Analysis of Many Globular Proteins Reveals Common Structural Patterns

Q Protein Architecture—Tertiary Structure of Large Globular Proteins For the beginning student, the very complex tertiary structures of globular proteins much larger than those shown in Figure 4-18 are best approached by fo-

10.0 8.0 6.0 4.0 2.0 0.0 -2.0 1H chemical shift (ppm)

FIGURE 2 A one-dimensional NMR spectrum of a globin from a marine blood worm. This protein and sperm whale myoglobin are very close structural analogs, belonging to the same protein structural family and sharing an oxygen-transport function.

10.0 8.0 6.0 4.0 2.0 0.0 -2.0 1H chemical shift (ppm)

FIGURE 2 A one-dimensional NMR spectrum of a globin from a marine blood worm. This protein and sperm whale myoglobin are very close structural analogs, belonging to the same protein structural family and sharing an oxygen-transport function.

for these distance constraints to be useful, the atoms giving rise to each signal must be identified. Complementary TOCSY experiments can help identify which NOE signals reflect atoms that are linked by covalent bonds. Certain patterns of NOE signals have been associated with secondary structures such as a helices. Modern genetic engineering (Chapter 9) can be used to prepare proteins that contain the rare isotopes 13C or 15N. The new NMR signals produced by these atoms, and the coupling with 1H signals resulting from these substitutions, help in the assignment of individual 1H NOE signals. The process is also aided by a knowledge of the amino acid sequence of the polypeptide.

To generate a three-dimensional structure, researchers feed the distance constraints into a computer along with known geometric constraints such as chirality, van der Waals radii, and bond lengths and angles. The computer generates a family of closely related structures that represent the range of conformations consistent with the NOE distance constraints (Fig. 3c). The uncertainty in structures generated by NMR is in part a reflection of the molecular vibrations (breathing) within a protein structure in solution, discussed in more detail in Chapter 5. Normal experimental uncertainty can also play a role.

When a protein structure has been determined by both x-ray crystallography and NMR, the structures cusing on structural patterns that recur in different and often unrelated proteins. The three-dimensional structure of a typical globular protein can be considered an assemblage of polypeptide segments in the a-helix and ^-sheet conformations, linked by connecting segments. The structure can then be described to a first approximation by defining how these segments stack on one generally agree well. In some cases, the precise locations of particular amino acid side chains on the protein exterior are different, often because of effects related to the packing of adjacent protein molecules in a crystal. The two techniques together are at the heart of the rapid increase in the availability of structural information about the macromolecules of living cells.

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