Lisa W. Kwok

Rapid compaction during RNA folding

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.

Conformational changes of calmodulin upon Ca2+ binding studied with a microfluidic mixer

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 Ca2+-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 ≈490 μs for the transitions in the C-terminal domain and ≈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.

Hinge stiffness is a barrier to RNA folding

Cation-mediated RNA folding from extended to compact, biologically active conformations relies on a temporal balance of forces. The Mg2 +-mediated folding of the Tetrahymena thermophila ribozyme is characterized by rapid nonspecific collapse followed by tertiary-contact-induced compaction. This article focuses on an autonomously folding portion of the Tetrahymena ribozyme, its P4-P6 domain, in order to probe one facet of the rapid collapse: chain flexibility. The time evolution of P4-P6 folding was followed by global and local measures as a function of Mg2 + concentration. While all concentrations of Mg2 + studied are sufficient to screen the charge on the helices, the rates of compaction and tertiary contact formation diverge as the concentration of Mg2 + increases; collapse is greatly accelerated by Mg2 +, while tertiary contact formation is not. These studies highlight the importance of chain stiffness to RNA folding; at 10 mM Mg2 +, a stiff hinge limits the rate of P4-P6 folding. At higher magnesium concentrations, the rate-limiting step shifts from hinge bending to tertiary contact formati

Mono- and trivalent ions around DNA: a small-angle scattering study of competition and interactions.

The presence of small numbers of multivalent ions in DNA-containing solutions results in strong attractive forces between DNA strands. Despite the biological importance of this interaction, e.g., DNA condensation, its physical origin remains elusive. We carried out a series of experiments to probe interactions between short DNA strands as small numbers of trivalent ions are included in a solution containing DNA and monovalent ions. Using resonant (anomalous) and nonresonant small angle x-ray scattering, we coordinated measurements of the number and distribution of each ion species around the DNA with the onset of attractive forces between DNA strands. DNA-DNA interactions occur as the number of trivalent ions increases. Surprisingly good agreement is found between data and size-corrected numerical Poisson-Boltzmann predictions of ion competition for non- and weakly interacting DNAs. We also obtained an estimate for the minimum number of trivalent ions needed to initiate DNA-DNA attraction.

Reconstructing three-dimensional shape envelopes from time-resolved small-angle X-ray scattering data

Modern computing power has made it possible to reconstruct low-resolution, three-dimensional shapes from solution small-angle X-ray scattering (SAXS) data on biomolecules without a priori knowledge of the structure. In conjunction with rapid mixing techniques, SAXS has been applied to time resolve conformational changes accompanying important biological processes, such as biomolecular folding. In response to the widespread interest in SAXS reconstructions, their value in conjunction with such time-resolved data has been examined. The group I intron from Tetrahymena thermophila and its P4-P6 subdomain are ideal model systems for investigation owing to extensive previous studies, including crystal structures. The goal of this paper is to assay the quality of reconstructions from time-resolved data given the sacrifice in signal-to-noise required to obtain sharp time resolution.

Focusing capillary optics for use in solution small-angle X-ray scattering

Measurements of the global conformation of macromolecules can be carried out using small-angle X-ray scattering (SAXS). Glass focusing capillaries, manufactured at the Cornell High Energy Synchrotron Source (CHESS), have been successfully employed for SAXS measurements on the heme protein cytochrome c. These capillaries provide high X-ray flux into a spot size of tens of micrometres, permitting short exposures of small-volume samples. Such a capability is ideal for use in conjunction with microfluidic mixers, where time resolution may be determined by beam size and sample volumes are kept small to facilitate mixing and conserve material.