Conformational Properties of Microtubule

Process in vitro Protein: Their Relation to the Self-Assembly

PETER M. BAYLEY, DAVID C. CLARK, and STEPHEN R. MARTIN, Biophysics Diuision, National Insti tute for Medical

Research, Mill Hill, London NW7 l A A , United Kingdom

Synopsis

Near- and far-uv CD spectra of microtubule protein preparations have been examined to study the possible role of protein conformation in relation to the kinetics of the self-assembly of these proteins into microtubules in uitro. Although tubulin can form conformations with high helical content under apolar solution conditions, this transformation is apparently not involved in self-assembly. There is no major perturbation of tubulin near-uv CD by reagents and solution conditions favoring assembly. Thus, in these preparations, tubulin, as dimer and as oligomer with MAPS, is effectively in the conformation in which it undergoes self- assembly. This conclusion is consistent with a hybrid model of assembly of microtubule protein involving direct incorporation of oligomeric species as an alternative to the conden- sation polymerization of tubulin dimer as the exclusive assembly mechanism.

INTRODUCTION

The ability to perform assembly of microtubules in uitro allows exami- nation of the effects on microtubule (MT) proteins of reagents and solution conditions which favor assembly. In the disassembled state, the prepa- rations contain tubulin dimer and specific oligomeric forms-tubulin plus microtubule associated proteins (MAPS)-whose relative proportions and hydrodynamic characteristics depend on MAP content, and solution con- ditions such as pH, buffer ion, ionic strength, solvent, and temperature.lj2 The observed “dynamic equilibrium” between tubulin and the oligomers has been taken to imply the dissociation of oligomers prior to the assembly process, which would then proceed according to the polymerization-con- densation mechanism of Oosawa, as postulated for the self-assembly of tubulin dimer either alone3 or seeded with nucleating materia1.l

The question of the conformation and stability of tubulin and the oli- gomeric species is central to the problem of the mechanism of MT protein self-assembly in uitro. Near-uv CD has shown the stability of the 30-S oligomer in glycerol-cycled MT protein under pH and buffer conditions favoring a~sembly.~ We have therefore extended the original observations to MT proteins (both porcine and bovine) prepared in the absence of glycerol and have examined (a) the secondary structure of tubulin under

Biopolymers, Vol. 22,87-91 (1983) 0 1983 John Wiley & Sons, Inc. CCC 0006-3525/83/010087-05$01.50

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BAYLEY, CLARK, AND MARTIN

A€ W-MT protein Multicomponent

0.095 rng/ml Analysis

0

-2

-4

-6' ' ' " I '

200 nm 2 7 0

P r t

.%

31 16

25 9 16 10

3 18 1 2

ap

30

39 27

27

31 -

Fig. 1. Far-uv CD of W-MT protein at 15°C as a function of trifluoroethanol concentration, and the corresponding multicomponent analysis ( r t = reverse turn; ap = aperiodic).

different solvent conditions, (b) the concentration and pH stability of MT protein, and (c) the kinetics of self-assembly of MT protein.

MATERIALS AND METHODS

Microtubule protein was prepared by assembly-disassembly procedures.2 We designate the glycerol-cycled bovine material (Shelanski) as S-MT protein, the glycerol-free bovine material (Wilson) as W-MT protein, and the glycerol-free porcine material (Borisy) as B-MT protein. These preparations contain tubulin plus MAPS (both HMW and tau-group pro- teins), the total MAP content being approximately 10-15% (S-MT protein) and 20-25% (W-MT and B-MT protein).

CD was recorded on the Jasco J41C spectropolarimeter, with data transfer to a PDP 11-23 computer for graphics output. Multicomponent analysis was performed (off-line on the DEC-20 computer) using the pro- cedure of P r~venche r .~ Kinetics of assembly were recorded on the Cary 118 spectrophotometer using absorbance a t 330 nm. Protein solutions in standard buffer (O.1M Mes, 0.5 mM Mg2+, 0.1 mM EGTA) plus 1 mM GTP initially at 15°C in a jacketed l-cm cylindrical cell were assembled at 37°C with half-life for the temperature change of 12 s. Absorbance data were digitized and analyzed by nonlinear least-squares routines as single- or double-exponential decays.

RESULTS AND DISCUSSION

Figure 1 shows the continous intensification in far-uv CD of MT protein up to 85% (v/v) TFE and the results of multicomponent analysis. The a-helical content increases progressively from 23 to 66%, an unusually large degree of perturbation for a globular protein of this size (Mr = 100,000). Control experiments with purified tubulin dimer indicated that this in- tensification was due to the dimer; a preparation of MAPS (HMW and tau) showed little helix and was unaffected by TFE. Conformational predic-

CONFORMATION OF MICROTUBULE PROTEIN 89

2.54 nm 320 250 nm m Fig. 2. Near-uv CD: pH and concentration dependence for W-MT protein (left: A, 4.0;

T = A, 0.2 mg/mL; pH 6.5) and B-MT protein (right: A, 5.4; A, 0.27 mg/mL; pH 6.95). 15°C.

tions6 indicate (for both a - and 0-tubulin sequences7) several regions with similar propensities for a-helix and P-sheet conformations, and it appears likely that the presence of TFE increases the relative probability of these a-helical regions. This transformation is not known to have any role in self-assembly in aqueous solution in uitro but might, in principle, indicate additional stable conformations that could be attained in less polar envi- ronments, possibly in association with membranes in uiuo.

The near-uv CD spectrum of tubulin shows intensification in the pres- ence of MAPS, correlating with the presence of 30-S oligomeric species. These spectra are shown for the glycerol-free MT protein preparations in Fig. 2. The sensitivity to dilution is pH dependent, as found for S-MT p r ~ t e i n . ~ At the lower pH range (6.51, there is little tendency to dissociate, reflecting the intrinsic stability of oligomeric species under these conditions of pH and concentration where efficient temperature-induced assembly is observed (see below). These findings eliminate the use of glycerol in the isolation procedure as a factor affecting the behavior of the oligomeric species for all these MT protein preparations.

Among other factors that enhance assembly, neither dimethyl sulfoxide at 10% ( V / V ) ~ nor glycerol (4M)9 produce a significant change in tubulin near-uv CD. Likewise, varying nucleotide concentration (GTP 10-3M) does not affect the protein CD at 280 nm.4 The near-uv CD of S-MT protein remains constant (in the presence of glycerol or sucrose) for up to 90 min at 37°C under nonassembly conditions, indicating the absence of a temperature-induced conformational transition.

In uitro assembly of MT protein induced at 37°C (Fig. 3) is effectively a single exponential at pH 2 7.0; at pH 6.5, the kinetics are clearly biphasic. These observations explain the discrepancy in reports of biphasic kinetics for S-MT proteinlo compared with single-exponential kinetics for B-MT protein1 We have previously shown the importance of pH in determining

90 BAYLEY, CLARK, AND MARTIN

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5

4

3

2 0 time ( 5 ) 500 0 time (5) 500

Fig. 3. Self-assembly of S-MT protein a t 37OC-see details in text. Arrow denotes start of kinetic analysis. Conditions: (left) Af:A, = 1.4, k f = 0.030 s-l, k , = 0.004 s-l, pH 6.50; (right) Af:A, = 0.6, k f = 0.048 s-’, k , = 0.007 s-l, pH 6.95. dA = A , - A ( t ) ; fast and slow phase amplitudes.

the oligomeric composition of S-MT protein? as found for B-MT pr0tein.l There is a clear correlation between the presence of stable oligomer and the biphasic assembly process. The nature of the kinetics is determined by solution conditions, particularly pH.

In assessing the role of protein conformation in this assembly process, far-uv CD shows that the secondary structure of tubulin dimer undergoes a significant alteration under the influence of a major change in the polarity of the surrounding medium, but this is not implicated in the in vivo as- sembly either in aqueous solution or in the presence of glycerol. Near-uv CD also shows little significant alteration associated with reagents and solution conditions known to favor assembly. We conclude that the preparations of MT proteins contain tubulin (as dimer and oligomeric species) in a conformation closely similar to that in which it undergoes self-assembly. This process involves the fine balance of interactions be- tween protein molecules and the temperature sensitivity of the interactions between protein and solvent.9 The correlation of near-uv CD intensity with the presence of 30-S oligomer shows the stability of this species at pH 6.5 and indicates that the oligomeric forms of microtubule proteins must be considered as participants in assembly. The assembly mechanism must therefore include at least two pathways: (a) the addition of oligomeric forms (e.g., 30-S species or oligomers derived from it) giving rise to the fast phase of assembly and (b) the polymerization condensation of tubulin dimer onto the growing microtubule. The relative importance of the pathways in this “hybrid model” must be dependent on (i) solution conditions, par- ticularly pH, but also buffer composition and ionic strength, and (ii) the nature, proportions, and properties of the MAPS that result from a given experimental procedure.

We acknowledge the assistance of Dr. R. W. Woody with the structure prediction of tub- ulin.

CONFORMATION OF MICROTUBULE PROTEIN 91

References

1. Scheele, R. B. & Borisy, G. G. (1979) in Microtubules, Roberts, K. & Hyams, J. S., Eds.,

2. Bayley, P. M., Charlwood, P. A,, Clark, D. C. & Martin, S. R. (1982) Eur. J . Biochern.

3. Timasheff, S. N. & Grisham, L. M. (1980) Annu. Reu. Aiochern. 49,565-591. 4. Martin, S. R., Clark, D. C. & Bayley, P. M. (1982) Biochern. J . 203,643-652. 5. Provencher, S. W. & Glockner, J. (1981) Biochemisiry 20,33-37. 6. Chou, P. Y. & Fasman, G. D. (1978) Annu. Reu. Biochern. 47,251-276. 7. Krauhs, E., Little, M., Kempf, T., Hofer-Warbinek, R., Ade, W. & Ponstingl, H. (1981)

8. Himes, R. H., Burton, P. R. & Gaito, J. M. (1977) J . Bid. Chem. 252,6222-6228. 9. Na, G. C. & Timasheff, S. N. (1981) J . Mol. Biol. 151,165-178.

Academic Press, New York, pp. 175-254.

121,579-585.

Proc. Natl. Acad. Sci. USA 78,4156-4160.

10. Barton, J. S. & Riazi, G. H. (1980) Biochim. Biophys. Acta 630,392-401.

Received June 20,1982 Accepted August 30,1982