Standfirst
Artificial scaffolds composed of carefully chosen protein domains permit protein complexes to be assembled in vitro, providing a line of sight to construct multiprotein architectures that mimic native complexes in vivo.
In the cell, proper functioning of individual proteins often requires close association with other proteins. It is difficult, however, to examine these complex relationships in the cell because of a lack of information regarding the proteins involved, and difficulty in defining and controlling the experimental conditions. This is particularly true when it comes to the operation of macromolecular complexes. In a recent issue of Science, new work from David Tirrell's laboratory demonstrates the use of genetically encoded molecular scaffolds to provide insights into the operation of multiprotein assemblies.
Studies of molecular motors have shown that the velocities measured in vitro are often much slower than in vivo velocities, indicating that some form of cooperativity is operating in vivo, likely mediated by multiprotein complexes. Several theoretical models have attempted to explain this discrepancy and complexes have been created using nonspecific immobilization techniques, but the lack of precise control over the number of motors has been intrinsically limiting.
A scaffold capable of controlling the numerical, spatial and elastic coupling of the molecular motors would be much better suited to exploring cooperative interactions between biomotors. Michael Diehl, who headed up the project to create the scaffolds, says, "Because of my background [in molecular electronics] my first inkling was to use nanowires, [but] we realized very quickly that we were still using a nonspecific immobilization." To overcome this problem they considered engineering artificial proteins.
By using artificial proteins, "We can specify the genetic code which allows us to have residue-level control over molecular architecture, and specific and acute control over spatial, numerical and elastic coupling," remarks Diehl. Tirrell's lab is accomplished at using artificial proteins to engineer new biomaterials, and they chose to use chains of leucine zipper motifs. Each chain of one, two or three motifs linked in series forms a scaffold for high-affinity binding by biomotors linked to complementary leucine zipper motifs.
Diehl knew it would be necessary to provide elasticity between the coupled molecular motors, so they inserted elastin between each leucine zipper motif in the scaffolds. He says, "We felt that elastin was going to be a very nice way to tune the elastic coupling because the viscoelastic properties of elastin are very well characterized."
The researchers chose kinesin to test the properties of their scaffold because it is a small molecular motor that has been extensively studied, and its properties are well established. Diehl et al. showed that in vitro assays using dimeric or trimeric scaffolds resulted in kinesin velocities almost twice as fast as single kinesin. By tuning the mechanical properties of the elastin domains, they also showed that coupling motors on the scaffold provided a means to reconfigure the mechanochemical mechanism of the motors.
Diehl says these scaffolds are not limited to use with molecular motors. "We can also put proteins that are coupled chemically on these scaffolds." In his lab at Rice University, Diehl is continuing to explore new uses for these scaffolds so it is almost certain that more useful applications of these scaffolds will be demonstrated shortly.