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]
Results
• Folding pathway includes an intermediate that reversible to
the unfolded state but irreversible to the native state. [Fig. A]
𝑈 ⇄ 𝐼 → 𝑁
• 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 ratelimiting. [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].