A Novel Method to Evaluate Ribosomal Performance in Cell-Free Protein Synthesis Systems.

Cell-free protein synthesis (CFPS) systems were designed to produce proteins with a minimal set of purified components, thus offering the possibility to follow translation as well as protein folding. In order to characterize the performance of the ribosomes in such a system, it is crucial to separately quantify the two main components of productivity, namely the fraction of active ribosomes and the number of synthesizing cycles. Here, we provide a direct and highly reliable measure of ribosomal activity in any given CFPS system, introducing an enhanced-arrest peptide variant. We observe an almost complete stalling of ribosomes that produce GFPem (~95%), as determined by common centrifugation techniques and fluorescence correlation spectroscopy (FCS). Moreover, we thoroughly study the effect of different ribosomal modifications independently on activity and number of synthesizing cycles. Finally, employing two-colour coincidence detection and two-colour colocalisation microscopy, we demonstrate real-time access to key productivity parameters with minimal sample consumption on a single ribosome level.

Conformational state distributions and catalytically relevant dynamics of a hinge-bending enzyme studied by single-molecule FRET and a coarse-grained simulation.

Over the last few decades, a view has emerged showing that multidomain enzymes are biological machines evolved to harness stochastic kicks of solvent particles into highly directional functional motions. These intrinsic motions are structurally encoded, and Nature makes use of them to catalyze chemical reactions by means of ligand-induced conformational changes and states redistribution. Such mechanisms align reactive groups for efficient chemistry and stabilize conformers most proficient for catalysis. By combining single-molecule Förster resonance energy transfer measurements with normal mode analysis and coarse-grained mesoscopic simulations, we obtained results for a hinge-bending enzyme, namely phosphoglycerate kinase (PGK), which support and extend these ideas. From single-molecule Förster resonance energy transfer, we obtained insight into the distribution of conformational states and the dynamical properties of the domains. The simulations allowed for the characterization of interdomain motions of a compact state of PGK. The data show that PGK is intrinsically a highly dynamic system sampling a wealth of conformations on timescales ranging from nanoseconds to milliseconds and above. Functional motions encoded in the fold are performed by the PGK domains already in its ligand-free form, and substrate binding is not required to enable them. Compared to other multidomain proteins, these motions are rather fast and presumably not rate-limiting in the enzymatic reaction. Ligand binding slightly readjusts the orientation of the domains and feasibly locks the protein motions along a preferential direction. In addition, the functionally relevant compact state is stabilized by the substrates, and acts as a prestate to reach active conformations by means of Brownian motions.

Light microscopy with doughnut modes: a concept to detect, characterize, and manipulate individual nanoobjects.

Higher order laser modes, mainly called doughnut modes (DMs) have use in many different branches of research, such as, bio-imaging, material science, single-molecule microscopy, and spectroscopy. The main reason of their increasing importance is that recently, the techniques to generate well-defined DMs have been refined or rediscovered. Although their potential is still not fully utilized, their specifically polarized field distribution gives rise to a wide field of applications. They are contributing to complete our fundamental knowledge of the optical properties of single emitting species, such as molecules, nanoparticles, or quantum dots, offering insight into the three-dimensional dipole or particle orientation in space. The perfect zero intensity in the focus center qualifies some DMs for stimulated emission depletion (STED) microscopy. For the same reason, they have been suggested for trapping and tweezing applications.

Probing dielectric interfaces on the nanoscale with elastic scattering patterns of single gold nanorods.

We study spatially isolated, individual gold nanorods placed at a planar interface between two dielectric media using confocal interference scattering microscopy in combination with higher order laser modes. Approaching refractive index matching conditions, we observe that the elastic scattering patterns of individual nanorods exhibit an exponential increase of both the scattering intensity and the signal-to-background ratio. In case refractive index matching conditions are fullfilled, the data acquisition rates are maximized and suitable for in-vivo biological measurements. In all cases, the characteristic two-lobe shape of the scattering patterns of single nanorods remains unchanged while the sign of the image contrast is a direct consequence of the refractive index variation occurring at the interface.

Topology measurements of metal nanoparticles with 1 nm accuracy by Confocal Interference Scattering Microscopy.

We present a novel scattering microscopy method to detect the orientation of individual silver nanorods and to measure their relative distances. Using confocal microscopy in combination with either the fundamental or higher order laser modes, scattering images of silver nanorods were recorded. The distance between two individual nanorods was measured with an accuracy in the order of 1 nm.We detected the orientation of isolated silver nanorods with a precision of 0.5 degree that corresponds to a rotational arch of about 1 nm. The results demonstrate the potential of the technique for the visualization of non-bleaching labels in biosciences.