Figure 3.

Hydrodynamic studies of the p27/Cdk2/cyclin A ternary complex. (a) Interference fringe displaced velocity data for p27/Cdk2/cyclin A were analyzed using the continuous sedimentation coefficient distribution, c (s), model with the c (s) distributions displayed in fringes/S versus sedimentation coefficient (s). Experiments were conducted at 20 °C at a rotor speed of 50,000 rpm and a starting protein concentration of 2 mg/ml (24 μM). The s-value of the complex was determined as 3.98 S with a best-fit weight-average frictional ratio (f/f₀)_w of 1.63 and a calculated molecular mass of 87,603 Da. This analysis was with regularization at a confidence level of p = 0.7 and at a resolution of sedimentation coefficients of n = 100. The peak at ~1.5 S marked “p27” corresponds to a small amount of unbound p27 and that marked “aggr.” corresponds to a small amount of unidentified protein aggregate. (b) Calculated sedimentation coefficients computed using the program HYDROPRO for p27/Cdk2/cyclin A structures from the MD trajectory. The red dotted line represents the standard value based the experimental s-value and the blue dotted line represents the average of the calculated s-values. (c) Contour plot of the two-dimensional c (s, f/f₀) distribution calculated using SEDFIT with an equidistant f/f₀ -grid from 1.0 to 2.2 with 0.1 steps, a linear s-grid from 1 to 8 S, a resolution of 100 s-values and regularization at one standard deviation. The differently colored contours represent c (s, f/f₀)-values from 0 fringes/S (white) to 1.0 fringes/S (red), with increasing color temperature indicating larger values.