FEATURE ARTICLE
Biomolecules and Nanotechnology
Evolution has forced innovative solutions to biomolecular problems. Some may inform the growing field of nanotechnology
David Goodsell
Biomolecular Flexibility and Dynamics
Engineers in our macroscopic world typically build rigid structures that stoically resist the forces of nature. Nature, however, has taken a different approach, developing machines that flex over the course of their action. Is a totally rigid nanostructure needed or even desired? Apparently not. In fact, biological molecules take advantage of flexibility for many aspects of their function. Many of these functions would be severely compromised, or not even possible, given a rigid molecule. Subtle motions can have surprisingly large effects on reaction rates or assembly. Biological molecules are perfectly placed to take advantage of these subtle motions. The step-by-step optimization provided by evolution allows a moderately active protein to be improved, through small changes modifying structure and flexibility, to yield a machine ideally tailored to fulfill its function.
This process is easy for evolution but far more difficult for biotechnological design. We design our machines in one step, instead of through many small random optimization steps, and we expect to get it right with a minimum of tweaking and redesign. Thus, to anticipate all of the subtle effects of motion, our design techniques must be accurate enough to predict conformation and flexibility of molecules at scales far smaller than the radius of an atom.
All biological molecules are flexible to some extent and are battered into different conformations by the constant pressure of surrounding water and the kinetic energy of their own atoms. At physiological temperatures, biological molecules constantly flex. Most of the interactions holding a protein together are conserved?covalent bonds remain connected, hydrogen bonds and salt bridges link portions of the chain?but entire elements of secondary structure flex, bending slightly or separating momentarily from the globule. These motions are often termed "breathing." Breathing is essential in the function of myoglobin, a deep red protein that stores oxygen in muscle cells. Oxygen is bound to myoglobin in a pocket that is completely buried within the protein. Looking at the static structures provided by x-ray crystallography, there are no channels leading into or out of the pocket. For the oxygen to enter and exit, the molecule must breathe, transiently forming channels that allow passage.
Many proteins use a carefully designed change of shape to regulate their action. These allosteric ("other shape") proteins are composed of several subunits, each of which performs identical functions. In the simplest model of their action, each subunit may adopt two conformations, one functionally active, the other less active. Regulation is performed by propagation of the shape change from one subunit to its neighbors. For instance, phosphofructokinase, a key enzyme in sugar metabolism, uses allosteric regulation to modify its action. Phosphofructokinase is composed of four identical subunits (a tetramer), each containing a reactive site for the sugar molecules. The tetramer also contains binding sites for the energy molecule adenosine triphosphate (ATP) in the cleft between subunits. When ATP binds to this second site, it forces the entire enzyme complex into a different shape, which is less active than the original form. In the cell, this regulation is used as a negative-feedback loop. ATP is one of the final products of the sugar-breaking process that the enzyme performs. When ATP is plentiful, it binds to the regulatory site in phosphofructokinase, shutting down its own synthesis. The enzyme that performs the opposite reaction, shown in Figure 8, is also allosterically regulated.
Many protein chains rely on "induced fit" to mediate their function. The chain may remain in a partially unfolded conformation that only completely folds when it binds to its target. Induced fit may be used to create doorways that allow ligands to enter protein cavities that are shielded from the surrounding environment. HIV-1 protease is an example. The active site is a cylindrical tunnel, with the cleavage machinery at its center. Somehow, a polypeptide must be threaded through this tunnel in order for the cleavage reaction to occur. This problem is solved through the use of two flexible flaps that cover the top of the tunnel. When free in solution, these flaps are disordered, opening a path to the active site. When the protease wraps around its target, the flaps close, forming a stable structure that positions the polypeptide accurately for cleavage.
Flexible linkages are common in the molecular world. Protein chains may be made more flexible through addition of many molecules of the amino acid glycine, which are less hindered in bond rotation because of the lack of a side chain, or through addition of many charged residues, which favor exposure to solvent over forming a compact globule. The rigid kink formed by proline, surprisingly, is also commonly found in flexible regions, because it does not fit comfortably within compactly folded structures. The immune system contains many examples of flexible linkages that enhance multivalent binding, as shown in Figure 9.
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