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In this chapter, we have emphasized that protein function is dependent on protein structure. Thus, to figure out exactly how a protein works, its three-dimensional structure must be determined. Determining a protein’s conformation requires sophisticated physical methods and complex analyses of the experimental data. Here we briefly describe three methods used to generate three-dimensional models of proteins.

X-ray Crystallography The use of x-ray crystallography to determine the three-dimensional structures of proteins was pioneered by Max Perutz and John Kendrew in the 1950s. In this technique, beams of x-rays are passed through a protein crystal, in which millions of protein molecules are precisely aligned with one another in a rigid crystalline array. The wavelengths of x-rays are about 0.1–0.2 nm, short enough to determine the positions of individual atoms in the protein. The electrons in the atoms of the crystal scatter the x-rays, which produce a diffraction pattern of discrete spots when they are intercepted by photographic film or an electronic detector (Figure 3-45). Such patterns are extremely complex—composed of as many as 25,000 diffraction spots, or reflections, whose measured intensities vary depending on the distribution of the electrons in the sample, which is, in turn, determined by the atomic structure and three-dimensional conformation of the protein. Elaborate calculations and modifications of the protein (such as the binding of heavy metals) must be made to interpret the diffraction pattern and calculate the distribution of electrons (called the electron density map). A portion of an electron density map of a protein can be seen in Figure 2-9. With the three-dimensional electron density map in hand, one then “fits” a molecular model of the protein to match the electron density, and it is these models that one sees in the various diagrams of proteins throughout this book (e.g., Figure 3-9). The process is analogous to reconstructing the precise shape of a rock from the ripples that it creates when thrown into a pond. Although sometimes the structures of parts of the protein cannot be clearly defined, using x-ray crystallography, researchers are systematically determining the structures of representative types of most proteins. To date, more than 90,000 detailed three-dimensional structures, including more than 35,000 distinct protein sequences, have been established using x-ray crystallography. These structures can be found in the Research Collaboratory for Structural Bioinformatics Protein Data Bank (http://www.rcsb.org/pdb/home/home.do), each with its own “PDB” entry.

Cryoelectron Microscopy Although some proteins readily crystallize, obtaining crystals of others—particularly large multisubunit proteins and membrane-associated proteins—requires a time-consuming, often robot-assisted trial-and-error effort to find just the right conditions, if they can be found at all. (Growing crystals suitable for structural studies is as much an art as a science.) There are several ways to determine the structures of such difficult-to-crystallize proteins. One is cryoelectron microscopy (Figure 3-46). In this technique, a dilute protein sample in an aqueous solution is applied in a thin layer to an electron microscope sample holder (a “grid”) and rapidly frozen in liquid helium to preserve its structure. It is then examined in the frozen, hydrated state in a cryoelectron microscope. Images of the protein are recorded on a very sensitive camera using a low dose of electrons to prevent radiation-induced damage to the structure. Since the individual proteins are in different orientations in the frozen sample, sophisticated computer algorithms analyze the images to sort them into groups with the same orientation. The average image of each orientation is calculated from images of the thousands of different molecules in each group, and then the computer assembles the average images, each of which show views of the protein from different orientations, to reconstruct the protein’s structure in three dimensions. Recent advances in this technology have produced structures in which the polypeptide backbone and amino acid side chains can be discerned. These structures help provide insight into the mechanisms underlying the protein’s function. The use of cryoelectron microscopy and other types of electron microscopy for visualizing cell structures is discussed in Chapter 4.

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NMR Spectroscopy The three-dimensional structures of small proteins containing as many as 200 amino acids can be studied routinely with nuclear magnetic resonance (NMR) spectroscopy, and specialized approaches can be used to extend the size range to somewhat larger proteins. In this technique, a concentrated protein solution is placed in a magnetic field, and the effects of different radio frequencies on the nuclear spin states of different atoms are measured. The spin state of any atom is influenced by neighboring atoms in adjacent residues, with closely spaced residues having a greater effect than distant residues. From the magnitude of the effect, the distances between residues can be calculated by a triangulation-like process; these distances are then used to generate a model of the three-dimensional structure of the protein. An important distinction between x-ray crystallography and NMR spectroscopy is that the former method directly determines the locations of the atoms, while the latter directly determines the distances between the atoms, from which the structure is deduced.

Although NMR does not require the crystallization of a protein—a definite advantage—this technique is usually limited to proteins smaller than about 50 kDa (although new techniques permit analysis of the dynamics in much larger proteins). However, NMR analysis can provide information about the ability of a protein to adopt a set of closely related, but not exactly identical, conformations and to move between those conformations (protein dynamics). This is a common feature of proteins, which are not absolutely rigid structures, but can “breathe” or exhibit slight variations in the relative positions of their constituent atoms. In some cases, these variations can have functional significance; for example, they may influence how proteins bind to one another. NMR structural analysis has been particularly useful in studying isolated protein domains, which can often be obtained as stable structures and tend to be small enough for this technique. To date, there are more than 10,000 NMR-determined structures available in the Protein Data Bank.

Another powerful approach to studying protein dynamics and protein-protein interactions is hydrogen/deuterium exchange mass spectrometry (HXMS). When a protein is placed in a deuterated water (D₂O) solution, the rate at which deuterium is exchanged for hydrogen in the amides in the peptide bonds depends on the accessibility of an amide to the solvent. Those amides exposed on the protein’s surface are highly accessible and exhibit rapid proton/deuterium exchange. Those amides buried in the center of the protein or in a protein-to-protein interface, as well as those participating in hydrogen bonds with other parts of the protein, exhibit slower proton/deuterium exchange rates. A change in protein conformation or binding to other molecules has the potential to alter the rate of hydrogen/deuterium exchange of one or more amides of a protein. MS analysis permits a hypersensitive assay of such conformational changes, allowing the identification of those parts of the protein that directly bind to other molecules or undergo such conformational changes.