Allosteric Control through Mechanical Tension

Conformational changes of proteins modulate their function. In allosteric control, the conformational change is induced by the binding of a signaling molecule. Here we insert a “molecular spring” on the enzyme guanylate kinase, to control the conformation of this protein. The stiffness of the spring can be varied externally, which allows one to exert a controlled mechanical tension between the two points on the protein’s surface where the spring is attached. We show that by applying and releasing the tension we can reversibly turn the enzyme off and on.

Figure 1

The protein-DNA chimera. The GK structure is PDB entry 1S4Q. The locations of the Cys mutations are shown in magenta. The distance between these two groups is 4.5 nm.

Figure 2

Reduction in GK activity upon hybridization of the chimera. Data are the average of 4–5 experiments; the error bars are $\pm 1$ standard deviation (SD). (a) Sample A shows a 2-fold effect, which rises to a 4-fold effect (sample B) upon purification on a sulfhydryl column, which retains molecules with unreacted Cys. (b) The 4-fold decrease in enzymatic activity going from ss to ds chimera, is reversed after adding DNAse, which degrades the DNA of the chimera, releasing the tension. The columns ss/DNAse and ss/Ca are controls.

Figure 3

The concentration of ATP remaining after a time $\tau$, $[A(\tau )]$, vs the initial GMP concentration $[G(0)]$, for the ss and ds chimera. $[A(\tau )]$ is a measure of the speed of the enzymatic reaction. Each experimental point represents the average of 4–6 measurements; error bars are $\pm 1$ SD. The ss data are fitted using Eq. (6), the ds data using (9); the corresponding parameter values are listed in Table .

Figure 4

The conformational change between the open (top: ligand-free) and closed (bottom) conformation of GK, from the PDB structures 1EX6 and 1GKY. While these structures are of the yeast protein, the structure of the TB protein used in this study is essentially identical. The structural location of the Cys mutations, where the mechanical stress is applied, is shown in red. The GMP binding site is colored blue, the ATP binding site green. Inspection of the structures suggests that the applied mechanical stress, between helices $\alpha 2$ and $\alpha 6$, favors the open conformation, and probably deforms the GMP binding site more than the ATP one. This correlates with the measured change in the binding constants for the two substrates upon applying the mechanical stress (see Table ).