Time-Resolved Small-Angle X-ray Scattering Studies of Macromolecular Dynamics

Lois Pollack

School of Applied & Engineering Physics, Cornell Univerity

Large biological molecules like proteins and RNA carry out their functions by folding to well-defined three-dimensional structures (illustrated in Figure 1). We are interested in the physical inter actions that direct this self-assembly process. By combining microfabricated flow cells (illustrated in Figure 2) with synchrotron small angle x-ray scattering, we have gained insight into the earliest steps of protein and RNA folding. A recent series of experiments indicates that the critical, first step of RNA folding is dominated by electrostatic interactions.

Figure 1 shows a cartoon of macro molecular folding. On the left, a denatured protein in a random coil conformation folds to its biologically active state. Denatured RNA (schematically shown second from right) possesses secondary structure. The addition of Mg (2 +) ions triggers tertiary structure formation. The Michel -Westhof model of the three dimensional structure of the Tetrahymenaibozyme is shown at the far right.

Figure 2 shows a cartoon of an RNA folding experiment. Folding is initiated with in the microfabricated flow cell by the rapid addition of small Mg 2 + ions. The RNA folds as it flows down the channel; the x-ray beam can be moved to probe the conformation at any position in the flow cell. Schematic conformations (see Russ ell et al., PNAS 99 (2002)) of the RNA are shown at different locations along the channel. Molecule a. is unfolded, molecules b. and c. are captured during folding.

The small angle scattering of an unfolded protein has a different angular dependence than that of a folded protein. To emphasize the changes, the scattering profiles are displayed as Kratky plots, where the intensity is multiplied by the square of the scatt ering angle. In this representation, the difference in scatter between unfolded and folded RNA are read ily app arent (Figure 3, bottom trace and top trace).

Using the continuous flow mixer, in conjunction with stopped flow and manual mixing, we can map out the entire course of folding of a large RNA, the Tetrahymenaribozyme, as shown in Figure 3. Singular value decomposition is used to provide a quantitative measure of folding progress by projecting each time resolved point onto a series of basis states. The relevant interactions are probed by monitori ng changes in the folding progression as a function of solution conditions or as the molecular sequence is varied.

Figure 3 demonstrates changes in Kratky plots as the RNA folds, from Russell et al., PNAS 99 (20 02).

Through a series of experiments to be described in the talk, we have found two phases of molecular compaction on the millisecond time scale. The most rapid compaction is largely independent of the presence of tertiary contacts in the molecule. On a time scale of tens of milliseconds, the tertiary contacts form and drive the molecule towards its final, folded state.This technique can be applied to study any solution-induced conformational change.

References:

RNA folding review articles:

P. B. Moore in ‘ The RNA World’ (1999).

Sosnick, T. and T. Pan (2003). " RNA folding: models and perspectives." Curr Opin. Struct Bi ol 13: 309-316.

Thirumalai, D., Lee, N., Woodson S., Klimov, D. K. (2001). " Early events in RNA folding." Annu Rev Phys. Chem 52: 751-62.

Tetrahymenaribozyme folding:

Treiber, D. K. and J. R. Williamson (2001). " Beyond kinetic traps in RNA folding." Curr. Opin Struct Biol 11(3 ): 309-314.

Time-resolved S AXS of Tetrahymenaribozyme folding:

Das, R., Kwok, L. W., Millett, I. S., Bai, Y., Mill s, T. T., Jacob, J., Mask el, G. S., Seifert, S., Mochrie, S. G.J., Thiyagarajan, P, Doniach, S., Pollack, L ., and Herschlag, D. (2003)." The fastest global events in RNA folding." J. Mol. Biol. 33 2(2): 31 1-31 9.

Russell, R., Millett, I. S ., Tate, M. W., Kwok, L. W., Nakatani, B., Gruner, S.M., Mochrie, S. G. J., Pande, V., Doniach, S., Herschlag, D., Pollack, L (2002). " Rapid Compaction During RNA Folding." Proc. Natl. Acad. Sci. U S A 99(7): 4266-4271.