been interpreted in terms of the so-called The present paper deals with two questions: (i) What is the microviscosity 77 by applying the Perrin equation structural information provided by the experimental limiting =ro FA value re, and (ii) to what extent can the same information rs = T1+0, [4] be obtained from steady-state measurements-i.e., from rs? The answers will be utilized to gain insight into the problem of ro being the maximal FA value in the absence of any rotational lipid-protein interaction in membranes. motion, r the fluorescence life time, and X the rotational relaxation time given by X = 77V/(kT), V the volume of the Fluorescence anisotropy and order parameter fluorophore, k the Boltzmann constant, and T the absolute To answer the first question a theoretical analysis is needed that temperature. The microviscosity 77 for lipid membranes so correlates r. to membrane properties. Such a study was carried determined was high in the ordered and low in the fluid phase, out by Kinosita et al. (10). For the case of both the absorption with an abrupt change at the phase transition. This behavior Abbreviations: FA, fluorescence anisotropy; LA, luminescence an- The publication costs of this article were defrayed in part by page isotropy; DMR, deuterium magnetic resonance; ESR, electron spin charge payment. This article must therefore be hereby marked "ad- resonance; DPH, diphenylhexatriene; Pam2PtdCho, dipalmitoyl vertisement" in accordance with 18 U. S. C. §1734 solely to indicate phosphatidylcholine; Myr2PtdCho, dimyristoyl phosphatidylcho- this fact. line. 6361 Downloaded by guest on September 30, 2021 6362 Biophysics: jihnig Proc. Natl. Acad. Sci. USA 76 (1979) and emission moment lying along the fluorophore axis their a lipid membrane as a function of temperature. The r., values result was for dipalmitoyl phosphatidylcholine (Pam2PtdCho) vesicles were measured by Kawato et al. (3) and by Lakowicz et al. (4), r= 2/5(P2(cos 0))2. [5] employing DPH as the fluorescence probe. Both sets of results Here P2(cos 0) = (3 cos20-1)/2 is the Legendre polynomial are presented in Fig. 2A and agree well. Because the absorption of second order; 6 is the angle between an instantaneous or- and emission moments of DPH are likely to lie along the mo- ientation of the fluorophore and the average orientation; and lecular axis, Eq. 7 can be utilized for evaluation, the result being the angular brackets denote the average over the orientations shown in Fig. 2B. The temperature behavior of the order pa- of the fluorophore within a certain time. This average and the rameter is as expected: the order is high in the ordered phase average orientation refer to the equilibrium state yielding r. and low in the fluid phase. The phase transition appears rela- The averaging time therefore is longer than the relaxation time tively broad, as known for vesicles. For comparison with the 0 needed to establish this equilibrium; it is, however, limited DMR order parameter, the results of Seelig and Seelig (17) for by the lifetime r because possible slower relaxation processes specifically deuterated Pam2PtdCho in liposomes are included. with >>» X are not detected. Introducing the probability dis- The FA result approaches the DMR order parameter in the tribution w(O) for the orientations, the average can be ex- fluid phase at the C12 position. According to the above argu- pressed as ments this is reasonable. In the ordered phase DMR order parameters are difficult to obtain. For perdeuterated soaps, Mely (P2(cos 0)) = 3o dcos 6 P2(cos 0) w(cos 0) [6] and Charvolin (18) found values between 0.7 and 0.9, which are in the same range as the FA result. For complete order (all 0 = 0) one gets (P2) = 1, and for FA complete disorder [w(6) = 1] (P2) = 0. Therefore (P2) is a Steady-State measure of the orientational order of the fluorophores within To answer the second question we turn to a phenomenological their lifetime, which is of the order of 10-8 sec. way of reasoning. From the experiments of Kawato et al. (8) In order to evaluate the experimental r. values for lipid and of Lakowicz et al. (4) it follows that rt in lipid membranes membranes in terms of the distribution w(O), Kinosita et al. shows a simple exponential decay to the limiting value r., employed a cone model as done earlier by Wahl (11), w(6) rt = (ro - r.) + r., being constant between 6 = 0 and 6 = O,, and determined 6C. exp(-t/0) [8] Two points can be made about this evaluation. Recent theo- with ro = 0.395 ± 0.01 (3) or ro = 0.39 (4). Within the experi- retical results indicate W(6) to be better described by a Gauss- mental error these values for ro agree with the theoretical result ian-like distribution (12). More important, however, is the fact ro = 2/sP2(cos X) if the angle X between the absorption and that Eq. 6 represents the definition of the orientational order emission moments is zero, as expected for DPH. Because It t parameter S known for lipid membranes both from theory (12, exp(-t/r) the integrations in Eq. 3 are easily performed, 13) and experiment-e.g., deuterium magnetic resonance yielding (DMR) (14). The r. values may then simply be evaluated for the order parameter. This order parameter obtained from FA rs= + r. [9] differs, however, from the DMR order parameter in two re- spects: (i) DMR measures the order parameter S, of the indi- The first term represents the kinetic contribution, the second vidual methylene segments v along the lipid chains, whereas the structural one. The Perrin formula, Eq. 4, is obtained if the FA measures the order parameter of a probe between the lipid chains; (ii) the order parameter from DMR is an average over 0. A

a time of about 10-4 sec, much longer than the FA averaging .3o o 0° time. The general problems connected with probe molecules 0. are the same for FA as with other methods such as electron spin resonance (ESR) (14). The widely used diphenylhexatriene rc 00..2 9 (DPH) molecules are known to be located deep in the lipid bilayer and to be oriented parallel to the lipid chains (15). Therefore, they detect the lipid chain order, and we will 0 .1 identify the probe order parameter with the lipid order parameter S, in which v simply indicates a mean position of the 1 probe along the chains. This approximation, leading to the re- B lationship 01.8 -o c

r" = 2/5 Sp2, [7] 0si.6- is useful when considering the effect of variables such as temperature and protein concentration; however, it has serious 0,1.4 -oC4c- limitations in studies of the intrachain variation of the lipid ,.~;C909 order. Incidentally, fluorescence labels covalently-bound to 01.2 -12* - specific positions on fatty acid molecules depict a profile of FA C15 along the chains comparable to the variation of the DMR order 0 parameter the difference between DMR 1 0 20 30 40 50 60 (16). Concerning and Temperature, 0C FA in averaging time, it becomes irrelevant if relaxation processes with >> r are absent. This should hold for lipid phases FIG. 2. (A) Limiting value r. for DPH in Pam2PtdCho mem- without a tilt of the chains- e.g., the fluid phase. branes: experimental results of Kawato et al. (0) and of Lakowicz et on from rs The correlation 7 between FA and the order parameter al. (o) sonicated vesicles, and the value calculated by using Eq. Eq. 10 (-). (B) Order parameter S, of Pam2PtdCho. FA result from in membranes has not been recognized in the past and affords rc. of Lakowicz et al. (o), and DMR results of Seelig and Seelig (*, new access to both lipid and protein order. A, V, 0) on unsonicated liposomes for lipid deuterated at the posi- As an example we determined the FA order parameter for tions indicated. Downloaded by guest on September 30, 2021 Biophysics: JAnig Proc. Natl. Acad. Sci. USA 76 (1979) 6363

1.0 0 00 0 10 0 0 0 0 000000 0 A 0 0 0.8 7., seec 5 0.6F C t 2 0 B- 0.4F 'xx \40Vt'ACub1 \ vs A-e13% 0, sec 0To To 0 00 1 00 °0 00 0.21- 0 00 I l7-lI___II 0~ ~ o I 0~ 0 0, 0 2 4 6 8 10 20 c - pH O 0 FIG. 4. FA order parameter S, of dimyristoylmethyl phosphatidic TIO acid vs. pH at the constant temperatures indicated (0C), in 0.1 M 10 _~~~~~~~~~~~8__0_----n0- o° I 0~~ 8 NaCI. a pH, negative surface charges are generated, leading to a de- 10 20 30 40 50 60 crease Temperature, 0C of the orientational order. The ordered-fluid transition can be triggered by changes in pH. The lower the fixed tem- FIG. 3. Fluorescence properties of DPH in Pam2PtdCho mem- perature the higher a surface charge is needed for trigger- branes measured by Kawato et al. (0) and by Lakowicz et al. (0). (A) ing-i.e., the transition pH increases with decreasing temper- Lifetime T, (B) rotational correlation time X, (C) ratio Tr/. ature. This relationship is the reversal of the variation of the transition temperature with pH studied by Trauble et al. (20). structural part rco is neglected. The relative weight of the two Because the spontaneous change of the order parameter is contributions is determined by r. and the ratio r/'. The values closely related to the latent heat (12), its decrease with in- of T and for DPH in Pam2PtdCho membranes were measured creasing pH should correspond to a decrease of the latent heat by Kawato et al. (3) and by Lakowicz et al. (4) and are shown with pH, which has indeed been found by calorimetry (21). An in Fig. 3 A and B. Here r is of the order of 10 nsec, X of the explanation of this phase transitional behavior was recently order of 1 nsec. The ratio r/T is plotted in Fig. 3C. In the fluid worked out based upon the tilting of the hydrocarbon chains phase it does not vary much with temperature due to the similar under the action of surface charges, which was observed by temperature dependencies of T and 0. Even at the phase x-ray diffraction in the ordered phase (22). It may be noted that transition this ratio remains constant. In the ordered phase there the FA order parameter in the ordered phase (at 30'C) does not is considerable scatter, but there the exact value of r/0 will turn change upon tilting of the hydrocarbon chains, which shows out to be irrelevant. As an average value we assume T10 = 8. that the FA order parameter measures the orientational order Then the kinetic contribution to rs in the ordered phase, using relative to the average axis irrespective of the orientation of this r. 0.3 in Eq. 9, is not more than 3%; in the fluid phase with axis. r. 0.05 it amounts to 40%. The earlier evaluation of rs by using Perrin's law, Eq. 4, would yield in the ordered phase X Lipid-protein interaction too large by a factor of 30, and correspondingly too large a value We now wish to apply the above method developed for the for A. This high microviscosity in the ordered phase obtained evaluation of steady-state FA measurements to study the in- earlier is incorrect; actually it was a reflection of the high order fluence of membrane proteins on the lipid order. In this case parameter. The true temperature dependence of q (apart from a further problem arises with FA, because the lateral distribu- a factor T) is the same as that of X in Fig. 3B. Both X and q seem tion of the fluorophores in the lipid-protein bilayer is usually to exhibit a maximum at the phase transition that represents a unknown. One may assume DPH to be distributed homoge- critical slowing down of the system's internal motion. neously in the lipid phase, but this assumption is not essential Taking the kinetic contribution to rs into account by as- for the following qualitative discussion. Using DPH, rs was suming r/T = 8 for DPH in a lipid phase, Eq. 9 can be trans- measured for the reconstituted system Ca2+,Mg2+-ATPase in formed into dimyristoyl phosphatidylcholine (Myr2PtdCho) by Gomez- Fernandez et al. (23). The order parameter can be determined r', = 9rs - '/20- [10] as above by using Eqs. 10 and 7, the resulting temperature To test this approximation, the result of Lakowicz et al. (4) for dependence for different lipid-protein ratios being shown in rs, obtained with the samples from the r. determination, was Fig. 5. Upon addition of protein the lipid order is decreased in evaluated by using Eq. 10. The resulting r. is included in Fig. the ordered phase and increased in the fluid phase. The spon- 2A. The agreement with the directly measured values of r. is taneous change at the phase transition becomes smaller and the convincing. This means that the lipid order parameter in phase transition becomes broadened, the midpoint remaining membranes can be obtained to a good approximation from approximately constant. steady-state FA measurements by using DPH. The effects of Ca2+,Mg2+-ATPase on the lipid order deduced We determined, for instance, the order parameter in mem- from steady-state measurements coincide with results for r. branes of ionizable lipids as a function of pH, in order to study obtained from time-resolved measurements for the effect of the electrostatic regulation of membrane structure. For the lipid cholesterol (5, 6) as well as of membrane protein (7, 8). Fur- dimyristoylmethyl phosphatidic acid, Strehlow (19) measured thermore, in these experiments the relaxation time 0 either rs at different pH values and constant temperature. Eqs. 10 and stayed constant or decreased due to cholesterol or membrane 7 yield the order parameter plotted in Fig. 4. With increasing protein, in agreement with data from ESR (for a review see ref. Downloaded by guest on September 30, 2021 6364 Biophysics: JAnig Proc. Natl. Acad. Sci. USA 76 (1979) 1.c

0.8

0.6F

0.4 F FIG. 6. Lipid order in a bilayer containing proteins (P). 0.2 ientational order relative to local mean axes which, as discussed for the case of surface charges, must not coincide with the membrane normal. The increase of the FA order parameter 0 10 20 30 40 50 therefore demands the lipid order around local axes to be in- Temperature, 0C creased. DMR, on the other hand, demands these local axes to FIG. 5. FA order parameter S, of Myr2PtdCho containing differ from the membrane normal. To satisfy these two de- Ca2+,Mg2+-ATPase vs. temperature at the molar lipid-to-protein mands we conclude that proteins induce a higher orientational ratios indicated. order of the surrounding lipid molecules in off-normal directions, as shown schematically in Fig. 6. 24) and fluorine magnetic resonance (25), indicating a partial Membrane protein order immobilization of lipids by proteins. These findings on sup- Proteins usually possess an intrinsic fluorescence and sometimes port our evaluation of rs, because according to Eq. 9 a decrease a long-lived chromophore that permits the determination of of would yield a decrease of rs so that the observed increase the orientational order of parts of the proteins by luminescence of rs has to be attributed to an increase of r. as done. anisotropy (LA), this being the generalization of FA over the As for the effect of surface charges discussed above, the de- entire scale of life times. In addition, specific labeling of proteins crease of the spontaneous change of the order parameter cor- is possible. An order parameter for protein segments was re- responds to a decrease of the latent heat, which has also been cently introduced by Rothschild and Clark (35). Because -r and observed (23). The decrease of the latent heat and the broad- X are not known beforehand and more than one relaxation ening of the phase transition are generally observed with pro- process may occur, one has to resort to time-resolved mea- teins. One is tempted to attribute these effects to a common surements of LA. Protein fluorescence still detects motions and origin, the increase of the lipid order in the fluid phase. Because order parameters on the nanosecond time scale, whereas the the fluid phase is more sensitive to external perturbations such long-lived chromophores are indicators on the microsecond and as proteins than the ordered phase is, an increase of the fluid millisecond time scales. phase order is sufficient alone, irrespective of the protein effect Nanosecond FA measurements were performed 10 years ago on the ordered phase, to account for the decrease of the spon- by Wahl et al. (9) on proteins in excitable membrane fragments. taneous change of the order parameter. These effects and By observing the FA of dansylated proteins, a relaxation process correlations are also consequences of the theoretical work of with 4 = 3 nsec was found starting at ri = 0.23 and leveling off Marcelja (26). at r . = 0.15. The fact that the initial value ri was not 0.4 as The influence of proteins on the lipid phase has already been expected (see the discussion following Eq. 8) was attributed to studied by other experimental techniques. ESR measurements a faster process. In this case, in order to determine the order on the cytochrome c oxidase/Myr2PtdCho system yielded parameter Sfast resulting from the nanosecond averaging process qualitatively the same result for the order parameter as found (indicated by the index fast in contrast to a slower process above (27). Similarly, the results of Raman studies with different discussed below), one has to use instead of Eq. 7 r = fast, proteins indicate a decrease of the conformational order of the yielding Sfast = 0.80. This is the order parameter of the protein hydrocarbon chains in the ordered phase and an increase in the groups in the neighborhood of the dansyl residues. The value fluid phase (28-32). DMR, however, for all proteins studied, 0.8, determined for a fluid membrane, shows that the labeled shows a decrease of the order parameter in the fluid phase (33, protein groups do fluctuate, but not extensively. If this is con- 34). Is this a contradiction or can this difference be interpreted sidered as a general feature, it would be in accordance with our to acquire additional insight into lipid-protein interaction? result on lipid-protein interaction above, stating that proteins To understand these results it is important to recall that the exert an ordering effect upon lipids in the fluid phase. It is re- DMR order parameter is a long-time average compared to the markable that the relaxation times for protein and lipid order other methods. Indeed, DMR was found to detect only one are comparable. One might imagine a coupling between pro- species of lipid molecules, implying that the lipid molecules tein and lipid dynamics to the extent that the cooperative order exchange between the immediate neighborhood of protein of the lipid phase imposes a certain order on protein fluctua- molecules and the bulk lipid phase within a time of 1o-4 sec. tions. Indeed, Wahl et al. found a temperature dependence of The measured order parameter is then the average over the r.-i.e., Sfast-which they attributed to structural changes in states of any one lipid molecule in the whole lipid phase. The the membrane, but further experiments are needed. mean axis of this averaging process is the membrane normal We now turn to the microsecond and millisecond LA of so that the DMR order parameter represents the orientational chromophores. Within this time scale, rotational motion of order of the lipid molecules relative to the membrane normal. whole protein molecules may occur. The rocking motion of the A decrease means that on the average lipid molecules become long molecular axes leads to a randomization of the initial or- more disoriented relative to the membrane normal. This must ientation of excited dipoles, as does the rotational diffusion be contrasted with the other techniques, in which the averaging around the long molecular axes if the dipole moment makes an process is shorter, for FA of the order of 10-8 sec. Within this angle -y with the long molecular axis. The first motion is ex- time lipid molecules can move by lateral diffusion only over pected to be slower than the second one. Generalization of Eq. distances on the order of angstroms, so that FA detects local 5 to this case leads to is order parameters and the measured order parameter the r =2/ S2ast (3 cos2" - 1)2 S2[11] average of them. The local order parameters represent the or- Downloaded by guest on September 30, 2021 Biophysics: jAnig Proc. Natl. Acad. Sci. USA 76 (1979) 6365 to the membrane plane. The assumption of a constant rj or Sf.A, however, is not necessarily fulfilled, as discussed above. In conclusion, it seems that further on - experiments protein I-- - 5r-..(I I LA are required to clarify the problems of composite protein I 11I% motion and protein-lipid interaction.

I /ll,I 1. Shinitzky, M. & Barenholz, Y. (1978) Biochim. Biophys. Acta 515, I 367-394. Oslow \ fastO 2. Chen, L. A., Dale, R. E., Roth, S. & Brand, L. (1977) J. Biol. Chem. 252, 2163-2169. 3. Kawato, S., Kinosita, K., Jr. & Ikegami, A. (1977) Biochemistry \J 16,2319-2324. 4. Lakowicz, F. & D. Membrane J. R., Prendergast, G. Hogen, (1979) Bio- normal chemistry 18, 508-519. 5. Veatch, W. R. & Stryer, L.(1977)J. Mol. Biol. 117,1109-1113. FIG. 7. Possible orientational motions of a protein segment. 6. Kawato, S., Kinosita, K., Jr. & Ikegami, A. (1978) Biochemistry 17,5026-5031. in which the absorption and emission moments are again as- 7. Glatz, P. (1978) Anal. Biochem. 87, 187-194. sumed to be parallel, usually lying along the chromophore axis, 8. Hildenbrand, K. & Nicolau, C. (1979) Biochim. Biophys. Acta 553,365-377. and Ssow = is parameter mo- (P2 (cos Oslow)) the order of the 9. Wahl, P., Kasai, M., Changeux, J.-P. & Auchet, J. C. (1971) Eur. lecular axis relative to its mean orientation-e.g., the membrane J. Biochem. 18, 332-341. normal. The three processes contributing to r. are pictured in 10. Kinosita, K., Jr., Kawato, S. & Ikegami, A. (1977) Biophys. J. 20, Fig. 7. The first term Sfat in Eq. 11 describes the average order 289-305. of the chromophore axes due to the fast intramolecular fluc- 11. Wahl, P. (1975) Chem. Phys. 7,210-219. tuations discussed above, the second term describes the order 12. Jahnig, F. (1979) J. Chem. Phys. 70, 3279-3290. due to rotational diffusion on the surface of a cone of half angle 13. Marcelja, S. (1974) Biochim. Biophys. Acta 367, 165-176. ly, and the third term Sslow describes the order due to the slow 14. Seelig, J. (1977) Q. Rev. Biophys. 10, 353-418. fluctuations of the long molecular axes. Neglecting the fluc- 15. Andrich, M. P. & Vanderkooi, G. (1976) Biochemistry 15, 1257-1261. = = 11 to an ex- tuations by putting Sfa~t Sslow 1, Eq. reduces 16. Thulborn, K. R., Treloar, F. E. & Sawyer, W. H. (1978) Biochem. pression known from FA of long molecules in solution if rota- Biophys. Res. Commun. 81, 42-49. tions around the short axes are forbidden (36). 17. Seelig, A. & Seelig, J. (1974) Biochemistry 13, 4839-4845. Vaz et al. (37) made time-resolved LA measurements on 18. Mely, B. & Charvolin, J. (1977) Chem. Phys. Lipids 19,43-55. cytochrome b5, in which the native heme group was replaced 19. Strehlow, U. (1978) Dissertation (Universitat Gottingen, Got- by a structurally similar chromophore, in Myr2PtdCho mem- tingen, Germany). branes. A relaxation process was observed with X = 9 psec in 20. Trauble, H., Teubner, M., Woolley, P. & Eibl, H. (1976) Biophys. the ordered lipid phase, and = 0.4 Atsec in the fluid phase. The Chem. 4, 319-342. A. & initial values and the final vlaues r.O were larger in the or- 21. Blume, Eibl, H. (1979) Biochim. Biophys. Acta, in press. ri 22. Jahnig, F., Harlos, K., Vogel, H. & Eibl, H. (1979) Biochemistry dered than in the fluid phase; their ratio, however, remained 18, 1459-1467. constant, r0,/r, = 0.6. Vaz et al. attributed the observed relax- 23. Gomez-Fernandez, J. C., Goni, F. M., Bach, D., Restall, C. & ation process to the rocking motion of the extramembranous Chapman, D. (1979) FEBS Lett. 98,224-228. part of the protein, which carries the chromophore, around the 24. Jost, P. C. & Griffith, 0. H. (1978) in Biomolecular Structure and membrane normal. In this case Eq. 11 reduces to ra = slOw Function, ed. Agris, P. F. (Academic, New York), pp. 25-54. For the order parameter of the long molecular axes we obtain 25. Longmuir, K. J., Capaldi, R. A. & Dahlquist, F. W. (1977) Bio- Sslo, = 0.77. This value is then independent of the lipid order, chemistry 16, 5746-5755. which may be conceivable because characterizes the order 26. Marcelja, S. (1976) Biochim. Biophys. Acta 455, 1-7. SS10W 27. W. & P. F. of an extramembranous segment. The de- Marsh, D., Watts, A., Maschke, Knowles, (1978) protein Biochem. Biophys. Res. Commun. 81, 397-402. pendence of ri on lipid order, however, reflects the partial 28. Chapman, D., Cornell, B. A., Eliasz, A. W. & Perry, A. (1977) J. hindrance of the rotational diffusion of the proteins in the or- Mol. Biol. 113,517-538. dered lipid phase. 29. Weidekamm, E., Bamberg, E., Brdiczka, D., Wildermuth, G., LA measurements in the millisecond regime were performed Macco, F., Lehmann, W. & Weber, R. (1977) Biochim. Biophys. by Heyn et al. (38) on bacteriorhodopsin in Pam2PtdCho Acta 464, 442-447. membranes, retinal being the chromophore. In the ordered lipid 30. Vogel, H. (1978) Dissertation (Universitat Gottingen, Gottingen, phase, rt was found constant in time, rt = 0.19. In the fluid Germany). phase, a relaxation was observed with = 0.5 msec and r. = 31. Curatolo, W., Verma, S. P., Sakura, J. D., Small, D. M., Shipley, & D. F. H. 0.037. Heyn et al. attributed this process to the rotational dif- G. G. Wallach, (1978) Biochemistry 17, 1802- 1807. fusion of the protein molecules around their long axes, assuming 32. Susi, H., Sampugna, J., Hampson, J. W. & Ard, J. S. (1979) Bio- that slow fluctuations do not occur in the millisecond re- chemistry 18, 297-301. gime-i.e., Sslow = 1. Here Eq. 11 reduces to r. = rj[(3 cos2'y 33. Oldfield, E., Gilmore, R., Glaser, M., Gutowsky, H. S., Hshung, - 1)/2]2 with rj = 2/5S2ast. In the ordered phase, rotational dif- J. C., Kang, S. Y., King, T. E., Meadows, M. & Rice, D. (1978) fusion is then inhibited and r. = ri. This would yield Sfast = Proc. Nati. Acad. Sci. USA 75,4657-4660. 0.69, but one has to be cautious with this result because the 34. Seelig, A. & Seelig, J. (1978) Hoppe Seyler's Z. Physiol. Chem. measured rt values were shown to depend on experimental 359, 1747-1756. factors (e.g., the light intensity due to partial bleaching). In 35. Rothschild, K. J. & Clark, N. A. (1979) Biophys. J. 25, 473- order to determine y from rc in the fluid phase, Heyn et al. 488. 36. R. & M. Rev. 1-19. rj to Rigler, Ehrenberg, (1976) Q. Biophys. 9, assumed be temperature independent and be given by the 37. Vaz, W. L. C., Austin, R. H. -c Vogel, H. (1979) Biophys. J. 26, value for the ordered phase. Then the two solutions 'y = 38° and 415-426. -Y = 780 are obtained, and Heyn et al. presented arguments in 38. Heyn, M. P., Cherry, R. J. & Muller, U. (1977) J. Mol. Biol. 117, favor of the second one, implying that retinal lies almost parallel 607-620. Downloaded by guest on September 30, 2021