[60] Time-resolved x-ray scattering of microtubule assembly using synchrotron radiation

[60]

X - R A Y S C A T T E R I N G OF M I C R O T U B U L E ASSEMBLY U S I N G S R

657

over, both the T M V and microtubule patterns have prominent nearmeridional reflections around 0.45 nm (layer lines 15 and 16 for TMV, layer line 9 for microtubules). This is suggestive of the spacing between strands of/]-pleated sheets running roughly parallel to the x - y plane (i.e., seen face-on w h e n viewing the particle from outside), and this is indeed found with T M V (see Fig. 3 of Bloomer et al.66). T h e s e conclusions are tentative and mainly serve to guide one's thinking about the tubulin molecule; but they concur with sequence comparisons showing that the N-terminal domain of tubulin contains several regions of homology with other nucleotide-binding proteins of known a/B structure. 67,68 H o w e v e r , details of the arrangement of the secondary structure elements will have to await a full crystal structure analysis of tubulin. Acknowledgments The data shown here are the result of collaborations with a number of colleagues whom I would like to thank for their help, advice, and encouragement: TMV, K.C. Holmes and U. Gallwitz (MPI Heidelberg); intermediate filaments, W. Renner (MPI Heidelberg), W. W. Franke (DKFZ Heidelberg), and K. Weber (MPI Grttingen); microfilaments, P. Matsudaira (Whitehead Institute, Cambridge, MA), W. Renner, and K. Weber; microtubules, J. Thomas and C. Cohen (Brandeis University, Waltham, MA), A. Harmsen and E.-M. Mandelkow (MPI Heidelberg). 67M. J. E. Sternberg and W. R. Taylor, FEBS Lett. 175, 387 (1984). 68E.-M. Mandelkow, M. Herrmann, and U, Rtihl, J. Mol. Biol. 185, 311 (1985).

[60] Time-Resolved X-Ray Scattering of Microtubule Assembly Using Synchrotron Radiation

By ECKrIARD MANDELI~OW and JOAN BORDAS

Introduction

Synchrotron Radiation: Properties, Applications, Sites S y n c h r o t r o n radiation (SR) has emerged as a versatile light source with applications to many problems in physics, chemistry, biology, and medicine. SR is emitted w h e n electrons or positrons are accelerated in a synchrotron; hence the name. Their energy loss is emitted as electromagMETHODS IN ENZYMOLOGY, VOL. 134

Copyright © 1986by Academic Press, Inc. All rights of reproduction in any form reserved.

658

ANALYSIS OF CYTOSKELETAL PROTEINS

[60]

netic radiation with several unique properties: SR is orders of magnitude brighter than other light sources, SR has a continuous wavelength distribution, ranging from X-rays to the infrared and beyond, SR is polarized in the plane of the orbit, and SR is pulsed, with pulse widths in the picosecond range and periods in the microsecond range. -The spectral brilliance is the most useful property, in particular for biologists, because it allows the weak scattering from biomolecules to be measured within a short time. This has opened the way to rapid data collection and to the observation of dynamic processes. Examples are muscle contraction, self-assembly of proteins, or conformational changes (see below). The continuous wavelength distribution is exploited when one is interested in the different absorption or scattering of certain atoms below and above their absorption edges. This has been applied to the spectroscopy of metalloenzymes (for a review, see Bordas~), anomalous diffraction from protein crystals (for solving the phase problem, see Greenhough and Helliwell2) or resonant scattering from solutions, 3 and X-ray microscopy (in order to enhance the contrast between cell components and solvent, see Schmahl et al.4). The discrete time structure allows the excitation of fluorophores (native or labeled) in the ultraviolet or visible region and the observation of fluorescence anisotropy decay (a measure of conformation, see Munro et al.5). Laboratories for biological research with SR have been created at several physics establishments dedicated to high energy physics or synchrotron radiation. Examples are the EMBL Outstation at DESY (Hamburg, FRG), and MRC/SERC Biological Support Laboratory at Daresbury Laboratory (Warrington, UK), LURE (Orsay, France), ADONE (Frascati, Italy), National Light Source at Brookhaven National Laboratory (Upton, NY), CHESS (Cornell University, Ithaca, NY), SSRL (Stanford University, Stanford, CA), the Photon Factory (Tsukuba, Japan), and VEPP (Novosibirsk, USSR). In this report we restrict ourselves to time-resolved X-ray scattering and illustrate the potential of the technique mainly with the model system of microtubule self-assembly. Information on other studies may be obtained from the annual reports of the above-mentioned laboratories or recent review books. 6-8 1 j. Bordas, in "Uses of Synchrotron Radiation in Biology" (H. Sturhmann, ed.), p. 107. Academic Press, New York, 1982. 2 T. J. Greenhough and J. R. Helliwell, Prog. Biophys. Mol. Biol. 41, 67 (1983). 3 H. B. Stuhrmann, Adv. Polym. Sci. 67, 123 (1985). 4 G. Sehmahl, D. Rudolph, B. Niemann, and O. Christ, Q. Rev. Biophys. 13, 297 (1980). I. H. Munro, I. Pecht, and L. Stryer, Proc. Natl. Acad. Sci. U.S.A. 76, 56 (1979). 6 H. Winick and S. Doniach, eds., "Synchrotron Radiation Research." Plenum, New York, 1980.

[60]

X-RAY SCATTERING OF MICROTUBULE ASSEMBLY USING SR

659

X-Ray Scattering from Solutions Our aim is to observe reactions in solution, i.e., in a disordered system. The appropriate theoretical framework is that o f " small angle X-ray scattering" (for reviews, see Guinier and Fournet 9 and Glatter and Kratkyl°). Strictly speaking this term is a misnomer, imposed by the weakness of conventional X-ray generators which allowed only the nearforward scattering to be observed. However, the X-ray scattering extends to high resolution and may be measured using SR. For our purposes a better term would therefore be "X-ray solution scattering." The structural information is contained in the dependence of the scattered intensity on the scattering vector S = 2 sin O/h which for small angles equals 20/h. Here 20 is the scattering angle, h is the X-ray wavelength (chosen around 0.15 nm), i.e., S is the scattering angle normalized with respect to the wavelength. This definition is also used in X-ray crystallography (Bragg spacing) or fiber diffraction, thus facilitating a direct comparison. The reciprocal of S is the resolution, i.e., we say that we know a structure at a resolution of 10 nm if the maximum scattering angle is such that S,~ax = (10 nm) -1 (note that the quantity h = 27rS is also in use).

A typical scattering curve contains a bell-shaped central maximum and rapidly decaying subsidiary fringes. The higher the angle, the better the structural resolution. Thus the very low-angle scattering contains information on higher order assemblies, followed by information on subunit assemblies, subunit shape, and finally internal structure. In contrast to crystallography this information cannot be extracted directly because of the disorder in the sample. The interpretation is usually done by comparing the experimental scattering curves with theoretical ones, calculated from model structures based on some reasonable assumption. The models are obtained from other physicochemical data, such as electron microscopy, ultracentrifugation, and X-ray fiber diffraction. These experiments, as well as computer modeling, are therefore a necessary complement to solution X-ray studies. If different particles coexist in solution their scattering is superimposed and becomes difficult to interpret. In static structural studies one tries to make the solutions as homogeneous as possible. However, in 7 H. B. Stuhrmann, ed., "Uses of Synchrotron Radiation in Biology." Academic Press, New York, 1982. 8 H. Bartunik & B. Chance, eds., "Structural Biological Applications of X-Ray Absorption. Scattering, and Diffraction." Academic Press, Orlando, Florida, 1986. 9 A. Guinier and G. Fournet, "Small-Angle Scattering of X-Rays." Wiley, New York, 1955. to O. Glatter and O. Kratky, eds., "Small Angle X-Ray Scattering." Academic Press, New York, 1982.

660

ANALYSIS OF CYTOSKELETAL PROTEINS

[60]

time-resolved experiments it is precisely the changing mixture of species which is of interest. This tends to reduce the structural information. It illustrates a general relationship: temporal information is gained at the expense of spatial information, and vice versa. In a self-assembly reaction we study a polydisperse solution whose constituents are built from identical subunits. Their scattering may be described as

I(S,t) = ~ [xk(t)pkik(S)]

(1)

k

Here the index k refers to the type of particle, xk is the fraction of subunits incorporated into aggregates of type k (analogous to the extent of the assembly reaction), pk is their degree of polymerization (number of subunits per particle), and ik is the shape function normalized to unity at S = 0. Form factors of model structures can be computed from Debye's formula which may be written in a normalized form as imod(S) =

isph(S)/P 2 [P + 2 ~] ~] sin(2zrSrij)/(27rSrij)] i

(2)

j>i

where r0 is the distance between a pair of subunits (id), isph is the transform of the subunit, and the sum extends over all pairs of units (i
[60]

X - R A Y SCATTERING OF MICROTUBULE ASSEMBLY USING S R

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I

661

~

'~0

,~..~

0 •~

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0 "---

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•

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.~! "7

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662

ANALYSIS OF CYTOSKELETAL PROTEINS

[60]

or turbidity at UV wavelengths. For certain particle dimensions and scattering angles the signal is proportional to the total mass of assembled material, as shown by Berne. 12However, light scattering is rather insensitive to structural details and therefore does not allow one to distinguish between different assembly forms.) There are several model-independent quantities which one can extract from the intensity decay of small-angle scattering. One is the radius of gyration which is a measure of the average size of a globular protein. Similarly, for rod-like and disk-like particles one obtains the radius of gyration of the cross-section or of the thickness. By extending the argument given above for the degree of polymerization it is possible to derive expressions for lateral aggregation of rods (degree of accretion), axial stacking of disks, or mixtures of different assembly forms.13 The analysis of X-ray scattering data from complex mixtures may be simplified in several ways. Difference scattering patterns are sensitive only to those species that change during a reaction, irrespectively of other inactive species. Cross-correlation of intensities at different angular regions reveals structural changes independently of kinetics. The dependence of the scattering on variables other than times (e.g., temperature) reveals different steps in a reaction. Examples will be given below. Instrumentation

X-Ray Instrumentation The details of various X-ray beam lines have been described elsewhere. 14-16Briefly, the radiation emitted by the electrons is focused and made monochromatic (h = 0.15 nm) by a set of mirrors and crystals (Fig, 2). The distance D between the specimen and the detector can be varied between a few centimeters and several meters. It determines the range of S values to be recorded. If L is the length of the detector the maximum scattering angles is (2*9)max = L/D rad, and Smax = L/(Dh). 12 B. J. Berne, J. Mol. Biol. 89, 755 (1974). ~3j. Bordas and E. Mandelkow, in "Structural Biological Applications of X-Ray Absorption, Scattering, and Diffraction" (H. Bartunik and B. Chance, eds.). Academic Press, Orlando, Florida, 1986. 14j. Hendrix, M. H. Koch, and J. Bordas, J. Appl. Crystallogr. 12, 467 (1979). 15j. Bordas, M. H. J. Koch, P. N. Clout, E. Dorrington, C. Boulin, and A. Gabriel, J. Phys. E 13, 938 (1980). 16 N. H. J. Koch and J. Bordas, Nucl. Instrum. Methods. 208, 461 (1983).

[60]

X-RAY SCATTERING OF MICROTUBULE ASSEMBLY USING S R

663

TANFJIENT POINT

MIRRORSYSTEM

BENT

x~7, ~

SAMPLE CELL

~, !i/%-.-.. ~:~IF,~ OR -- \jh~ :~'\~. ~~ ' !~I DETECTOR

SOLUTIONSCATTER!NGPATTERN

FxG. 2. Diagram of a synchrotron radiation X-ray instrument (XI3 at EMBL Hamburg). The radiation is emitted tangentially from the storage ring, made monochromatic (L = 0.15 nm) and focused by X-ray mirrors and a crystal monochromator, passes through the sample chamber containing the protein solution, and is detected at a distance up to several meters by a detector. Since the particles in solution are randomly oriented the scattering pattern takes the form of a series of concentric maxima (inset). From Bordas et al.24 Instrumentation f o r Kinetic Measurements X-Rays are strongly absorbed by most materials. This means that the observation chambers must be covered with very thin windows (e.g., mica sheets 20-40/.~m thick) with concomitant stability problems. Commercial chambers made for UV or visible light observation are not usable. Reactions are studied by perturbing the chemical potential and monitoring the approach to a new equilibrium. The perturbation may be a change in temperature (T-jump) or solvent conditions (rapid mixing, stopped flow). Temperature Jump. Usual temperature relaxation experiments operate with small temperature changes (a few degrees) which can be induced within milliseconds (e.g., by laser heating or by Peltier devices). By contrast, the study of microtubule assembly and disassembly requires a large j u m p which must be applicable in both directions (e.g., 0 to 37 ° and back). We are using a specimen c h a m b e r thermostatted by four water baths to achieve T-jumps o f a few seconds, z7 The same apparatus can be used for 17 W. Penner, E.-M. Mandelkow, E. Mandelkow, and J. Bordas, Nucl. Instrum. Methods

208, 535 (1983).

664

ANALYSIS OF CYTOSKELETAL PROTEINS

[60]

slow near-equilibrium temperature scans (about l°/min). The advantage of temperature changes is their reversibility. Several cycles of assembly and disassembly can be performed without refilling the cell and without perturbing the sample otherwise (e.g., mechanical shearing). Rapid Mixing. Several types of mixing device have been constructed for different applications of time-resolved X-ray scattering (e.g., Renner et al., 17Fowler et al. 18and other designs available at several synchrotron facilities). They share the need for very thin chamber windows. Differences depend on several factors, e.g., the quantity of protein available and the type of reaction studied, and the desired resolution range. If the protein is abundant and recoverable one can use continuous flow mixing while stopped-flow is more appropriate in other cases. One usually requires many repetitive experiments in order to accumulate a sufficient scattering signal. The apparatus used for microtubule assembly has been designed for small quantities of protein (e.g., for mixing of tubulin and MAPs). Microtubule disassembly cannot be studied conveniently with mixing because of the high viscosity of a microtubule solution, and because shearing in the mixing cell distorts the results since the rates depend on the microtubule number concentration. Another way to perturb an equilibrium would be by pressure jump which is appropriate for reactions involving volume changes (e.g., entropy-driven self-assembly). This has been applied to microtubule assembly using observation with UV light. 19The method has not yet been used with time-resolved X-ray scattering of solutions because the required pressures (a few hundred bar) are not compatible with the need for thin windows and reasonable scattering volumes (several cubic millimeters). Detectors and Data Acquisition

In order to record the X-ray intensities as a function of the scattering angle one requires position-sensitive detectors. Most of the devices currently in use operate on the principle of detecting the charges created in a gas (mixture of Ar or Xe with CO2) when it is ionized by an X-ray photon. The charges are amplified by an acclerating voltage, and the position at the anode wire(s) is detected electronically (for a recent review, see Bordas et al.2°). The details of detectors vary, depending on the problem at hand. For instance, for circularly symmetric patterns (e.g., solution scattering) a is A. G. Fowler, A. M. Foote, M. F. Moody, P. Vacchett, S. W. Provencher, A. Gabriel, J. Bordas, and M. H. J. Koch, J. Biochem. Biophys. Methods 7, 317 (1983). ~9y . Engelborghs, K. Heremans, L. de Maeyer, and J. Hoebeke, Nature (London) 259, 686 (1976). 20 j. Bordas, R. Fourme, and M. H. J. Koch, Nucl. Instrum. Methods 201, 1 (1982).

[60]

X-RAY SCATTERING OF MICROTUBULE ASSEMBLY USING S R

665

linear detector oriented along a radial line is sufficient31 When the pattern is weak the statistical accuracy can be improved by integrating over all angles at a given radial distance (quadrant detector, J. Hendrix and J. Bordas, unpublished). In the case of oriented fibers whose scattering is confined to layer lines the data are collected efficiently by a linear wireper-wire detector, zz Finally, for diffraction patterns from crystals containing discrete reflections the intensities must be detected as a function of the x - y coordinates on the detector plane and require two-dimensional area detectors. 23 The detector system has to be associated with a data acquisition system which simultaneously records the position and the time of an arriving photon. The existing data acquisition systems are based on the principles described in Bordas et al. is and Boulin et al. 23 A characteristic feature of time-resolved experiments is the large amount of data storage and processing. In the simplest case of a linear detector the scattering curve is typically subdivided into 256 pixels or channels, each 16 bit deep. A single experiment of several minutes duration, sampled every few seconds, requires about 256 time frames, resulting in 128 kbytes of data. This amount is stored in a computer during the measuring period and retrieved at a later stage for analysis, using appropriate programs, display devices, etc. Radiation Effects Unless one is specifically interested in radiation chemistry the damage caused by X rays is an undesirable side effect. It deserves particular attention, given the high flux of X-ray photons incident on the specimen. Fortunately, experience shows that it is less severe than expected. With 0.15 nm X-rays, the dose in rads is given by D = 1.3 × 10-VNinc(1 - e - t ) / V

(3)

where Nine = incident flux (photons/sec), t = sample thickness (mm), and V = irradiated volume (/~1).1~For a thickness of 1.5 mm, volume of 50 txl, and a flux of 5 × 1011 photons/sec the dose rate is typically 1000 rad/sec (10 Gy/sec). Damage of biological samples in SR experiments sets in around 10 t2 to 1013 photons//xl at 0.15 nm wavelength. It increases considerably at 21 A. Gabriel, Reo. Sci. lnstrum. 48, 1303 (1977). 22 j. Hendrix, H. Fuerst, D. Hartfiel, and D. Dainton, Nucl. lnstrurn. Methods 201, 139 (1982). 23 C. Boulin, D. Dainton, E. Dorrington, G. Eisner, A. Gabriel, J. Bordas, and M. H. J. Koch, Nucl. Instrum. Methods 201, 209 (1982).

666

ANALYSIS OF CYTOSKELETAL PROTEINS

[60]

shorter wavelengths, judging from experiments with live muscle (C. Nave, G. Diakun, and J. Bordas, unpublished). In the experiments with microtubules the permissible dose was 2-3 orders of magnitude higher than that derived from radiation damage studies using laboratory sources (compare Bordas et al. 24 and Zaremba and Irwin25). This suggests that the critical factor is not only the total radiation dose but also the dose rate. The onset of radiation damage can be delayed by including radical scavengers such as dithiothreitol, superoxide dismutase, and catalase (reviewed by Durchschlag and Zipper26). Applications of Time-Resolved X-Ray Scattering The initial motivation for developing the method came from research on muscle contraction. Static X-ray patterns from muscle using SR were first obtained by Rosenbaum et al. 27 at DESY, Hamburg (for a review, see Rosenbaum and Holmes28). Following the improvement in instrumentation ~4,15the first time-resolved experiments with SR were those of Huxley et al. 29 on muscle, Mandelkow et al. 3° on microtubules, and Moody et al. 31 on aspartate transcarbamylase. In the following sections we give a brief survey of the types of problems that can be addressed. Microtubule A s s e m b l y Static Solution Scattering o f Microtubule Protein in Different States o f A s s e m b l y . Before attempting a time-resolved study it is necessary to

ascertain that the reference states are well defined and interpretable. Electron microscopy shows that cold microtubule protein (0-5 °) contains ring-like structures of about 36 nm diameter, while heating to 37° induces the formation of microtubules of about 24 nm (Figs. 3 and 4). The difference in diameter and state of aggregation is reproduced by the X-ray patterns (Fig. 3, bottom right). They contain a central maximum (partially invisible because of the rectangular beam stop intercepting the unscat24 j. Bordas, E.-M. Mandelkow, and E. Mandelkow (1983). J. Mol. Biol. 164, 89 (1983). 2~ T. G. Zaremba and R. D. Irwin, Biochemistry 20, 1323 (1981). 26 H. Durchschlag and P. Zipper, Radiat. Environ. Biophys. 24, 99 (1985). 27 G. Rosanbaum, K. C. Holmes, and J. Witz, Nature (London) 230, 434 0971). G. Rosenbaum and K. C. Holmes, in "Synchrotron Radiation Research" (H. Winick and S. Doniach, eds.), p. 533. Plenum, New York, 1980. 29 H. E. Huxley, A. R. Faruqi, J. Bordas, M. H. J. Koch, and J. R. Milch, Nature (London) 284, 140 (1980). 3o E.-M. Mandelkow, A. Harmsen, E. Mandelkow, and J. Bordas, Nature (London) 287, 595

(1980). ~ M, F. Moody,P. Vachette,A. M. Foote, A. Tardieu,M. H. J. Koch, and J. Bordas, Proe. Natl. Acad. Sei. U.S.A. 779 4040 (1980).

[60]

X-RAY SCATTERING OF MICROTUBULE ASSEMBLY USING S R

667

tered radiation) and a series of side maxima centered at reciprocal spacings $1, $2, etc. The maxima are distributed over rings because the particles are randomly oriented. Since the structures are hollow the diameter is close to D = 1.83/S1. The diameter observed by X-rays is the mean diameter of the hydrated particles, weight averaged over all particles in solution. By contrast, electron microscopy yields inner or outer diameters of single particles flattened on the grid. This explains the small but measurable differences between the two sets of data. The patterns can be explained by small-angle scattering theory, but in the case of rotationally symmetric partices it is equally possible to interpret them by fiber diffraction theory. This applies particularly to microtubules whose oriented fiber pattern is known 32 (Fig. 3, top). The solution pattern is generated by rotating the fiber pattern and averaging over all orientations. In the solution pattern the contributions from different layer lines are superimposed on each other. However, this loss of information can be partially retrieved by reference to the fiber pattern. This becomes important for the interpretation of higher angle reflections. With SR the X-ray patterns are obtained within minutes on film and within seconds on a detector. By contrast, they require hours or days with laboratory X-ray sources. In the earlier fiber diffraction experiments the microtubules had to be stabilized by high concentrations of glycerol, and patterns from rings could not be obtained reliably. Thus one advantage of SR is that it allows the study of labile proteins, tubulin being a case in point. Substructure of Tubulin Rings. Much of the earlier interest in tubulin rings was generated from the fact that microtubule assembly appeared to be facilitated when they were initially present. Thus several models of microtubule assembly were proposed, based on rings as nucleation centers (reviewed by Kirschner33). Apart from the kinetic aspects the models differed in terms of the assumed substructure and the structural transitions implied by it. There were three basic ring models, all based on electron microscopy, in which the axis of the tubulin dimer was oriented either radially, out of the plane of the ring, or along the circumference. XRay studies of rings at intermediate resolution distinguished between the models and supported the latter one. Thus rings may be viewed as coiled variants of the protofilaments from which microtubules are built. This argues against certain classes of ring structure and the assembly models associated with t h e m ) 4 32 E. Mandelkow, J. Thomas, and C. Cohen, Proc. Natl. Acad. Sci. U.S.A. 74, 3370 (1977). 33 M. W. Kirschner, Int. Rev. Cytol. 54, 1 (1978). 34 E. Mandelkow, E.-M. Mandelkow, and J. Bordas, J. Mol. Biol. 167, 179 (1983).

668

ANALYSIS OF CYTOSKELETAL PROTEINS

[60]

_ ~ - ~protofiloment "f~ formation ./

~ ~ _L~-3 -3. lo \ 1 0 ~

I--I

,I iii I l//7~,o\~"Xo~o,, 13\~k/ I kI \2 I=0

mFm/bIY /.' I ~ '

\ \_

7/

/ I =-I

\

i

i/

J


i

I 0.05

S(nm-~)---,..

0.1

FIo. 3. Diagram illustrating the principle of a time-resolved X-ray experiment and the relationship between the fiber diffraction and solution scattering patterns of microtubules which provide the basis of the data interpretation. Top: Diagram of the oriented fiber pattern, with layer lines at orders of 4 nm. Numbers indicate Bessel terms corresponding to

[60]

X-RAY SCATTERING OF MICROTUBULE ASSEMBLY USING SR

20

220

° **O 0

669

370

- - D i n .

~"

0 0

~ o

F1G. 4. Assembly scheme illustrating the central role of tubulin oligomers in the assembly of microtubules at high temperature (tight) and in the assembly of tings at low temperature (left). The oligomers are short stretches of protofilaments. Electron micrographs of negatively stained particles correspond to the subreactions determined by X-ray scattering (left, low temperature, rings; center, intermediate temperature, oligomers; right, high temperature, microtubules). Rings are absent from purified tubulin, but oligomers persist (see Fig. 5). Adapted from Mandelkow e t a l ? ° and Bordas et al. 2~

Radius of Gyration of Tubulin. The size and shape of the tubulin molecule are important for an understanding of its assembly states. The bond lengths are known from the periodicities revealed by optical diffraction of electron micrographs or by X-ray fiber diffraction, but this leaves the size of the individual subunit uncertain. We therefore measured the radius of gyration of tubulin dimers purified by phosphocellulose chromatography and obtained a value of 3.1 nm (Fig. 5). This value is larger than expected, considering the molecular weight of 2 × 50,000 and axial repeat different structural features (e.g., zero order = diameter, thirteenth order = separation of protofilaments, etc.; for an interpretation, see Mandelkow et al.32). If the microtubules were randomly oriented each reflection would be distributed around a circle (dashed). Bottom fight: Solution scattering pattern of microtubule protein at low and high temperature, obtained on X-ray film within a few minutes. The first two subsidiary maxima (labeled i and 2) indicate the diameter (about 24 nm for microtubules, 36 nm for rings). The innermost part of the pattern is obscured by the shadow of a lead stop intercepting the unscattered beam. The dashed line indicates the position of the linear detector used for time-resolved experiments. Bottom left: Two cycles of temperature-induced assembly and disassembly monitored by a linear detector. Note the increase in the central scatter (left) with increasing degree of polymerization, and the concomitant shift of the subsidiary maxima. Adapted from Mandelkow e t al. 3° and Bordas e t al. 24

670

ANALYSIS OF CYTOSKELETAL PROTEINS -3.6

I

m

I

'

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[60]

I

"¢

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-4.0

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I

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S2 Inm-21 FIG. 5. Gunier plot of a solution of tubulin purified by phosphocellulose chromatography. The straight section corresponds to the tubulin dimer with a radius of gyration of 3.1 nm. The increase at low spacings is due to oligomers. From Bordas e t al. 24

of 4 nm (measured along the protofilaments of microtubules). It means either that the subunit is more elongated than expected, or that the distance between the monomers in the unpolymerized dimers is larger than in the polymer, indicating a possible conformational difference between the two states. 24 Oiigomers of Tubulin. The study of PC-tubulin showed that part of the tubulin dimers were associated into oligomers (smaller than rings). They are seen even at low temperature, and in conditions that do not support self-assembly (Fig. 5). Similar oligomers are seen during temperatureinduced assembly of microtubule protein (see below). Since rings are coiled protofilaments the oligomers, too, may be viewed as short stretches of protofilaments. They represent an intermediate level of organization of tubulin which is probably the key to understanding microtubule nucleation.

Assembly of Microtubule Protein Induced by Temperature-Jump. When a solution of microtubule protein is placed in the X-ray beam and heated rapidly from 0 to 37 ° one observes a transition of the pattern of the initial cold state to the final warm state. The reponse of the intensity depends on the scattering angle (Fig. 3, bottom left). At the lowest angles one observes an initial decrease, followed by a strong increase; other regions of the pattern either show a similar or an opposite behavior or are indifferent ("isosbestic points"). Analysis of the patterns by difference plots (Fig. 6) shows that the beginning of the reaction is characterized by a

[60]

X-RAY SCATTERING OF MICROTUBULE ASSEMBLY US1NG SR

671

3

FIG. 6. Difference pattern between pattern of initial cold solution and the pattern observed shortly after the temperature jump. The difference can be fitted against the scattering expected from rings, indicating that rings disappear during the initial phase. From Mandelkow e t a l ) °

disappearance of the ring structures (prenucleation phase), followed by microtubule growth. 3° This implies that rings are not directly involved in microtubule nucleation, as had been assumed in some assembly models. Rather, the role of nucleation units must be ascribed to smaller entities, i.e., tubulin oligomers (see Fig. 4). After reaching the level typical of assembled microtubules, the low-angle scattering often keeps rising slowly (postassembly phase), indicating a further increase in the degree of organization (e.g., cross-linking or bundling). Assembly of PC-Tubulin. The assembly of purified tubulin is usually rather inefficient and requires special solution conditions such as high Mg2÷ and glycerol. In normal solution conditions the protein can be driven into self-assembly by raising its concentration, e.g., to 10-20 mg/ ml or more. 35 In this case the initial cold solution does not contain rings, but a mixture of oligomers and dimers (Fig. 5). The change of the X-ray pattern during the assembly phase is similar to that observed with microtubule protein. However, the prenucleation phase shows very little initial decrease because there are no rings to be dissolved. Both with microtubule protein and with tubulin the onset of assembly is characterized by an increase in the oligomeric structures. 35 E.-M. Mandelkow, M. Herrmann, and U. Rfihl, J. M o l . Biol. 185, 311 (1985).

672

ANALYSIS OF CYTOSKELETAL PROTEINS

[60]

..E

5

E

.A ~me

3 5

I

t

f

6

7

8

I total

"-

FzG. 7. Correlation between total scattering (x axis) and scattering at first subsidiary maximum (y axis) du.ring a temperature scan. Transitions between subreactions appear as breaks between straight sections. From Bordas and Mandelkow. 11

Assembly by Slow Temperature Scan. In the T-jump experiments the solution is perturbed rapidly, and one observes the approach to a new equilibrium. This method is suitable for measuring rate constants and overall behavior, but it does not reveal subreactions which may be too shortlived. By contrast, when the heating takes place as slowly as possible the reaction proceeds close to equilibrium and reveals a number of different steps. 24 The transitions are revealed by cross-correlating different parts of the scattering pattern (Fig. 7) and analyzing the structural changes by difference plots (Fig. 6). For example, ring dissolution takes place in two stages, the stage of nucleation is characterized by the presence of oligomers smaller than rings, there is a pronounced hysteresis between the assembly and disassembly branches during a complete cycle (Fig. 8), and the process of ring reformation after returning to low temperature is separate from microtubule breakdown. Assembly by Rapid Mixing. A common argument against T-jump studies in vitro is that living cells do not change their temperature when forming microtubules. We therfore polymerized microtubule protein prewarmed to 37 ° by rapid mixing with GTP) 7 The changes in the X-ray patterns were similar to those observed after a T-jump, i.e., initial breakdown of rings and nucleation characterized by oligomers. This suggests that a similar pathway of assembly can be induced in various ways as long as the conditions for starting the reaction are the same (37°, GTP). Differences between the experiments are found mainly in the relative rates and magnitudes of the subreactions. This result justifies the use of T-jumps as the most convenient method for perturbing the chemical equilibrium.

[60]

X-RAY SCATTERING OF MICROTUBULE ASSEMBLY USING S R

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FIG. 8. Temperature dependence of the central scatter during a slow temperature scan (1-2°/min). Letters indicate transitions between subreactions of the overall process. Note prenucleation events (A-C), nucleation (C-D), elongation (D-EL and postassembly events (E-F), and pronounced hysteresis. From Bordas and Mandelkow. tt

Kinetic Overshoot. By varying the initial conditions it is possible to initiate rapid assembly after a T-jump to 37°, followed by transient disassembly and reassembly at constant temperature. The structures formed during the overshoot are microtubules as well as a variable fraction of polymorphic aggregates; at the final equilibrium one finds mainly microtubules. 36 The extent of the overshoot appears to be controlled largely by the distribution of dimers and oligomers present at the onset of assembly. Implications for Microtubule Assembly. The results from the X-ray experiments have to be interpreted in conjunction with other structural approaches, in particular electron microscopy and image reconstruction of negatively stained or frozen-hydrated specimens) 7 Some of the points relevant to an understanding of the assembly process are summarized below. X-Ray scattering distinguishes between different assembly states of tubulin. It is sensitive to transitions that are difficult to detect by light scattering (Figs. 1 and 3). Microtubule assembly consists of several stages: prenucleation events, nucleation, elongation, and postassembly events (Fig. 8). The tubulin rings observed at low temperature are not nucleation centers but rather storage forms of the protein (Fig. 4). 36 E. Mandelkow, E.-M. Mandelkow, and J. Bordas, Trends Biochem. Sci. 8, 374 (1983). 37 E.-M. Mandelkow and E. Mandelkow, J. Mol. Biol. 181, 123 (1985).

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ANALYSIS OF CYTOSKELETAL PROTEINS

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The rings disintegrate into smaller units (tubulin dimers and oligomers) prior to microtubule assembly (prenucleation events, Fig. 6). Rings may be viewed as coiled protofilaments, and thus the oligomeric breakdown products of rings constitute short stretches of protofilaments. Oligomers play an important role in nucleation and may contribute to elongation as well. Assembly at constant temperature (37°, addition of GTP) shows similar phases to temperature-induced assembly. Assembly of purified tubulin differs from that of microtubule protein mainly by the absence of the initial ring breakdown (rings are stabilized by MAPs and are therefore absent from pure tubulin in normal buffer conditions). Nucleation is characterized by lateral association of (protofilamentous) oligomers, whereas elongation consists of addition of dimers and/or oligomers to the prototilaments. Thus both nucleation and elongation are nonhelical (two-dimensional assembly). The relative rates of lateral association and elongation can be varied, depending on solution and initial conditions. This generates the polymorphism of tubulin assembly. The polymorphic forms are overshoot aggregates rather than assembly intermediates. The experiments illustrated above were obtained with a time resolution of a few seconds, spatial resolution between 3 and 60 nm, and protein concentrations around 10 mg/ml or more. Each of these parameters can be varied, but an improvement in one factor will usually be at the expense of another. For example, subunit rearrangements and conformational changes require higher spatial resolution. The concomitant loss of time resolution may be offset by higher X-ray intensities or more efficient detection. Conversely, interactions between microtubules (e.g., crosslinking or bundling) and between microtubules and MAPs can be observed at lower spatial resolution and/or lower protein concentrations. Comprises between these constraints have to be found, depending on the problem under study.

Assembly of Other Proteins in Solution Most of the studies make use of the fact that SR provides time resolutions in a range of spatial resolutions not accessible to visible or UV light. We mention a few typical examples. Aspartate Transcarbamylase. In a stopped flow experiment the dissociation of this oligomeric enzyme into its subunits was studied with a time resolution of 1 sec. 3~

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Hemoglobin. In this study the reassociation of CO-liganded hemoglobin subunits into tetramers was observed, 38 using the same stopped-flow device as above. Chromatin. The salt-induced condensation of chicken erythrocyte chromatin was measured in the presence or absence of histone H5. It was found that a 30 nm superstructure was already present in uncondensed nucleofilaments, explaining their rapid condensation upon increasing the ionic strength. 39-41 Actin. The assembly of filaments from solutions of actin or actinprofilin complexes was induced by T-jump or rapid mixing with salts. Changes in the degree of polymerization were visible almost instantaneously, in contrast to the results from viscosimetry which show an initial lag period. 42 Collagen. The formation of collagen fibrils in solution was induced by temperature jump. The X-ray scattering increased immediately, whereas corresponding light scattering experiments showed a pronounced lag. This suggests that there are oligomeric assembly intermediates (not visible by light scattering) prior to the accretion and cross-linking of the fibrils .43 Membranes

The distribution of proteins in biological membranes can be studied by packing them into parallel layers and observing their meridional diffraction. Reactions are induced by changing the solution conditions. An example is the study of the sarcoplasmic reticulum membrane. 44 Its Ca 2÷ uptake was stimulated by ATP generated in situ from caged-ATP by laser flash photolysis. The time resolution was below I sec. Tissues Muscle Fibers. Because of the high degree of order the diffraction patterns from muscle are ideally suited for time-resolved X-ray experi3s y . Inoko, H. Kihara, and M. Koch, Biophys. Chem. 17, 171 (1983). 39 L. Perez-Grau, J. Bordas, and M. H. J. Koch, Nucleic Acids Res. 12, 2987 (1984). 4o j. Bordas, L. Perez-Grau, M. H. J. Koch, M. C. Vega, and C. Nave, Eur. Biophys. J. 13, 157 (1986). 4~ j. Bordas, L. Perez-Grau, M. H. J. Koch, M. C. Vega, and C. Nave, (1985b). Eur. Biophys. J. 13, 175 (1986). 42 Z. Sayers, M. H. J. Koch, J. Bordas, and U. Lindberg, Eur. Biophys. J. 13, 99 (1985). 43 G. Suarez, A. L. Oronsky, J. Bordas, and M. H. J. Koch, Proc. Natl. Acad. Sci. U.S.A. 82, 4693 (1985). J. K. Blasie, L. Herbette, D. Pierce, D. Pascolini, A. Scarpa, and S. Fleischer, Ann. N. Y. Acad. Sci. 402, 478 (1982).

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ments (this was exploited by Huxley and Brown 45 even before SR became available to biologists). The contraction of striated muscle was investigated in a series of studies, using millisecond time resolution. 29,46-4s The aim is to correlate the mechanical events (e.g., tension) with molecular interactions, e.g., between myosin cross-bridges and actin filaments, and their dependence on nucleotides. A similar approach can be used to study the contraction of smooth muscle. It is less well ordered but also slower so that time resolutions of seconds are sufficient. 49 Collagen-Containing Tissue. The studies concerned with these samples were not always time resolved in the strict sense. However, SR was necessary to obtain X-ray patterns quickly in order to cover a range of different states (induced, for example, by stretching) within a short time. Examples of studies on tendons are those of Bordas et al. 5° or Nemetschek et al.,S1 using the sharp meridional reflections to monitor the interactions between subfibrils. In contrast to tendons the fibrils of the cornea are randomly oriented. Thus the reflections are distributed around circles but occur at similar spacings. In addition there are changes in intensity that are related to the transparency of the lens. 52 This survey illustrates the rapid increase of time-resolved X-ray studies with SR in recent years. We have not dealt with other SR experiments which make use of the continuous wavelength distribution (e.g., for EXAFS, anomalous scattering), time structure (fluorescence decay), or imaging capabilities (X-ray microscopy). A similar increase of applications may be expected in these areas. Acknowledgment We are grateful to our colleagues M. Koch, J. Hendrix, E. Dorrington, and R. Klaering (EMBL Hamburg) as well as W. Renner and P. Maier (MPI Heidelberg) for their help in many aspects of the experiments described here. Special thanks are due to E.-M. Mandelkow for her continuous engagement throughout this project.

45 H. E. Huxley and W. Brown, J. Mol. Biol. 30, 383 (1967). H. E. Huxley, R. M. Simmons, A. R. Faruqi, M. Kress, J. Bordas, and M. H. J. Koch, Proc. Natl. Acad. Sci. U.S.A. 78, 2297 (1981). 47 H. E. Huxley, A. R. Faruqi, M. Kress, J. Bordas, and M. H. J. Koch, J. Mol. Biol. 158, 637 (1982). H. E. Huxley, R. M. Simmons, A. R. Faruqi, M. Kress, J. Bordas, and M. H. J. Koch, J. Mol. Biol. 169, 469 (1983). 49 j. Lowy and F. R. Poulsen, Nature (London) 299, 308 (1982). 5o j. Bordas, I. H. Munro, and A. M. Glazer, Nature (London) 262, 541 (1976). 5t T. Nemetschek, K. Jelinek, E. Knrrzer, E. Mosler, H. Nemetschek-Gansler, H. Riedl, and V. Schilling, J. Mol. Biol. 167, 461 (1983). 52 K. Meek, G. Eiliott, Z. Sayers, S. Whitburn, and M. Koch, J. Mol. Biol. 149, 477 (1981).