Applications of optical tweezers in protein-protein interaction analysisRan 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]

Results• Folding pathway includes an intermediate that reversible to

the unfolded state but irreversible to the native state. [Fig. A]

• I is somewhat more stable than U, but N is much more stable than I.• Estimated through force calculations: 3.6pN causes U and I to be

equally populated; I is 10nm shorter than U

Results• 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 isequivalent topulling in 1nmof 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 becauseClpP digests the polypeptideso that “slipping” would simplycause ClpX to let go of the entiresubstrate.

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].