Dynamic light scattering studies of .alpha.IIb.beta.3 solution conformation

Abstract

The prototypical integrin receptor, alpha IIb beta 3, isolated from the membrane fraction of human blood platelets by solubilization in Triton X-100 (reduced) and affinity chromatography on lentil lectin-agarose, has been further purified by gel filtration chromatography in octyl glucoside to obtain the intact receptor complex in a form suitable for hydrodynamic measurements. The molecular weight [(6.0 +/- 0.2) x 10(3)] and Stokes radius (2.3 +/- 0.1 nm) of detergent micelles formed in 0.03 M octyl glucoside have been determined by classical light scattering intensity and dynamic light scattering measurements, respectively. An algorithm has been developed which explicitly considers the contribution of detergent micelles to the intensity autocorrelation function of particles suspended in detergent. This procedure has been validated with polystyrene particles of known radius, as well as with the soluble protein fibrinogen. Application of these procedures to dynamic light scattering data obtained with alpha IIb beta 3 resulted in a translational diffusion coefficient (Dto(20,w)) of (2.78 +/- 0.31) x 10(-7) cm2 s-1, corresponding to a Strokes radius (Rs) of 7.67 +/- 0.85 nm for the integrin/octyl glucoside complex. Light scattering intensity measurements gave a molecular weight of (2.26 +/- 0.22) x 10(5) for the polypeptide moiety of the complex, in excellent agreement with the 2.28 x 10(5) value calculated from primary structure data. As a spherical, hydrated alpha IIb beta 3 complex, with bound detergent, would exhibit a Stokes radius of approximately 5 nm, these data indicate considerable asymmetry in the solution conformation of alpha IIb beta 3.

Modeling the αIIbβ3 integrin solution conformation

“…These calculated values are in significantly better agreement with the experimental value, R, = 7.7 k 0.8 nm (Hantgan et al, 1993), obtained by dynamic light scattering with a I l b @ 3 in octyl glucoside. However, the dynamic light scattering data reported in our previous study (Hantgan et al, 1993) exhibited significant concentration dependence, indicative of ci1IbP3 self association. In fact, extending the concentration range of the dynamic light-scattering measurements to 0.28 mg/mL a l I b @ 3 yielded R, = 6.75 f 0.17 nm (this work).…”

“…Further, because experimental solution data have been obtained in two different detergents, octyl glucoside (Hantgan et al, 1993) and Triton X-100 (Jennings & Phillips, 1982;Rivas et al, 1991), models were constructed using both types of micelles. To avoid unnecessary complications not warranted by the resolution of the electron micrographs, the cytoplasmic portions of each q , b & subunit were included within the terminal bead(s).…”

“…To avoid unnecessary complications not warranted by the resolution of the electron micrographs, the cytoplasmic portions of each q , b & subunit were included within the terminal bead(s). The radii of these beads were therefore calculated by summing the hydrated volumes of the transmembrane and cytoplasmic portions of both chains to the experimentally determined volumes of the detergent micelles (Hantgan et al, 1993) (Table 1).…”

“…The anhydrous volume of the transmembrane residues was added to the octyl glucoside micelle volume, calculated from its experimentally determined radius of 2.3 nm (Hantgan et al, 1993), and a new radius was then calculated for this bead (Table 3). It is interesting to note that the resulting diameter of this bead, -4.9 nm, is fully consistent with the average thickness of the plasma membrane, 4-5 nm (Alberts et al, 1989), albeit somewhat larger (10-20%) than the 3.9-4.4-nm calculated length for the putative helical transmembrane segments of the a and subunits.…”

“…Models were built by representing each polypeptide chain as an array of nonoverlapping interconnected spheres; the hydrodynamic theory for multisubunit particles (Garcia de la Torre & Bloomfield, 198 1 ; Garcia de la Torre, 1989) has been utilized to calculate the Stokes radius (R,) and sedimentation coefficient (s&+,) for each model, for comparison with experimental data (Jennings & Phillips, 1982;Rivas et al, 1991;Hantgan et al, 1993). These calculations include explicit consideration of the contributions of the detergent micelles (either Triton X-100 or octyl glucoside) required for solubilization of the isolated integrin.…”