Following translation of individual proteins by single ribosomes in real-time using dual-trap optical tweezers

Abstract

Gradient force traps (optical tweezers) have found many applications in physics and the life sciences [1-3] ever since Arthur Ashkin's first description of optical trapping of dielectric particles in liquid [4], Near-infrared (NIR) gradient force traps made it possible to indirectly apply pN-scale forces on large molecules, nm-sized motor enzymes and ribozymes (e.g. ribosomes), internal parts of cells and whole living cells in their native environment without causing optical damage [5, 6]. The use of optical traps in single molecule biophysics has greatly enhanced our understanding of a wide range of molecular motors found within the cell [7|. Optical trap force spectroscopy has enabled researchers to carry out precise measurements of the minuscule forces and displacements that govern many microbiological processes at the single molecule level [8|. Protein folding and biosynthesis (ribosome translation) in vivo has been researched extensively since the 1960s [9], when Anfinsen postulated that a protein's native structure; is determined only by its amino acid sequence [10]. The protein folding problem is not just of purely scientific interest however, since aggregation of misfolded proteins (polypeptides) is observed in a wide range of pathological disorders in ageing organisms [11]. The aggregation of the protein hTau40 in neurons, for example, has been linked to Alzheimer's disease [12]. A better understanding of the mechanisms of protein synthesis and folding in vivo is highly desirable. The process of co-translational protein synthesis and folding in the crowded environment of the cell is difficult to study using ensemble methods due to the stochastic nature of ribosome translation. In this thesis a custom built high-resolution dual-trap optical tweezers instrument enabled the study of translation kinetics and folding pathways of individual ribosome-bound nascent polypeptide chains (proteins) in real-time, for the first time to our knowledge, using sub-micron-sized surface-modified polystyrene beads as force, displacement and fluid flow sensors. By effectively decoupling the instrument from the environment and by using back-focal-plane interferometry with differential detection, sub-nm displacements and sub-pN forces on ms timescales could be measured without drift over long periods of time [13]...