Joanna Andrecka

A nano-positioning system for macromolecular structural analysis

Very often, the positions of flexible domains within macromolecules as well as within macromolecular complexes cannot be determined by standard structural biology methods. To overcome this problem, we developed a method that uses probabilistic data analysis to combine single-molecule measurements with X-ray crystallography data. The method determines not only the most likely position of a fluorescent dye molecule attached to the domain but also the complete three-dimensional probability distribution depicting the experimental uncertainty. With this approach, single-pair fluorescence resonance energy transfer measurements can now be used as a quantitative tool for investigating the position and dynamics of flexible domains within macromolecular complexes. We applied this method to find the position of the 5′ end of the nascent RNA exiting transcription elongation complexes of yeast (Saccharomyces cerevisiae) RNA polymerase II and studied the influence of transcription factor IIB on the position of the RNA.

Single-molecule tracking of mRNA exiting from RNA polymerase II

Single-pair fluorescence resonance energy transfer was used to track RNA exiting from RNA polymerase II (Pol II) in elongation complexes. Measuring the distance between the RNA 5′ end and three known locations within the elongation complex allows us determine its position by means of triangulation. RNA leaves the polymerase active center cleft via the previously proposed exit tunnel and then disengages from the enzyme surface. When the RNA reaches lengths of 26 and 29 nt, its 5′ end associates with Pol II at the base of the dock domain. Because the initiation factor TFIIB binds to the dock domain and exit tunnel, exiting RNA may prevent TFIIB reassociation during elongation. RNA further extends toward the linker connecting to the polymerase C-terminal repeat domain (CTD), which binds the 5′-capping enzyme and other RNA processing factors.

Nano positioning system reveals the course of upstream and nontemplate DNA within the RNA polymerase II elongation complex

Crystallographic studies of the RNA polymerase II (Pol II) elongation complex (EC) revealed the locations of downstream DNA and the DNA-RNA hybrid, but not the course of the nontemplate DNA strand in the transcription bubble and the upstream DNA duplex. Here we used single-molecule Fluorescence Resonance Energy Transfer (smFRET) experiments to locate nontemplate and upstream DNA with our recently developed Nano Positioning System (NPS). In the resulting complete model of the Pol II EC, separation of the nontemplate from the template strand at position +2 involves interaction with fork loop 2. The nontemplate strand passes loop β10-β11 on the Pol II lobe, and then turns to the other side of the cleft above the rudder. The upstream DNA duplex exits at an approximately right angle from the incoming downstream DNA, and emanates from the cleft between the protrusion and clamp. Comparison with published data suggests that the architecture of the complete EC is conserved from bacteria to eukaryotes and that upstream DNA is relocated during the initiation–elongation transition.

Label-free, all-optical detection, imaging, and tracking of a single protein

Optical detection of individual proteins requires fluorescent labeling. Cavity and plasmonic methodologies enhance single molecule signatures in the absence of any labels but have struggled to demonstrate routine and quantitative single protein detection. Here, we used interferometric scattering microscopy not only to detect but also to image and nanometrically track the motion of single myosin 5a heavy meromyosin molecules without the use of labels or any nanoscopic amplification. Together with the simple experimental arrangement, an intrinsic independence from strong electronic transition dipoles and a detection limit of <60 kDa, our approach paves the way toward nonresonant, label-free sensing and imaging of nanoscopic objects down to the single protein level.

Direct observation and control of supported lipid bilayer formation with interferometric scattering microscopy.

Supported lipid bilayers (SLB) are frequently used to study processes associated with or mediated by lipid membranes. The mechanism by which SLBs form is a matter of debate, largely due to the experimental difficulty associated with observing the adsorption and rupture of individual vesicles. Here, we used interferometric scattering microscopy (iSCAT) to directly visualize membrane formation from nanoscopic vesicles in real time. We observed a number of previously proposed phenomena such as vesicle adsorption, rupture, movement, and a wave-like bilayer spreading. By varying the vesicle size and the lipid-surface interaction strength, we rationalized and tuned the relative contributions of these phenomena to bilayer formation. Our results support a model where the interplay between bilayer edge tension and the overall interaction energy with the surface determine the mechanism of SLB formation. The unique combination of sensitivity, speed, and label-free imaging capability of iSCAT provides exciting prospects not only for investigations of SLB formation, but also for studies of assembly and disassembly processes on the nanoscale with previously unattainable accuracy and sensitivity.

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

Significance We use high spatiotemporal resolution single-molecule microscopy to directly visualize the structural transitions underlying each step of the molecular motor kinesin-1 at physiological stepping rates. Our results identify a one-head–bound intermediate in the stepping cycle that is initiated by ATP binding and is terminated by ATP hydrolysis. These results supersede previous functional studies because they identify the transitions that must occur to produce a step as opposed to transitions that may occur if the motor is studied under controlled conditions. We thus show that kinesin utilizes a two-step powerstroke mechanism to walk at maximum velocity. The single-molecule methods developed here are broadly applicable for resolving protein conformational changes as small as 2 nm with millisecond temporal resolution. To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head–bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.

Structural dynamics of myosin 5 during processive motion revealed by interferometric scattering microscopy

Myosin 5a is a dual-headed molecular motor that transports cargo along actin filaments. By following the motion of individual heads with interferometric scattering microscopy at nm spatial and ms temporal precision we found that the detached head occupies a loosely fixed position to one side of actin from which it rebinds in a controlled manner while executing a step. Improving the spatial precision to the sub-nm regime provided evidence for an ångstrom-level structural transition in the motor domain associated with the power stroke. Simultaneous tracking of both heads revealed that consecutive steps follow identical paths to the same side of actin in a compass-like spinning motion demonstrating a symmetrical walking pattern. These results visualize many of the critical unknown aspects of the stepping mechanism of myosin 5 including head–head coordination, the origin of lever-arm motion and the spatiotemporal dynamics of the translocating head during individual steps. DOI: http://dx.doi.org/10.7554/eLife.05413.001

Interferometric Scattering Microscopy for the Study of Molecular Motors

Our understanding of molecular motor function has been greatly improved by the development of imaging modalities, which enable real-time observation of their motion at the single-molecule level. Here, we describe the use of a new method, interferometric scattering microscopy, for the investigation of motor protein dynamics by attaching and tracking the motion of metallic nanoparticle labels as small as 20nm diameter. Using myosin-5, kinesin-1, and dynein as examples, we describe the basic assays, labeling strategies, and principles of data analysis. Our approach is relevant not only for motor protein dynamics but also provides a general tool for single-particle tracking with high spatiotemporal precision, which overcomes the limitations of single-molecule fluorescence methods.

Quantitative mass imaging of single molecules in solution

The cellular processes underpinning life are orchestrated by proteins and their interactions. Structural and dynamic heterogeneity, despite being key to protein and drug function, continues to pose a fundamental challenge to existing analytical and structural methodologies used to study these associations. Here, we use interferometric scattering microscopy to mass-image single biomolecules in solution with <2% mass error, up to 19-kDa resolution and 1-kDa precision. Thereby, we resolve oligomeric distributions at high dynamic range, detect small-molecule binding, and mass-image biomolecules composed not only of amino acids, but also heterogeneous species, such as lipo- and glycoproteins. These capabilities enable us to characterize the molecular mechanisms of processes as diverse as oligomeric selfassembly, glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry (iSCAMS) provides spatially resolved access to the dynamics of biomolecular interactions ranging from those involving small molecules to mesoscopic assemblies, one molecule at a time.

Extending the Nano-Positioning System (NPS)

Single-Pair Fluorescence Resonance Energy Transfer (FRET) experiments reveal structural and dynamic information about macro-molecules by monitoring the change in FRET efficiency between fluorescent dyes attached to a macro-molecule. The Nano-Positioning System (NPS) developed recently [1] uses data from several of such experiments to infer the position of a dye attached to protein sites unresolved by x-ray crystallography. Briefly, we perform probabilistic data analysis that allows us to calculate the distribution of possible dye positions in a simple and objective way without relying on ad-hoc procedures.Up to now NPS was limited to the triangulation of just one fluorescently labelled position based on FRET measurements to several other positions known from crystal structure [1,2]. Here, we discuss ways to extend the present model beyond this basic triangulation principle. In particular, we show how to gain three dimensional distance information by analysing triangulation networks where FRET is measured between arbitrary labelling sites in absence of other structural information.[1] A. Muschielok, J. Andrecka, A. Jawhari, F. Bruckner, P. Cramer & J. Michaelis, Nat. Meth. 5, 965-971 (2008)[2] J. Andrecka, B. Treutlein, M.A. Izquierdo Arcusa, A. Muschielok, R. Lewis; A.C.M. Cheung, P. Cramer, and J. Michaelis, NAR doi:10.1093/nar/gkp601 (2009)