beam of x-rays x-ray source protein crystal diffracted beams x-ray diffraction pattern obtained from the protein crystal beam stop (A) (B) (C)Figure 8–21 X-ray crystallography. (A) A narrow beam of x-rays is directed at a well-ordered crystal (B). Shown here is a protein crystal of ribulose bisphosphate carboxylase, an enzyme with a central role in CO2 fixation during photosynthesis. The atoms in the crystal scatter some of the beam, and the scattered waves reinforce one another at certain points and appear as a pattern of diffraction spots (C). This diffraction pattern, together with the amino acid sequence of the protein, can be used to produce an atomic model (D). The complete atomic model is hard to interpret, but this simplified version, derived from the x-ray diffraction data, shows the protein’s structural features clearly (α helices, green; β strands, red). The components pictured in A to D are not shown to scale. (B, courtesy of C. Branden; C, courtesy of J. Hajdu and I. Andersson; D, adapted from original provided by B. Furugren.)
NMR Can Be Used to Determine Protein Structure in Solution
Nuclear magnetic resonance (NMR) spectroscopy has been widely used for many years to analyze the structure of small molecules, small proteins, or protein domains. Unlike x-ray crystallography, NMR does not depend on having a crystalline sample. It simply requires a small volume of concentrated protein solution that is placed in a strong magnetic field; indeed, it is the main technique that yields detailed evidence about the three-dimensional structure of molecules in solution.
Certain atomic nuclei, particularly hydrogen nuclei, have a magnetic moment or spin: that is, they have an intrinsic magnetization, like a bar magnet. The spin aligns along the strong magnetic field, but it can be changed to a misaligned, excited state in response to applied radiofrequency (RF) pulses of electromagnetic radiation. When the excited hydrogen nuclei return to their aligned state, they emit RF radiation, which can be measured and displayed as a spectrum. The nature of the emitted radiation depends on the environment of each hydrogen nucleus, and if one nucleus is excited, it influences the absorption and emission of radiation by other nuclei that lie close to it. It is consequently possible, by an ingenious elaboration of the basic NMR technique known as two-dimensional NMR, to distinguish the signals from hydrogen nuclei in different amino acid residues, and to identify and measure the small shifts in these signals that occur when these hydrogen nuclei lie close enough together to interact. Because the size of such a shift reveals the distance between the interacting pair of hydrogen atoms, NMR can provide information about the distances between the parts of the protein molecule. By combining this information with a knowledge of the amino acid
Figure 8–22 NMR spectroscopy.
An example of the data from an NMR machine. This two-dimensional NMR spectrum is derived from the C-terminal domain of the enzyme cellulase. The spots represent interactions between hydrogen atoms that are near neighbors in the protein and hence reflect the distance that separates them. Complex computing methods, in conjunction with the known amino acid sequence, enable possible compatible structures to be derived.
Ten structures of the enzyme, which all satisfy the distance constraints equally well, are shown superimposed on one another, giving a good indication of the probable three-dimensional structure. (Courtesy of
P.
Kraulis.) sequence, it is possible in principle to compute the three-dimensional structure of the protein (Figure 8–22).
For technical reasons, the structure of small proteins of about 20,000 daltons or less can be most readily determined by NMR spectroscopy. Resolution decreases as the size of a macromolecule increases. But recent technical advances have now pushed the limit to about 100,000 daltons, thereby making the majority of proteins accessible for structural analysis by NMR.