Supplementary Data s20

Supplementary data

Appendix I.

In order to ascertain the confidence of fitted parameters, an experiment with actin and α- actinin complex were polymerised with 0.65mM Mg2+. The results were obtained on four experiments on aliquots of the same preparations.

1 . 1 N o r m a l i s e d f l u o r e s c e n c e r e c o v e r y 1

B l u e : a c t i n ( 1 0 µ M ) 0 . 9 R e d : - a c t i n i n : a c t i n ( 0 . 9 : 1 ) B u f f e r G + M g 2 + 0 . 8

0 . 4 - 5 0 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 R e c o v e r y t i m e ( s e c ) Figure IIA shows the fluorescence recovery of actin (blue symbols) and α-actinin:actin complex (red symbols) measured in steady-state (1h after Mg2+ addition). Actin concentration is 10µM in both complexes, α-actinin concentration is 9µM. Error bars are standard errors obtained for each recovery moment. Like all experiments reported, in the article, fittings each recovery curve to two exponentials were obtained using Kaleidagraph 4.0 (Synergy Inc, USA). Determination of fHM, DHM, fLM and DLM are provided by the fitting as well as their fitting errors. Also displayed are the corresponding correlation values (R). Appendix II.

1.- Assuming that α-actinin has a molecular mass of about 105 (twice as much of that of actin) and that the G-actin: α-actinin complex is a sphere, the equivalent radius of such sphere would be of the order of 82Å.

5 MC = 1.5x10 4 MG = 4.3x10 3 MC/MG ≈ 3.5 = (rC/rG)  rC/rG = 1.5 Assuming a radius of 54Å for the actin monomer, the radius of the complex would be something close to 82Å

That would mean an expected difussion coefficient of D = 54/82 x 7.5x10-7cm2/s = 4.9x10-7cm2/s, too fast to be accounted in curves like those in Fig 2.

2.- Assuming that the complex is an exaggerated linear combination of α-actinin as a rod of 40Å x 500Å, plus the globule of actin monomer 50Å x 50Å: A rod of ≈ 50Å x 550Å in total we can use the expression for rods (García de la Torre J, Bloomfield VA (1981) Hydrodynamic properties of complex, rigid, biological macromolecules: theory and applications. Quarterly Rev.Biophys. 14:81-139):

D = kT/(3L) ln(L/d), where  is the viscosity of the solvent (10-3 N/m2 x s), L and d are the length and diameter of the rod.

This expression gives a value of D = 1.878x10-7 cm2/s, which still is too fast to account for the fluorescence recoveries in Fig 2.