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September 2001
Vol. 4, No. 9, p 15.
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Microscopically measuring Kd

Speaking volumes. AFM shows that UvrD comes in monomers (left) and dimers. (Adapted from J. Am. Chem. Soc. 2001, 123, 5632–5635.)
Protein–protein interactions mediate most cellular processes. The strength of binding between two proteins is typically described by the dissociation constant (Kd ) and can be determined by many methods. Atomic force microscopy (AFM) can now be added to this list.

AFM is a nanometer-resolution technique that permits imaging of samples in solution at physiological temperatures. This relatively new microscopy can also measure the forces exerted on individual molecules with nanoNewton accuracy, as in the mechanical unfolding of a protein or in the strength of interaction between a ligand and receptor.

The high-resolution topographical imaging capabilities of AFM have been used to determine the oligomerization states of proteins by relating the observed volume of an individual molecule to its molecular weight. Glenn Ratcliff and Dorothy Erie of the University of North Carolina (Chapel Hill) recently took this one step further and used AFM to measure the Kd of E. coli DNA helicase II (UvrD) (J. Am. Chem. Soc. 2001, 123, 5632–5635). “We have been studying mismatch repair, in which UvrD participates,” explains Erie. “UvrD was good for proof of concept that AFM can be used to measure Kd’s.”

Ratcliff and Erie first established that each molecule’s observed volume correlates linearly with its molecular weight. After this calibration, they measured the molecular weights of UvrD proteins deposited on a mica surface and found two distinct molecular weight populations that could be fitted with Gaussian curves. One population was UvrD monomers, the other dimers. By counting the number of individual monomers and dimers formed when various concentrations of UvrD were deposited, the researchers calculated a Kd that agreed closely with values determined by other techniques. This is the first time that a Kd has been measured by visualizing individual molecules.

“It will be difficult to measure weak binding constants,” says Erie, “because we are limited on the high end of the concentration scale [because] the surface will be completely covered with protein.” But, she adds that “Kd’s can be measured under different solution conditions, small amounts of protein are required, and low concentrations of higher-order species are detectable.”

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