Jen Chao 20.309 Presentation November 20, 2008 Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates Schwesinger et al, 2000 Jen Chao 20.309 Presentation November 20, 2008

Outline Background Methods Results Further Research

Motivation Correlation between unbinding force and solution kinetics or thermodynamics Prediction of off-rates Combinatorial chemistry, genomics research The existence of a correlation between unb. Force and sol. Kin/thermo would be very impt. To find solution properties of protein-ligand systems at molecular level, predicting off-rates could be very valuable for combinatorial chemistry biology. Combinatorial chemistry involves the rapid synthesis or the computer simulation of a large number of different but structurally related molecules.

Background Atomic Force Microscopy (AFM) Antibodies specific for fluorescein Parameters: KD = koff/kon AFM is high-resolution, really sensitive type of scanning probe microscope, used for imaging, measuring and manipulating matter at the nanoscale. It uses a probe and piezoelectric elements to make tiny movements to measure things such as Van der Waals forces, capillary forces, chemical bonding, electrostatic forces, What they wanted to do was: So they can use the AFM too seek the correlation looking at KD (eq. behavior), kon and koffs (spont. Assoc/dissoc) because those are the parameters of prime interest in describing any biological ligand-rec sys.

Methods 3 fluorescein-binding antibodies, point mutations KD, kon, koff for each protein in solution Dependence on Pulling velocity Nonlinearity They chose 3 unrelated fluorescein-binding ab’s in the form of monomeric single-chain Fv fragments. For each prot, determined KD @ eq, Kon, Koff, and temp dependence of Koff in solution Tested the dependence of unbinding forces on pulling velocity to determine 1) whether theres any nonlinearlity that would indicate a change of unbinding mech/transition state w/ pulling speed 2) to extrapolate to the unbinding rate at zero force.

Off/On-Rate Measurements Competitive dissociation assay 1nM fluorescein solution Analog mixed in Stopped-flow fluorimeter 5 different concentrations of scFv Used a fluor. Analog of fluorescein which occupies the binding site after dissoc. Of fluorescein to PREVENT REBINDING Used a 1 nM flour. Soln. and equilibrated it w/ protein until fluorescence reached stable baseline The prot/fluor. Complex mixed with the analog and the increasing fluorescence of FREE fluor. Measured Used a stop flow fluorimeter and 5 different concentrations of scFv fragments were mixed with the SAME conc. Of fluorescein (.2um) Kobs plotted against conc. Of scFv fragments, kon found (koff small can be neglected)

Activation E, Loading Rates EA ~ off-rate temperature dependence 10, 15, 20, 25 degrees C Force-distance measurements Constant loading rate Loading rate dependent Act. Energies were determined by measuring off-rates at 4 diff. temps Force-distance measurements with const. loading rate Bound scFv fragments to a gold chip and unbinding forces for all of the fragments were consecutively measured with the AFM tip Loading rate was varied by changing the retract velocity of the piezo, approach v stayed the same. Loading rate determined directly from the slope of the measured force v. time curves

Results Remarkable correlation of the unbinding forces to the off-rates. Off vs. On Rate Off-rate follows Arrhenius Law: V and EA are correlated By using both related and unrelated antibodies, they found a correlation of the unbinding forces to the off-rates across all systems Indicating that in the AFM exp., the molecular systems are following a dissoc. Path similar to the one at zero force Among the kinetic/eq. constants determined, off-rate measurements expected to be the most accurate; dep only on temp On-rate also temp dep but ALSO concentration of active protein plays a role Follow arrhenius – v “pre-exponential factor that’s temp indep. But contains components related to ENTROPY” Limits: too difficult to interpret this factor in any mechanistic way b/c complexity of unbinding process of ligand from prot. Pkt. An increase in the activation energy coincides with a decrease of the v. may be b/c unbinding process can either be dominated by enthalpic or entropic components

Further research Some protein mutants break earlier along unbinding path Recommend How mutants affect interaction Geometry Protein properties Conclusion: Correlation between off-rate and unbinding force Explain why and what the mutation does – shift the molecule closer to the transition state