Presentation on theme: "Applications of optical tweezers in protein-protein interaction analysis Ran Yang."— Presentation transcript:
Applications of optical tweezers in protein-protein interaction analysis Ran Yang
What are optical tweezers? Highly focused laser beam holds a dielectric object (e.g. bead) in place using a strong electric field Use Hooke’s law to estimate the force needed to horizontally displace the bead
The Ribosome Modulates Nascent Protein Folding
Problem The transition from ribosome-bound nascent proteins to functional native proteins has only been characterized through computational analysis. How do proteins attain their native state? Can we observe their intermediates?
Methods Optical tweezers apply force between the ribosomal subunit and the nascent chain. T4 lysozyme Synthesis requires interaction between C and N termini Added 41aa sequence to C-terminus to allow complete T4 chain to emerge from the ribosome Apply force to unfold T4 polypeptide then allow refold
Results Protein in solution always refolds correctly, but not the ribosomal-bound T4. Ribosome-bound protein refolds slower Increasing the C-extension to 60aa leads to slightly faster refold Electrostatic interactions between ribosomal surface and charged residues in nascent chain slow down refolding [Fig. D]
The rate of ribosome-bound I-N transition is much lower than that of the free protein. [Fig. A] Ribosome-bound U is more compact than free U. Ribosomal interactions decelerate formation of the native state and stabilizes the intermediate. [Fig. C]
Results If the full polypeptide doesn’t emerge from the ribosome, there is no refolding. If T4 is fragmented and released from the ribosome, the proteins will fold stably, but they are probably not all functional. [Fig. A, B] The ribosome may prevent misfolding of incomplete proteins, as a molecular chaperone
Conclusions What is the function of the ribosome with respect to protein transitions from nascent to native state? Ribosomes slow folding of polypeptide chains that have not been completely synthesized by attracting positively charged residues. Ribosomes compact polypeptide chains and limits nascent chain interactions. Ribosomes may complement the activity of other molecular chaperones.
ClpX(P) Generates Mechanical Force to Unfold and Translocate Its Protein Substrates
Problem AAA unfoldases degrade damaged polypeptides using ATP hydrolysis to unfold and translocate it to the AAA peptidase chamber. ClpX is an ATPase that recognizes degradation target via ssrA tag, unfolds target protein, and ports it to the peptidase ClpP, which hydrolyzes polypeptides By what mechanism does ClpXP unravel the 2’ and 3’ structures of proteins?
Methods ClpXP immobilized on polystyrene beads with X exposed, allowing binding to ssrA Substrate (GFP) fused to ssrA-tagged titin I27 (red chain) and to dsDNA (blue chain) Observe ClpX binding to ssrA-tagged substrate when bringing beads close enough together, with ATP Fixed positions of traps allows observation of ClpX motor force by the movement of the beads
Results Sudden extension followed by retraction of the GFP show unfolding and polypeptide transport respectively. [Fig. B] Smaller rips are attributed to the polypeptide slipping along the motor. ClpX pulls in the polypeptide at roughly 8nm/s or about 80aa/s It seems that GFP unfolds basically all at once (red arrow). The 220aa extension agrees with calculated length of unfolded – folded GFP
Results What if you pull on the beads to create an opposing force? ClpX stall force is about 20pN, i.e. this is the maximum force ClpX can use to unravel 2’ and 3’ protein structures Below 13pN, translocation velocity is about constant, suggesting ClpX generates mechanical force and that chemical steps are rate- limiting. [Fig. A] If you pull even harder, you see the polypeptide translocated in fixed-length steps. [Fig. B] One rotation of ClpX motor is equivalent to pulling in 1nm of polypeptide
Results There is a short-lived intermediate state when unraveling GFP [Fig. E, red circle] From the observed lengths of the two “halves” of the rip, we can predict the structure of the intermediate Residue 130, occurring at the end of a β-sheet is a good candidate [Fig. D, F]
Results Increasing the external force increases the number of pauses during translocation, but not the length of the pauses. If you slow down the system, it is more likely to pause. Translocation and pausing could be kinetically competing processes (but why should this be the case?) Slipping (green circles) after failing to unravel a substrate is most likely caused by temporarily releasing the substrate. ClpXP complexes are much less prone to slipping, possibly because ClpP digests the polypeptide so that “slipping” would simply cause ClpX to let go of the entire substrate.
Conclusions ClpX can generate enough force to unravel protein substrates. A motor translocates the polypeptide to ClpP in fixed-length steps (not fixed-aa steps), suggesting that it largely ignores the contours of the substrate itself. High external forces slow down the ClpX motor, causing more frequent pauses, possibly because ClpX stochastically fails to turnover the next step. ClpX and ClpXP both form the same intermediate, indicating that unraveling is a function of the substrate, not ClpX.
General Conclusions Optical tweezers allows analysis of forces in protein-protein interactions. Ribosomal function on nascent polypeptides Effect of protein motors on polypeptides Reminder: Must be careful when making assumptions from these data, e.g. what the GFP intermediate looks like based on the length of rips in the folded -> unfolded transition [Fig. 4].