H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 41
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Freeman andCompany, p. 64; part (b) courtesy of J. Berger.]Gel electrophoresis separates proteins on the basis oftheir rates of movement in an applied electric field. SDSpolyacrylamide gel electrophoresis can resolve polypeptidechains differing in molecular weight by 10 percent or less(see Figure 3-32).■Liquid chromatography separates proteins on the basisof their rates of movement through a column packed withspherical beads. Proteins differing in mass are resolved ongel filtration columns; those differing in charge, on ionexchange columns; and those differing in ligand-bindingproperties, on affinity columns (see Figure 3-34).■Various assays are used to detect and quantify proteins.Some assays use a light-producing reaction or radioactivity to generate a signal. Other assays produce an amplifiedcolored signal with enzymes and chromogenic substrates.■error effort to find just the right conditions.
The structuresof such difficult-to-crystallize proteins can be obtained by cryoelectron microscopy. In this technique, a protein sample israpidly frozen in liquid helium to preserve its structure andthen examined in the frozen, hydrated state in a cryoelectronmicroscope.
Pictures are recorded on film by using a low doseAntibodies are powerful reagents used to detect, quantify, and isolate proteins. They are used in affinity chromatography and combined with gel electrophoresis in■Review the ConceptsWestern blotting, a powerful method for separating anddetecting a protein in a mixture (see Figure 3-35).Autoradiography is a semiquantitative technique for detecting radioactively labeled molecules in cells, tissues, orelectrophoretic gels.■Pulse-chase labeling can determine the intracellular fateof proteins and other metabolites (see Figure 3-36).■Three-dimensional structures of proteins are obtained byx-ray crystallography, cryoelectron microscopy, and NMRspectroscopy.
X-ray crystallography provides the most detailed structures but requires protein crystallization. Cryoelectron microscopy is most useful for large protein complexes, which are difficult to crystallize. Only relativelysmall proteins are amenable to NMR analysis.■PERSPECTIVES FOR THE FUTUREImpressive expansion of the computational power of computers is at the core of advances in determining the threedimensional structures of proteins. For example, vacuumtube computers running on programs punched on cardswere used to solve the first protein structures on the basisof x-ray crystallography.
In the future, researchers aim topredict the structures of proteins only on the basis ofamino acid sequences deduced from gene sequences. Thiscomputationally challenging problem requires supercomputers or large clusters of computers working in synchrony. Currently, only the structures of very smalldomains containing 100 residues or fewer can be predictedat a low resolution. However, continued developments incomputing and models of protein folding, combined withlarge-scale efforts to solve the structures of all protein motifs by x-ray crystallography, will allow the prediction ofthe structures of larger proteins. With an exponentially expanding database of motifs, domains, and proteins, scientists will be able to identify the motifs in an unknownprotein, match the motif to the sequence, and use this headstart in predicting the three-dimensional structure of theentire protein.New combined approaches will also help in in determining high-resolution structures of molecular machines such asthose listed in Table 3-1.
Although these very large macromolecular assemblies usually are difficult to crystallize andthus to solve by x-ray crystallography, they can be imaged ina cryoelectron microscope at liquid helium temperatures andhigh electron energies. From millions of individual “particles,” each representing a random view of the protein complex, the three-dimensional structure can be built. Becausesubunits of the complex may already be solved by crystallography, a composite structure consisting of the x-ray-derivedsubunit structures fitted to the EM-derived model will be generated.
An interesting application of this type of study wouldbe the solution of the structures of amyloid and prion pro-97teins, especially in the early stages in the formation of insoluble filaments.Understanding the operation of protein machines will require the measurement of many new characteristics of proteins. For example, because many machines do nonchemicalwork of some type, biologists will have to identify the energy sources (mechanical, electrical, or thermal) and measure the amounts of energy to determine the limits of aparticular machine. Because most activities of machines include movement of one type or another, the force poweringthe movement and its relation to biological activity can bea source of insight into how force generation is coupled tochemistry. Improved tools such as optical traps and atomicforce microscopes will enable detailed studies of the forcesand chemistry pertinent to the operation of individual protein machines.KEY TERMS helix 61activation energy 74active site 75allostery 83amyloid filament 73autoradiography 93 sheet 61chaperone 69conformation 60cooperativity 83domain 63electrophoresis 87homology 68Km 76ligand 73liquid chromatography 90molecular machine 59motif 63motor protein 79peptide bond 60polypeptide 61primary structure 61proteasome 71protein 61proteome 60quaternary structure 66rate-zonal centrifugation 87secondary structure 61tertiary structure 62ubiquitin 71Vmax 76x-ray crystallography 95REVIEW THE CONCEPTS1.
The three-dimensional structure of a protein is determined by its primary, secondary, and tertiary structures.Define the primary, secondary, and tertiary structures. Whatare some of the common secondary structures? What are theforces that hold together the secondary and tertiary structures? What is the quaternary structure?2. Proper folding of proteins is essential for biologicalactivity. Describe the roles of molecular chaperones andchaperonins in the folding of proteins.3.
Proteins are degraded in cells. What is ubiquitin, andwhat role does it play in tagging proteins for degradation?What is the role of proteasomes in protein degradation?98CHAPTER 3 • Protein Structure and Function4. Enzymes can catalyze chemical reactions. How do enzymes increase the rate of a reaction? What constitutes theactive site of an enzyme? For an enzyme-catalyzed reaction,what are Km and Vmax? For enzyme X, the Km for substrateA is 0.4 mM and for substrate B is 0.01 mM.
Which substrate has a higher affinity for enzyme X?5. Motor proteins, such as myosin, convert energy into amechanical force. Describe the three general properties characteristic of motor proteins. Describe the biochemical eventsthat occur during one cycle of movement of myosin relativeto an actin filament.6. The function of proteins can be regulated in a number ofways. What is cooperativity, and how does it influence protein function? Describe how protein phosphorylation andproteolytic cleavage can modulate protein function.7. A number of techniques can separate proteins on thebasis of their differences in mass.
Describe the use of two ofthese techniques, centrifugation and gel electrophoresis. Theblood proteins transferrin (MW 76 kDa) and lysozyme (MW15 kDa) can be separated by rate zonal centrifugation or SDSpolyacrylamide gel electrophoresis. Which of the two proteins will sediment faster during centrifugation? Which willmigrate faster during electrophoresis?8. Chromatography is an analytical method used to separate proteins. Describe the principles for separating proteinsby gel filtration, ion-exchange, and affinity chromatography.9.
Various methods have been developed for detectingproteins. Describe how radioisotopes and autoradiographycan be used for labeling and detecting proteins. How doesWestern blotting detect proteins?10. Physical methods are often used to determine proteinconformation. Describe how x-ray crystallography, cryoelectron microscopy, and NMR spectroscopy can be used todetermine the shape of proteins.A N A LY Z E T H E DATAProteomics involves the global analysis of protein expression.
In one approach, all the proteins in control cells andtreated cells are extracted and subsequently separated usingtwo-dimensional gel electrophoresis. Typically, hundreds orthousands of protein spots are resolved and the steady-statelevels of each protein are compared between control andtreated cells. In the following example, only a few proteinspots are shown for simplicity.
Proteins are separated in thefirst dimension on the basis of charge by isoelectric focusing(pH 4–10) and then separated by size by SDS polyacrylamidegel electrophoresis. Proteins are detected with a stain suchas Coomassie blue and assigned numbers for identification.a. Cells are treated with a drug (“ Drug”) or left untreated(“Control”) and then proteins are extracted and separatedby two-dimensional gel electrophoresis. The stained gels areshown below.
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