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The Shape and Structure of Proteins
How We Know: Probing Protein Structure
Coiled-coils
a
helices and
b
sheets: how do we know thatthese structures exist? How can we see them? And howcan we determine which of these or other structures a par-ticular protein adopts—and whether a protein’s shapechanges as it does its job?Most proteins are small—too small to be seen in any detail,even with a powerful electron microscope. To follow thepaths that the string of amino acids takes inside a good-sized protein molecule, you need to be able to see theatoms that form the individual amino acids. For that job,you generally need X-rays. Like light, X-rays are a form of electromagnetic radiation. But they have a wavelength of 0.1 nanometer (nm)—the approximate diameter of ahydrogen atom—so they allow you to look at very smalland detailed structures.Before you can examine your protein with X-rays, you haveto isolate it in a pure form. You must also determine itsamino acid sequence, so that the X-ray data (or other typesof structural information) are easier to interpret. With thatin mind, we will now review how to go about solving thethree-dimensional structure of a protein.
Isolation
Maybe you are interested in studying a set of proteins thatis involved in copying DNA, processing RNA, or degradingdamaged proteins. Each of these cellular processes is car-ried out by a molecular machine that contains many dif-ferent proteins, as we discuss in Chapters 6 and 7.If you know the identity of any one of the proteins in sucha complex, you may be able to identify some of the otherproteins in the complex by protein affinity chromatography.Essentially, you break open cells, separate all of the solu-ble proteins by centrifugation, and then pass those proteinsthrough a column matrix that contains either the pure tar-get protein, or an antibody that binds this protein tightly. Ineither case, protein complexes will collect on the columnand often can be eluted with salt or by changing the pH of the solution. Proteins that are physically associated withthe target protein can thereby be identified and isolated.These techniques are described in greater detail in Panels4–3 through 4–6 (pp. 160–165).The next step involves visualizing, and then isolating,these proteins by electrophoresis through a polymer gel,which separates polypeptides on the basis of their sizes.If the purification yields a great many proteins, or if theproteins are very similar in size, they can be resolvedusing two-dimensional gel electrophoresis, which sepa-rates proteins by both size and overall electrical charge(see Panel 4–5, pp. 163). Both techniques yield a num-ber of bands or spots, each one containing a differentprotein.Once a protein has been selected for further study, you areready to determine its amino acid sequence. The smallamount of protein that is present in the gel actually pro-vides enough material for this analysis.
Identification
Before a protein is sequenced (i.e., the order of its aminoacids is determined), it is generally broken into smallerpieces using a selective protease. The enzyme trypsin, forexample, cleaves polypeptide chains on the carboxyl sideof lysine or arginine residues. So if a protein has ninelysines and seven arginines, digestion with trypsin shouldcut it into 17 peptide fragments.Mass spectrometry can then be used to determine the exactmass of each peptide fragment—information that will allowyou to identify your protein from the list of all proteins pro-duced by the organism as determined from the DNAsequence of its genome. The process works like this. Thepeptides from the tryptic digest are dried onto a metal plate.The sample is then blasted with a laser, which heats thepeptides, causing them to become “ionized”—ejected fromthe plate in the form of a gas. Accelerated by a powerfulelectric field, the peptide ions then fly toward a detector, andthe time it takes them to get there is related to their massand their charge. (The larger the peptide is, the slower itmoves, while the more highly charged it is, the faster itmoves.) Knowledge of the exact mass of each of the proteinfragments produced by trypsin cleavage serves as a “finger-print,” which allows the identification of the protein’s gene(Figure 4–11) and thus its amino acid sequence.You now need to produce enough protein to do a structuralanalysis. Using recombinant DNA technology (which wediscuss in detail in Chapter 10), you insert the gene intocells, usually bacteria, and get the cells to produce largeamounts of the protein. Once the protein is purified (by thetechniques described in Panel 4–3, pp. 160–161), you areready to attempt to solve its structure.
Interrogation
The toughest part is yet to come. To determine a protein’sstructure using X-ray crystallography, you first need to coaxthe protein into forming crystals—large, highly orderedarrays in which every protein has the same conformationand is perfectly aligned with its neighbors. Growing suchcrystals is still something of an art, as it requires a trial-and-error process of determining the proper conditions forforming the highest-quality crystals—selecting the rightions, the optimal temperature, and so on.
With crystals in hand, you are ready for the X-ray analysis.When a narrow beam of X-rays is directed at a protein crys-tal, the atoms in the protein molecules scatter the incom-ing X-rays. These scattered waves either reinforce or can-cel one another, producing a complex diffraction patternthat is collected by electronic detectors. The position andintensity of each spot in the diffraction pattern containsinformation about the position of the atoms in the proteincrystal (Figure 4–12).Because these patterns are so complex—even a small pro-tein can generate 25,000 discrete spots—computers areused to interpret them. By combining information obtainedfrom such maps with the amino acid sequence of the pro-tein, you can generate an atomic model of the protein’sstructure. To determine if the protein undergoes conforma-tional changes in its structure when it binds a ligand thatboosts its activity, you might subsequently try crystallizingit in the presence of the ligand. With crystals of sufficientquality, even small atomic movements can be detected bycomparing the structures obtained in the presence andabsence of stimulatory or inhibitory ligands.There is a different way to solve the structure of your pro-tein, one that does not require obtaining protein crystals.If the protein is small—say, 40,000 daltons or less—youcan determine its structure by nuclear magnetic reso-nance (NMR) spectroscopy. This technique takes advan-tage of the fact that the nuclei of many atoms are intrin-sically magnetic and that their behavior is influenced bysurrounding atoms. In NMR spectroscopy, a solution of pure protein is placed in a strong magnetic field and thenbombarded with radio waves of different frequencies. Thehydrogen nuclei in the protein will generate an NMR sig-nal that can be used to determine the distances betweenthe amino acids and between different parts of the pro-tein. Again, combined with the known amino acidsequence, an NMR spectrum can allow you to computethe three-dimensional structure of the protein (Figure4–13).If the protein is larger than 40,000 daltons, you can try tobreak the polypeptide up into its constituent functionaldomains and then perform this type of NMR analysis oneach domain.Recently, X-ray crystallography was used to determinethe structure of the ribosome, a complex cellular machinemade of several RNAs and more than 50 proteins. In thefuture, improvements in X-ray crystallography and NMRspectroscopy should permit rapid analysis of many moreproteins and protein machines. And once enough struc-tures have been determined, we may be able to generatealgorithms for accurately predicting structure based solelyon the amino acid sequence itself. After all, it is thesequence of the amino acids alone that determines howeach protein folds up into its three-dimenionsal structure.
Figure 4–11Mass spectrometry can be used to identifyproteins by determining the precise masses of peptidesderived from them.
In this example, the protein of interestis excised from a two-dimensional polyacrylamide gel andthen digested with trypsin. The peptide fragments areloaded onto the mass spectrometer and their exact massesmeasured. Sequence databases are then searched to findthe protein whose calculated tryptic digest profile matchesthese values. (Micrograph courtesy of Patrick O’Farrell.)
Chapter 4: Protein Structure and Function
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single protein spot excised from gelGENOME SEQUENCE DATABASE SEARCHEDFOR MATCHES WITH THEORETICAL MASSESCALCULATED FOR ALL TRYPSINRELEASED PEPTIDESIDENTIFICATION AND ISOLATIONOF CORRESPONDING GENENCPEPTIDES RELEASEDBY TRYPTIC DIGESTIONAND THEIR MASSESMEASURED USING AMASS SPECTROMETER
a b u n d a n c e
01600
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