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Force measurements using optical tweezers


Single ribosome held at the uL4 ribosomal protein and the N-terminal end of the nascent chain via biotin-streptavidin interactions between two optically trapped polystyrene beads with streptavidin-DNA handles

Protein synthesis is directly linked to protein folding. Translation kinetics plays a crucial role in forming secondary structural elements that lead to tertiary structure formation. However, how these two processes are coordinated is still not fully understood. Single-molecule techniques are essential for the study of protein synthesis and subsequent folding, since these are asynchronous processes that are extremely difficult to be observed using ensemble methods. The use of optical tweezers as a single-molecule manipulation technique offers an additional valuable tool to study protein synthesis and co-translational folding at the ribosome in real-time.
Optical tweezers can answer additional questions that fluorescent techniques cannot touch, such as what are the variations of the forces during synthesis of the nascent polypeptide chain at the ribosome, how are these forces dependent on the secondary structure and the whole folding process and how they potentially affect the rate of synthesis.
In this respect, we immobilize translating ribosomes on a polystyrene bead and we bind the N-terminus of the nascent polypeptide chain to a DNA handle attached to a second polystyrene bead. The two beads are trapped either by a single laser trap and a micropipette that can move with respect to the trap or a dual laser trap where one trap is fixed and the other is steerable.
Full synthesis of proteins with different tertiary structures and intermediate folding states can be clearly observed. This way the intricate relationship between a protein’s amino acid sequence, its co-translational elongation rate and folding can be analyzed and explained. The way opens to eventually observe the folding and unfolding of the nascent chain with and without the interactions of chaperones or other cellular factors. (Katranidis et al., FEBS Letters. 2011, 585, 1859-1863, Wruck et al., PNAS 2017, submitted)

Contact: Dr. Alexandros Katranidis