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L. Pollack1, M.W. Tate1, N.C. Darnton2, J.B. Knight2, S.M. Gruner1, W.A.Eaton3and R.H. Austin2
1Laboratory of Atomic and Solid State Physics, Cornell University
2Physics Department, Princeton University
3Laboratory of Chemical Physics, National Institutes of Health, Bethesda MD
[Proc. Nat. Acad. Sci. in press]

Many questions are now being asked about the dynamics of large protein molecules, including how they fold and move. The stability and folding speed of a protein depend on the structures of the denatured as well as the native state. Computer simulations suggest that slow folding amino acid sequences collapse to compact structures with non-native topologies before folding, while faster counterparts both collapse and fold simultaneously.

To address questions about protein dynamics, a new technique has been developed to aide measuring sub-millisecond time-dependent changes in the conformation of large protein molecules. Proteins can be forced to change conformational state using a solvent exchange technique, during which proteins are subject to an abrupt change in the acidity of their buffer solution. To achieve both rapid mixing and allow x-ray measurements to study time-dependent changes, this group has micro-fabricated a tiny 100-micron by 390-micron deep mixing device onto a silicon wafer using the Cornell Nanofabrication Facility. Small-angle x-ray scattering measurements inside the flow cell monitor the radius of gyration of the macromolecules as a function of time before, during and after solvent exchange. A recent experiment studied cytochrome-C as it folds and unfolds in response to pH changes occurring at intermediate time intervals of 150-500 microseconds. The results showed that the protein first collapses to compact denatured structures before folding very quickly to the native state.


Schematic of the nanofabricated flow cell used to measure protein folding. The x-ray beam is moved along the output flow to measure the protein shape at a variety of time intervals after mixing.