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Byline: Gerard C.L. Wong, Materials Science and Engineering Department and Physics Department, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801; email: gclwong@uiuc.edu; Lois Pollack, School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853; email: lp26@cornell.edu Keywords: DNA, RNA, F-actin Author Notes: Present address: Department of Bioengineering, University of California at Los Angeles, Los Angeles, California 90095-1600; email: gclwong@seas.ucla.edu Abstract Charges on biological polymers in physiologically relevant solution conditions are strongly screened by water and salt solutions containing counter-ions. However, the entropy of these counterions can result in surprisingly strong interactions between charged objects in water despite short screening lengths, via coupling between osmotic and electrostatic interactions. Widespread work in theory, experiment, and computation has been carried out to gain a fundamental understanding of the rich, yet sometimes counterintuitive, behavior of these polyelectrolyte systems. Examples of polyelectrolyte association in biology include DNA packaging and RNA folding, as well as aggregation and self-organization phenomena in different disease states.

A microfluidic mixer is applied to study the kinetics of calmodulin conformational changes upon Ca2+ binding. The device facilitates rapid, uniform mixing by decoupling hydrodynamic focusing from diffusive mixing and accesses time scales of tens of microseconds. The mixer is used in conjunction with multiphoton microscopy to examine the fast [Ca.sup.2+]-induced transitions of acrylodan-labeled calmodulin. We find that the kinetic rates of the conformational changes in two homologous globular domains differ by more than an order of magnitude. The characteristic time constants are [approximately equal to] 490 [micro]s for the transitions in the C-terminal domain and [approximately equal to] 20 ms for those in the N-terminal domain of the protein. We discuss possible mechanisms for the two distinct events and the biological role of the stable intermediate, half-saturated calmodulin. hydrodynamic focusing | multiphoton microscopy | fluorescence correlation spectroscopy

We describe a microfluidic mixer that is well-suited for kinetic studies of macromolecular conformational change under a broad range of experimental conditions. The mixer exploits hydrodynamic focusing to create a thin jet containing the macromolecules of interest. Kinetic reactions are triggered by molecular diffusion into the jet from adjacent flow layers. The ultimate time resolution of these devices can be restricted by premature contact between co-flowing solutions during the focusing process. Here, we describe the design and characterization of a mixer in which hydrodynamic focusing is decoupled from the diffusion of reactants, so that the focusing region is free from undesirable contact between the reactants. Uniform mixing on the microsecond time scale is demonstrated using a device fabricated by imprinting optical-grade plastic. Device characterization is carded out using fluorescence correlation spectroscopy (FCS) and two-photon microscopy to measure flow speeds and to quantify diffusive mixing by monitoring the collisional fluorescence quenching, respectively. Criteria for achieving microsecond time resolution are described and modeled.

We have used small angle x-ray scattering and computer simulations with a coarse-grained model to provide a time-resolved picture of the global folding process of the Tetrahymena group I RNA over a time window of more than five orders of magnitude. A substantial phase of compaction is observed on the low millisecond timescale, and the overall compaction and global shape changes are largely complete within one second, earlier than any known tertiary contacts are formed. This finding indicates that the RNA forms a nonspecifically collapsed intermediate and then searches for its tertiary contacts within a highly restricted subset of conformational space. The collapsed intermediate early in folding of this RNA is grossly akin to molten globule intermediates in protein folding.

Time-resolved small-angle x-ray scattering was used to measure the radius of gyration of cytochrome c after initiation of folding by a pH jump. Submillisecond time resolution was obtained with a microfabricated diffusional mixer and synchrotron radiation. The results show that the protein first collapses to compact denatured structures before folding very fast to the native state.

Anomalous small-angle X-ray scattering (ASAXS) has provided information about the spatial distribution of ions. An extension of ASAXS is provided that can be used for counting the number of ions around nucleic acids.

Photoexcitation is coupled with time-resolved small-angle X-ray scattering (SAXS) in order to monitor conformational changes accompanying light activation of the blue-light signal transduction protein Vivid (VVD). The application of a continuous flow cell to couple SAXS and photoexcitation has elucidated millisecond conformational changes without signal averaging by photocycle repetition, thus avoiding the sample damage associated with repeat exposures and facilitating measurement of samples with slow photocycles.