Chemical reaction rate coefficients and free energies are usually time-independent quantities. Protein folding in vitro is one such reaction with a fixed energy landscape. However, in the milieu of the cell, the energy landscape can be modulated in space and time by fluctuations in the intracellular environment such as cytoskeletal rearrangements, changes in biomolecule concentrations, and large scale cellular reorganization. We studied the time dependence of the folding landscape of a FRET-labeled enzyme, yeast phosphoglycerate kinase (PGK-FRET). Living U2OS cells served as our test tube, and the mammalian cell cycle, a process strictly regulated in time, served as our clock. We found that both the rate of folding and the thermodynamic stability of PGK-FRET are cell cycle-dependent. We also assayed folding rates of PGK-FRET in spatial proximity to and far away from mitotic chromosomes. Our results show that expedited folding in DNA-rich regions cannot account for the faster rate of PGK-FRET folding in mitotic cells.

Chemical reactions are usually observed either by relaxation of a bulk sample after applying a sudden external perturbation, or by intrinsic fluctuations of a few molecules. Here we show that the two ideas can be combined to measure protein folding kinetics, either by periodic thermal modulation, or by creating artificial thermal noise that greatly exceeds natural thermal fluctuations. We study the folding reaction of the enzyme phosphoglycerate kinase driven by periodic temperature waveforms. As the temperature waveform unfolds and refolds the protein, its fluorescence color changes due to FRET (Förster resonant Energy Transfer) of two donor/acceptor fluorophores labeling the protein. We adapt a simple model of periodically driven kinetics that nicely fits the data at all temperatures and driving frequencies: The phase shifts of the periodic donor and acceptor fluorescence signals as a function of driving frequency reveal reaction rates. We also drive the reaction with stochastic temperature waveforms that produce thermal fluctuations much greater than natural fluctuations in the bulk. Such artificial thermal noise allows the recovery of weak underlying signals due to protein folding kinetics. This opens up the possibility for future detection of a stochastic resonance for protein folding subject to noise with controllable amplitude.

Biological systems are often studied under the most "physiological" conditions possible. However, purposeful perturbation of biological systems can provide much information about their dynamics, robustness, and function. Such perturbations are particularly easy to apply at the interface of molecular biophysics and cellular biology, at which complex and highly regulated networks emerge from the behavior of individual biomolecules. Due to the size of diffusion coefficients and the length scale of biomolecules, the fastest timescales at this interface extend to below a microsecond. Thus perturbations must be induced and detected rapidly. We focus on examples of proteins and RNAs interacting with themselves (folding) or one another (binding, signaling). Beginning with general principles that have been learned from simple models and perturbation experiments in vitro, we progress to more complex environments that mimic aspects of the living cell, and finally rapid perturbation experiments in living cells. On the experimental side we highlight in particular two classes of rapid perturbation methods (nanoseconds to seconds) that have been traditionally employed in biophysical chemistry, but that will become increasingly important in cell biology and in vivo: fast relaxation techniques and phase-sensitive modulation techniques. These techniques are now increasingly married with imaging to produce a spatiotemporal map of biomolecular stability, dynamics and, in the near future, interaction networks inside cells. Many important chemical processes occur on biologically fast timescales, and yet have important ramifications for slower biological networks.

Fast and reliable incision closure is critical in any surgical intervention. Common solutions are sutures and clips or adhesives, but they all present difficulties. These difficulties are especially pronounced in classical and robot-assisted minimally-invasive interventions. Laser soldering methods present a promising alternative, but their reproducibility is limited. We present a system that combines a previously reported laser soldering system with a robotic system, and demonstrate its feasibility on the incision-closure of ex-vivo mice skins. In this demonstration, we measured tearing forces of ~2.5N, 73% of the tearing force of a mouse skin without an incision. This robot-assisted laser soldering technique has the potential to make laser tissue soldering more reproducible and revolutionize surgical tissue bonding.