Zooming In on Molecular Machines

Timothy Sage

Both the largest and the smallest molecules that we know are crucial to the life process. The cells of all organisms are crammed with proteins, giant molecular machines constructed according to information recorded on DNA, another macromolecule. On the other hand, we require a continual supply of diatomic oxygen (O2) to efficiently generate adenosine triphosphate (ATP), the fuel that drives the operation of many proteins. Many proteins involved in the usage of O2 contain heme, a flat, intermediate-size molecule.

The most familiar heme-containing protein is hemoglobin, which transports O2 from the lungs to the tissues and gives oxygenated blood its distinctive red color. Myoglobin (Figure 1) is a closely related protein that stores O2 in muscle tissue, lending its color to “red” meat. As in other proteins, a linear sequence of amino acids spontaneously assembles to form the major component of the molecule. Most of the atomic “foliage” has been stripped away on the right-hand side of Figure 1, revealing this backbone. Oxygen (yellow) binds at an iron atom located at the center of the heme (red).

Medical motivations for understanding these oxygen-carrying molecules include the need to develop safe, effective blood substitutes and to treat diseases such as sickle-cell anemia, which is caused by a defective hemoglobin. The increasing industrial application of enzymes (protein catalysts) and the development of tools to produce large quantities of virtually any desired protein also provide a technological impetus to learn how to engineer proteins with novel, yet predictable, properties. Detailed investigation of the relatively small protein myoglobin (“only” about two thousand atoms!) provides dual insights into the physical properties of nature’s smallest machines and into how proteins manipulate smaller molecules.

Although proteins are larger than most molecules, they are still about ten million times smaller than a breadbox. Since we cannot “see” an object this small directly, we use spectroscopy to map out the intricate behavior of these molecular machines. In spectroscopy, we measure the absorption or scattering of light to determine the frequency of characteristic resonances (oscillatory motions) of the electrons and nuclei in a molecule. Variations in these frequencies indicate changes in a protein’s structure just as the tone of a bell reflects its size and shape. Practical spectroscopic measurements usually reflect the average behavior of a large number of molecules, since an individual protein molecule can only interact with a very small number of photons.

The detailed arrangement of atoms shown in the two figures comes from investigations of crystals, repetitive arrays containing many trillions of proteins. Conventionally, laborious analysis of the scattering of X rays from a crystal leads to a model for the atomic arrangement in the protein. Measurements of the absorption of infrared light by protein crystals in my laboratory reveal significant variations in molecular frequency among individual proteins. In contrast to a simple crystal, such as sodium chloride (table salt), which is often visualized as a nearly infinite stack of identical perfect spheres, our results emphasize that a protein crystal must be thought of more as a stack of cannon balls, each slightly different, none perfectly round or free of imperfections. Conventional static models of the atomic arrangement in proteins gloss over these crucial variations in molecular conformation or shape among individual protein molecules. In some cases, this can lead to significant errors, as we have found in the binding of carbon monoxide to myoglobin.

Carbon monoxide (CO) is toxic because it can displace oxygen from the binding site of myoglobin, hemoglobin, and other heme proteins. The active sites of these proteins are carefully tailored to minimize binding of the low levels of CO produced in normal metabolism. (They cannot cope with the inhalation of large amounts of CO, which usually proves fatal.) It is important to understand how heme proteins make the crucial distinction between the life-giving O2 and the poisonous CO, since it is essential for artificial blood substitutes to mimic this feat. One role of the protein atoms surrounding the heme is to accommodate the O2 molecule, which tips toward the heme plane when it binds (Figure 1), and to exclude the poisonous CO, which binds upright.

Some textbooks claim that these atoms deter CO binding by packing so tightly around the binding site that CO is highly distorted from this preferred upright orientation. The structures that various research teams deduce by scattering X rays, however, are highly inconsistent. We have developed a new technique to determine the geometry of the bound CO. By applying polarized light at the correct frequency, we cause the C-O bond to oscillate, and determine the direction that these two atoms move. We find that the CO is nearly upright and conclude that it is the need to displace the surrounding protein atoms that discourages CO binding.

A useful machine must have moving parts, and understanding the dynamics of proteins is another ongoing challenge. Water molecules continually bombard the protein and rupture some of the bonds that maintain the protein structure shown in Figure 1. Based on experience with macroscopic machines, this might sound rather disruptive — think how you react when your car hits a pothole! In fact, this process is indispensable to the normal operation of these microscopic machines. Characteristic functions of the protein, such as changing its shape or reconfiguring smaller molecules, rely on the energy provided by these random collisions.

The challenge of understanding these processes is that they are driven by haphazard fluctuations and thus occur at different times in each one of a group of identical proteins. In some proteins, including heme proteins such as myoglobin, absorption of a photon provides enough energy to initiate a reaction. An intense light pulse from a laser will start a reaction in a large number of proteins simultaneously, something like the starter pistol in a footrace. This allows us to investigate the course of a reaction even using spectroscopic observations that detect the average behavior of large numbers of molecules.

Myoglobin happens to be a “man in the street” example of the importance of protein dynamics, since O2 has no apparent pathway through the atoms surrounding its binding site in the average structure in Figure 1. Spectroscopic measurements in my laboratory and in collaboration with Paul Champion, chair of the physics department, are beginning to provide some insight into how oxygen may enter and leave the protein. Results from a number of research teams have detected occasional fluctuations of the protein that change the frequency of CO bound at the heme. Our work showed that it was possible to stabilize these fluctuations in crystals, and subsequent analysis of X-ray scattering from such crystals by the O2 group headed by George Phillips at Rice University showed that the fluctuations in the shape of the protein open a specific channel that may allow O2 to enter and leave the protein (Figure 2). More recently, we determined that the gate opens and closes about a million times a second, rapidly enough to allow small molecules such as O2 and CO out of the protein.

Many scientific disciplines imply a restricted field of view: for example, geologists study the earth and its history; chemists study molecules and their interactions. One of the joys of being a physicist is the traditional entitlement to turn one’s scientific attention to virtually anything in the physical universe. As a faculty member at Northeastern, I feel privileged to be able to contribute to our understanding of the large molecules that constitute the machinery of life.

Timothy Sage is an assistant professor of physics. He received his B.S. from Carnegie-Mellon University and his Ph.D. from the University of Illinois at Urbana-Champaign. His research is funded by a five-year FIRST Award from the National Institutes of Health.