289

Biochimica et Biophysica Acta, 559 (1979) 289 327 © Elsevier/North-Holland Biomedical Press

BBA 85198

ROTATIONAL AND LATERAL DIFFUSION OF MEMBRANE PROTEINS

RICHARD J. C t t E R R Y

Eidgendssische Technische Hochschule, Laboratorium fiir Biochemie, ETH-Zentrum, CH-8092 Ziirieh (Switzerlandj (Received May 23rd, 1979)

Contents I.

Introduction

I1.

Measurement of rotational diffusion by llash photolysis . . . . . . . . . . . . . . . . . . . A. Principles o f rotational diffusion measurements with triplet probes . . . . . . . . . . . B. E x p e r i m e n t a l methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Flash photolysis apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Analysis of the data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Protein ro tation in the h u m a n e r y t h r o c y t e membran e . . . . . . . . . . . . . . . . . . . 1. Ro tational mobility of band 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Band 3 dimers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Comparison with lateral diffusion measurements . . . . . . . . . . . . . . . . . . . . 4. Interaction of band 3 with peripheral proteins . . . . . . . . . . . . . . . . . . . . . E. (Mg 2+ + Ca2+)-ATPase in sarcoplasmic reticulum . . . . . . . . . . . . . . . . . . . . . . t7. Intrinsic ch ro mophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Rhodopsin and bacteriorhodopsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. C y t o c h r o m e e oxidase and c y t o c h r o m e P 4 5 0 . . . . . . . . . . . . . . . . . . . . .

............................................

290 290 291 292 292 293 294 297 297 298 299 299 300 301 301 305

III.

Saturation transfer electron paramagnetic resonance . . . . . . . . . . . . . . . . . . . . . A. Principles of saturation transfer methods . . . . . . . . . . . . . . . . . . . . . . . . . . B. Applications to membrane proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Comparison of saturation transfer-EPR with optical me t hods for measuring slow rotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

306 306 307

IV.

Fluorescence photobleaching recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. E x p e r i m e n t a l m e t h o d s and analysis of recovery kinetics . . . . . . . . . . . . . . . . . B. Lateral diffusion coefficients of membrane proteins . . . . . . . . . . . . . . . . . . . . 1. Lectin receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Surface antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Other measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Control of m e m b r a n e mobility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1). Lipid diffusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

311 312 313 314 314 314 316 317

31 ()

Abbreviations: eosin-SCN, eosin 5-isothiocyanate; IA-eosin, 5-iodoacetamido-eosin; eosin-MA, eosin 5-maleimide: fluorescein-SCN, fluorescein isothiocyanate; FPR, fluorescence photobleaching recovery; saturation transfer-l'PR, saturation transfer electron paramagnetic resonance; ELDOR, electron-electron double resonance; Fab, antigen binding fragment.

290 V. VI.

Probe-inducedperturbations of membrane structure . . . . . . . . . . . . . . . . . . . . . Miscellaneousdiffusion measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

317 318

VII. Evaluation of diffusion coefficients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

320

VIII. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

322

Note added in proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

323

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

323

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

323

I. Introduction The mobility of membrane proteins was first demonstrated in the pioneering experiments of Frye and Edidin [1], Taylor et al. [2] and Cone [3,4]. These observations have undoubtedly exerted considerable influence on current concepts of membrane structure. Subsequently, further evidence was accumulated that a variety of membrane proteins are capable of lateral motion in the plane of the membrane. Typically, this involved demonstrating that proteins, initially randomly distributed in the plane of the membrane, become aggregated by antibodies, lectins or simply by changes in temperature or pH. Some of the observed phenomena appear to be simply a consequence of protein diffusion. Capping of cells by iectins and antibodies, however, is a more complex process requiring metabolic energy. Investigations of these aggregation phenomena together with other observations of protein mobility published prior to about mid 1975 have already been extensively reviewed (5-7). More recently, the emphasis has shifted to the development of techniques which give quantitative measurements of protein diffusion and are of general applicability. Essentially three principal methods have emerged, two for measuring rotational diffusion and one for measuring lateral diffusion. It is to be hoped that these methods will lead to an improved understanding of membrane dynamics and provide useful information concerning structural interactions between membrane components. The purpose of this article is to review current techniques for measuring protein diffusion and to evaluate the data so far obtained.

II. Measurement of rotational diffusion by flash photolysis Optical spectroscopy has long proved useful for investigating rotational motion of macromolecules in aqueous solution through the technique of fluorescence polarization [8,9]. The method is successful because fluorescence lifetimes, which are typically ~10 -8 s, are not too different from the rotational relaxation times to be measured. When the rotational relaxation time is slower than about 10 -6 s, however, the method fails because the fluorescent emission decays before any detectable rotation can occur. This indeed was the result found for electroplax membrane proteins labeled with a fluorescent probe by Wahl et al. [10], while Cone's [3] measurement of rhodopsin rotation in the rod outer segment membrane yielded a relaxation time of 20/~s at 20°C. Assuming that membranes are at least 100 times more viscous than water, it may be calculated that the relaxation times of intrinsic globular membrane proteins will inevitably be in the microsecond time range or longer. In order to measure such slow rotation by optical spectroscopy, it is necessary to use a spectroscopic state having a long lifetime. Cone's transient dichroism measurements with

291 rhodopsin were in fact possible because excitation produces long-lived spectroscopic intermediates. In 1973, it was proposed that a general method for measuring rotation of membrane proteins might be based on the long lifetime of the triplet state of probe molecules [11 ]. The idea of using triplet states to measure slow rotational diffusion had been put forward earlier [12,13] but few experiments were reported and no applications to biological systems had been attempted. Following the development of suitable probes [14,15], the measurement of protein rotation in the human erythrocyte membrane was reported [16]. Triplet probes have also been recently used to study rotation of large macromolecular complexes in aqueous solution [ 17,18]. The above experiments were all carried out using the flash photolysis technique. The use of steady state and modulation methods has also been proposed [19,20]. An alternative approach to measuring slow rotational diffusion is fluorescence autocorrelation spectroscopy [21], which although using fluorescence for detection, is essentially measuring fluctuations in angular distribution of molecules in the ground state. So far no applications to rotational motion in membranes have been reported and the method will therefore not be further considered here.

11,4. Principles of rotational diffusion measurements with triplet probes Spectroscopic methods of measuring rotation depend on photoselection whereby an oriented population of excited molecules is optically selected from an initial random distribution. This is achieved by excitation with plane polarized light, so that those molecules whose transition dipole moment for absorption is parallel or at a small angle to the electric vector of the incident light are preferentially excited. Signals arising from the excited molecules in general reflect their anisotropic distribution so that emission signals are polarized and absorption signals are dichroic. When excitation is by a brief pulse of light, the initial emission or absorption anisotropy decays as the molecules again become randomized by Brownian rotation. From the rate of decay, rotational relaxation times may be determined. Fig. 1 shows the lower electronic energy levels of an organic molecule with an even number of electrons and illustrates some of the principal optical transitions which occur. Triplet states cannot normally be populated to any appreciable extent by direct absorption and the absorption spectrum arises from the singlet-singlet transitions So-S1, So-$2, etc. Usually higher singlet states degrade rapidly and non-radiatively to the lowest excited singlet state $1. The subsequent return to the ground state may occur either directly or via the triplet state. In selected molecules, the S~-T~ transition (intersystem crossing) effectively competes with the S1-So transition. Because the Tl-So transition is spin forbidden, the lifetime of the lowest triplet state (typically >10 -a s) is much longer than that of the S~ state (typically 10-8-10 -9 s). Quenching, especially by oxygen, may considerably shorten the observed triplet lifetime. In principle, triplet states may be detected by observing the phosphorescent emission arising from radiative T1-So transitions. In fact, the first experimental measurement of rotational motion using triplet states was obtained by observing the phosphorescence depolarization of a benzene derivative at low temperature in a highly viscous medium [12[. Phosphorescence from aromatic amino acids in proteins at room temperature has also been observed [22]. In general, however, phosphorescence may be difficult to detect at room temperature in fluid solutions, where the non-radiative transition has a much higher probability. In flash photolysis experiments, triplet st:.~tes are detected from

292 $3

T3 $2 -i-2

$1

~ "~"~,,,.~.

INTERSYSTEM '~'-,.. CROSSING

I

So

4,

Fig. 1. Electronic energy level scheme. Solid arrows represent radiative transitions, and dashed arrows non-radiative transitions (vibrational states are omitted).

absorbance changes in the sample. At appropriate wavelengths, an absorbance increase occurs due to the transition from the lowest triplet state to a higher triplet state. Alternatively, a ground state depletion signal may be observed. This is a decrease in absorbance in the singlet-singlet absorption band due to removal of molecules to the triplet state. When the triplet state is populated following flash excitation of the So-S1 transition with plane polarized light, the resulting transient absorbanee changes are dichroic due to photoselection. The time dependence of dichroism enables rotational motion to be investigated. Because of the long lifetime of the triplet state, it is possible to detect rotation times as slow as milliseconds, whereas fluorescence methods are confined to times shorter than ~1 tts (except in the case of delayed fluorescence [23]). Of course, not only triplet states, but any photoproduct with a suitably long lifetime can be used in a similar way to measure slow rotational motion.

liB. Experimental methods Full details of the experimental methods of measuring rotational diffusion using triplet probes were presented elsewhere [24]. Hence, only the m ~ points are summarized here.

liB(I). Probes The most useful triplet probes available at the present time are derivatives of eosin, illustrated in Fig. 2. The reactive group enables eosin to be coupled eovalently to proteins; in the case of eosin-5-isothiocyanate (eosin-SCN) the reaction is preferentially ~ t h amino groups, while 5-iodoacetarnido-eosin (IA-eosin) and eosin-5-maleimide (eosin-MA) react preferentially with sulphydryl groups. The amount of bound eosin in a given sample

293

Br

_o ;f

Rotational and lateral diffusion of membrane proteins.

289 Biochimica et Biophysica Acta, 559 (1979) 289 327 © Elsevier/North-Holland Biomedical Press BBA 85198 ROTATIONAL AND LATERAL DIFFUSION OF MEMBR...
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