Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease.

ClpXP is an ATP-fueled molecular machine that unfolds and degrades target proteins. ClpX, an AAA+ enzyme, recognizes specific proteins, and then uses cycles of ATP hydrolysis to denature any native structure and to translocate the unfolded polypeptide into ClpP for degradation. Here, we develop and apply single-molecule fluorescence assays to probe the kinetics of protein denaturation and degradation by ClpXP. These assays employ a single-chain variant of the ClpX hexamer, linked via a single biotin to a streptavidin-coated surface, and fusion substrates with an N-terminal fluorophore and a C-terminal GFP-titin-ssrA module. In the presence of adenosine 5'-[gamma-thio]triphosphate (ATPgammaS), ClpXP degrades the titin-ssrA portion of these substrates but stalls when it encounters GFP. Exchange into ATP then allows synchronous resumption of denaturation and degradation of GFP and any downstream domains. GFP unfolding can be monitored directly, because intrinsic fluorescence is quenched by denaturation. The time required for complete degradation coincides with loss of the substrate fluorophore from the protease complex. Fitting single-molecule data for a set of related substrates provides time constants for ClpX unfolding, translocation, and a terminal step that may involve product release. Comparison of these single-molecule results with kinetics measured in bulk solution indicates similar levels of microscopic and macroscopic ClpXP activity. These results support a stochastic engagement/unfolding mechanism that ultimately results in highly processive degradation and set the stage for more detailed single-molecule studies of machine function.

Force of an actin spring.

Cellular movements are produced by forces. Typically, cytoskeletal proteins such as microtubules and actin filaments generate forces via polymerization or in conjunction with molecular motors. However, the fertilization of a Limulus polyphemus egg involves a third type of actin-based cellular engine--a biological spring. During the acrosome reaction, a 60-microm long coiled and twisted bundle of actin filaments straightens and extends from a sperm cell, penetrating the vitelline layer surrounding the egg. A subtle overtwist of 0.2 degrees /subunit underlies the mechanochemical basis for the extension of this actin spring. Upon calcium activation, this conformational strain energy is converted to mechanical work, generating the force required to extend the bundle through the vitelline layer. In this article, we stall the extension of the acrosome bundle in agarose gels of different concentrations. From the stall forces, we estimate a maximum force of 2 nN and a puncturing pressure of 1.6 MPa. We show the maximum force of extension is three times larger than the force required to puncture the vitelline layer. Thus, the elastic strain energy stored in the acrosome bundle is more than sufficient to power the acrosome reaction through the egg envelope.

Geometric confinement influences cellular mechanical properties II -- intracellular variances in polarized cells.

During migration, asymmetrically polarized cells achieve motion by coordinating the protrusion and retraction of their leading and trailing edges, respectively. Although it is well known that local changes in the dynamics of actin cytoskeleton remodeling drive these processes, neither the cytoskeletal rheological properties of these migrating cells are well quantified nor is it understand how these rheological properties are regulated by underlying molecular processes. In this report, we have used soft lithography to create morphologically polarized cells in order to examine rheological differences between the front and rear zone of an NIH 3T3 cell posed for migration. In addition, we trapped superparamagnetic beads with optical tweezers and precisely placed them at specific locations on the immobilized cells. The beads were then allowed to endocytose overnight before magnetic tweezers experiments were performed to measure the local rheological properties of the leading and trailing edges. Our results indicate that the leading edge has an approximately 1.9 times higher shear modulus than the trailing edge and that this increase in shear modulus correlates with a greater density of filamentous actin, as measured by phalloidin-staining observed through quantitative 3D microscopy.

Single M13 bacteriophage tethering and stretching.

The ability to present biomolecules on the highly organized structure of M13 filamentous bacteriophage is a unique advantage. Where previously this viral template was shown to direct the orientation and nucleation of nanocrystals and materials, here we apply it in the context of single-molecule (SM) biophysics. Genetically engineered constructs were used to display different reactive species at each of the filament ends and along the major capsid, and the resulting hetero-functional particles were shown to consistently tether microscopic beads in solution. With this system, we report the development of a SM assay based on M13 bacteriophage. We also report the quantitative characterization of the biopolymer's elasticity by using an optical trap with nanometer-scale position resolution. Expanding the fluctuating rod limit of the wormlike chain to incorporate enthalpic polymer stretching yielded a model capable of accurately capturing the full range of extensions. Fits of the force-extension measurements gave a mean persistence length of approximately 1,265 nm, lending SM support for a shorter filamentous bacteriophage persistence length than previously thought. Furthermore, a predicted stretching modulus roughly two times that of dsDNA, coupled with the system's linkage versatility and load-bearing capability, makes the M13 template an attractive candidate for use in tethered bead architectures.

Interlaced optical force-fluorescence measurements for single molecule biophysics.

Combining optical tweezers with single molecule fluorescence offers a powerful technique to study the biophysical properties of single proteins and molecules. However, such integration into a combined, coincident arrangement has been severely limited by the dramatic reduction in fluorescence longevity of common dyes under simultaneous exposure to trapping and fluorescence excitation beams. We present a novel approach to overcome this problem by alternately modulating the optical trap and excitation beams to prevent simultaneous exposure of the fluorescent dye. We demonstrate the dramatic reduction of trap-induced photobleaching effects on the common single molecule fluorescence dye Cy3, which is highly susceptible to this destructive pathway. The extension in characteristic fluorophore longevity, a 20-fold improvement when compared to simultaneous exposure to both beams, prolongs the fluorescence emission to several tens of seconds in a combined, coincident arrangement. Furthermore, we show that this scheme, interlaced optical force-fluorescence, does not compromise the trap stiffness or single molecule fluorescence sensitivity at sufficiently high modulation frequencies. Such improvement permits the simultaneous measurement of the mechanical state of a system with optical tweezers and the localization of molecular changes with single molecule fluorescence, as demonstrated by mechanically unzipping a 15-basepair DNA segment labeled with Cy3.

Excitation nonlinearities in emission reabsorption laser-induced fluorescence techniques.

The effects of the nonlinear behavior of fluorescent intensity with excitation intensity on emission reabsorption laser-induced fluorescence (ERLIF) are investigated. Excitation nonlinearities arise mainly as a consequence of the depletion of the ground-state population stemming from the finite lifetime of molecules in the excited state. These nonlinearities hinder proper suppression of the excitation intensity information in the fluorescence ratio, degrading measurement accuracy. A method for minimizing this effect is presented. This method is based on the approximation of the fluorescence intensity nonlinearities by a power law. Elevating the two-dimensional fluorescent intensity maps to the appropriate exponent allows for proper suppression of excitation intensity in the fluorescence ratio. An overview of the principles and constitutive equations behind ERLIF film-thickness measurements, along with a characterization of the fluorescence's nonlinear behavior, is presented. The power law approximation and processing scheme used to mitigate this behavior are introduced. Experimental proof of the validity of the approximation and processing scheme is provided.