Elasticity of Globular Proteins Measured from the ac Susceptibility

We introduce a new method to measure the elastic constants of globular proteins. Gold nanoparticles, tethered to a gold surface by the protein, are driven by an ac electric field while their displacement is synchronously detected by evanescent wave scattering, yielding the mechanical response function of the macromolecular sample in the frequency domain. We apply the method to measure the stiffening of an enzyme upon binding its substrate.

Figure 1

Schematics of the experimental method. (a) The flow chamber, electric excitation, and optical readout. (b) The DNA construction used to test the method. (c) Geometry of the protein attached by the 171 and 75 sites. The protein and the radius of the GNP are drawn to scale.

Figure 2

Amplitude of the response function of the DNA sample before (squares) and after (circles) hybridization. The solid and dashed lines are fits with Eq. (4), yielding $f_c(ss)=328\pm 6\mathrm{Hz}$ and $f_c(ds)=675\pm 39\mathrm{Hz}$. We have also plotted (dotted line), for comparison, the function Eq. (4) for the hybridized case, using the corresponding amplitude $z_0(ds)$ but the cutoff frequency of the unhybridized case $f_c(ss)$.

Figure 3

Amplitude of the frequency response of the GK sample in the absence (squares) and presence (circles) of the substrate GMP. Lines are fits with Eq. (4), showing the protein’s stiffness increases by 20% upon binding GMP.

Figure 4

Specific vs nonspecific binding of GK to the gold surfaces, under various conditions. The $p\mathrm{H}$ of the buffer increases left to right from 4.0 (A) to 10.0 (E). Upper row ($+$): GNPs are tethered by the GK mutant with Cys at 171 and 75. Lower row ($-$): control with a mutant without Cys. The ratio of specific (thiol-gold) to nonspecific binding reaches a maximum 1.4 at $p\mathrm{H}$ 7.0 (D).