Each step of the kinesin motor involves a force-generating molecular rearrangement. Although significant progress has been made in elucidating the broad features of the kinesin mechanochemical cycle, molecular details of the force generation mechanism remain a mystery. Recent molecular dynamics simulations have suggested a mechanism in which the forward drive is produced when the N-terminal cover strand forms a -sheet with the neck linker to yield the cover-neck bundle. We tested this proposal by comparing optical trapping motility measurements of cover strand mutants with the wild-type. Motility data, as well as kinetic analyses, revealed impairment of the force-generating capacity accompanied by a greater load dependence in the mechanochemical cycle. In particular, a mutant with the cover strand deleted functioned only marginally, despite the fact that the cover strand, the Nterminal ''dangling end,'' unlike the neck linker and nucleotidebinding pocket, is not involved with any previously considered energy transduction pathway. Furthermore, a constant assisting load, likely in lieu of a power stroke, was shown to rescue forward motility in the cover strand deletion mutant. Our results support a stepping mechanism driven by dynamic cover-neck bundle formation. They also suggest a strategy to generate motors with altered mechanical characteristics by targeting the force-generating element.biological motor ͉ force generation ͉ optical trap ͉ power stroke ͉ motor protein T ranslocating motors, such as kinesins and certain myosins, form a distinct class of proteins that ''walk'' along biofilament tracks to perform a wide range of vital cellular processes (1). A fundamental, yet poorly understood aspect of these motors is the energy transduction mechanism that converts the chemical energy of ATP binding, hydrolysis, and product release into mechanical work. Kinesins and myosins appear to have a common nucleotide sensor (2, 3) yet have evolved different energy conversion mechanisms to achieve a variety of motile properties. In myosin, a series of structural changes leading to the rotation of its lever arm have been identified (4), but the details of the force generation have yet to be established.Likewise, the force generation mechanism of kinesin (herein, we mainly consider Kinesin-1) is not known. Until recently, the only mechanical element considered was the neck linker (NL), which connects the N-terminal motor head to the ␣-helical stalk. This Ϸ12-residue segment is disordered and flexible in the absence of ATP and ''docks'' when ATP binds to the motor head (5). Mutations in the NL impair the motility while preserving ATPase activity and microtubule (MT) binding (6, 7). However, a mechanism for its contribution to force generation is not available, and, in lieu of this, affinity-driven zippering of the NL to the motor head has been assumed. Unlike the structurally well-defined lever arm of myosin, the NL is short and flexible when detached. Furthermore, it interacts only weakly with the motor head (8), drawing f...
Sensitive, flexible, and rapid: A post‐hybridization labeling scheme was combined with a high‐throughput microfluidic scanning system to demonstrate the use of graphically encoded gel particles for rapid microRNA quantification (see picture). A versatile particle encoding scheme allows for scalable multiplexing that provides attomole sensitivity with a simple and efficient workflow.
We demonstrate the use of graphically encoded hydrogel microparticles for the sensitive and high-throughput multiplexed detection of clinically relevant protein panels in complex media. Combining established antibody capture techniques with advances in both microfluidic synthesis and analysis, we detected 1-8 pg/mL amounts of three cytokines (interleuken-2, interleuken-4, and tumor necrosis factor alpha) in single and multiplexed assays without the need for filtration or blocking agents. A range of hydrogel porosities was investigated to ensure rapid diffusion of targets and reagents into the particle as well as to maintain the structural integrity of particles during rinsing procedures and high-velocity microfluidic scanning. Covalent incorporation of capture antibodies using a heterobifunctional poly(ethylene glycol) linker enabled one-step synthesis and functionalization of particles using only small amounts of valuable reagents. In addition to the use of three separate types of single-probe particles, the flexibility of the stop-flow lithography (SFL) method was leveraged to spatially segregate the three probes for the aforementioned target set on an individual encoded particle, thereby demonstrating the feasibility of single-particle diagnostic panels. This study establishes the gel-particle platform as a versatile tool for the efficient quantification of protein targets and significantly advances efforts to extend the advantages of both hydrogel substrates and particle-based arrays to the field of clinical proteomics.
This protocol describes the core methodology for the fabrication of bar-coded hydrogel microparticles, the capture and labeling of protein targets and the rapid microfluidic scanning of particles for multiplexed detection. Multifunctional hydrogel particles made from poly(ethylene glycol) serve as a sensitive, nonfouling and bio-inert suspension array for the multiplexed measurement of proteins. Each particle type bears a distinctive graphical code consisting of unpolymerized holes in the wafer structure of the microparticle; this code serves to identify the antibody probe covalently incorporated throughout a separate probe region of the particle. The protocol for protein detection can be separated into three steps: (i) synthesis of particles via microfluidic flow lithography at a rate of 16,000 particles per hour; (ii) a 3-4-h assay in which protein targets are captured and labeled within particles using an antibody sandwich technique; and (iii) a flow scanning procedure to detect bar codes and quantify corresponding targets at rates of 25 particles per s. By using the techniques described, single- or multiple-probe particles can be reproducibly synthesized and used in customizable multiplexed panels to measure protein targets over a three-log range and at concentrations as low as 1 pg ml(-1).
The detailed design of a robust and inexpensive optical trap system is presented. The system features high-sensitivity back focal plane position detection, mechanically controlled specimen stage movement, and fluorescence imaging to provide broad experimental applications. Three educational experimental modules are described to cover basic concepts in optical trapping and biophysics at a level appropriate for undergraduate students.
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