H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 40
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In thisexperimental protocol, a cell sample is exposed to a radiolabeled compound—the “pulse”—for a brief period of time,then washed with buffer to remove the labeled pulse, and finally incubated with a nonlabeled form of the compound—the “chase” (Figure 3-36). Samples taken periodically areassayed to determine the location or chemical form of theradiolabel. A classic use of the pulse-chase technique was instudies to elucidate the pathway traversed by secreted proteins from their site of synthesis in the endoplasmic reticulumto the cell surface (Chapter 17).Mass Spectrometry Measures the Massof Proteins and PeptidesA powerful technique for measuring the mass of moleculessuch as proteins and peptides is mass spectrometry.
ThisLaserMetaltarget1 Ionization++2 AccelerationSampleIntensity94+3DetectionLightest ionsarrive atdetector firstTime▲ EXPERIMENTAL FIGURE 3-37 The molecular weight ofproteins and peptides can be determined by time-of-flightmass spectrometry. In a laser-desorption mass spectrometer,pulses of light from a laser ionize a protein or peptide mixturethat is absorbed on a metal target ( 1 ). An electric fieldaccelerates the molecules in the sample toward the detector( 2 and 3 ). The time to the detector is inversely proportionalto the mass of a molecule. For molecules having the samecharge, the time to the detector is inversely proportional to themass.
The molecular weight is calculated using the time of flightof a standard.3.6 • Purifying, Detecting, and Characterizing Proteinstechnique requires a method for ionizing the sample, usuallya mixture of peptides or proteins, accelerating the molecular ions, and then detecting the ions.
In a laser desorptionmass spectrometer, the protein sample is mixed with an organic acid and then dried on a metal target. Energy from alaser ionizes the proteins, and an electric field accelerates theions down a tube to a detector (Figure 3-37). Alternatively, inan electrospray mass spectrometer, a fine mist containing thesample is ionized and then introduced into a separationchamber where the positively charged molecules are accelerated by an electric field. In both instruments, the time offlight is inversely proportional to a protein’s mass and directly proportional to its charge. As little as 1 1015 mol(1 femtomole) of a protein as large as 200,000 MW can bemeasured with an error of 0.1 percent.Protein Primary Structure Can Be Determinedby Chemical Methods and from Gene SequencesThe classic method for determining the amino acid sequenceof a protein is Edman degradation.
In this procedure, the freeamino group of the N-terminal amino acid of a polypeptideis labeled, and the labeled amino acid is then cleaved fromthe polypeptide and identified by high-pressure liquid chromatography. The polypeptide is left one residue shorter, witha new amino acid at the N-terminus. The cycle is repeated onthe ever shortening polypeptide until all the residues havebeen identified.Before about 1985, biologists commonly used the Edmanchemical procedure for determining protein sequences.
Now,however, protein sequences are determined primarily byanalysis of genome sequences. The complete genomes of several organisms have already been sequenced, and the database of genome sequences from humans and numerousmodel organisms is expanding rapidly. As discussed in Chapter 9, the sequences of proteins can be deduced from DNAsequences that are predicted to encode proteins.A powerful approach for determining the primary structure of an isolated protein combines mass spectroscopy andthe use of sequence databases.
First, mass spectrometry isused to determine the peptide mass fingerprint of the protein.A peptide mass fingerprint is a compilation of the molecularweights of peptides that are generated by a specific protease.The molecular weights of the parent protein and its proteolytic fragments are then used to search genome databasesfor any similarly sized protein with identical or similar peptide mass maps.Peptides with a Defined Sequence Can BeSynthesized ChemicallySynthetic peptides that are identical with peptides synthesized in vivo are useful experimental tools in studies of proteins and cells.
For example, short synthetic peptides of10–15 residues can function as antigens to trigger the production of antibodies in animals. A synthetic peptide, when95coupled to a large protein carrier, can trick an animal intoproducing antibodies that bind the full-sized, natural proteinantigen. As we’ll see throughout this book, antibodies are extremely versatile reagents for isolating proteins from mixtures by affinity chromatography (see Figure 3-34c), forseparating and detecting proteins by Western blotting (seeFigure 3-35), and for localizing proteins in cells by microscopic techniques described in Chapter 5.Peptides are routinely synthesized in a test tube frommonomeric amino acids by condensation reactions that formpeptide bonds. Peptides are constructed sequentially by coupling the C-terminus of a monomeric amino acid with the Nterminus of the growing peptide.
To prevent unwantedreactions entailing the amino groups and carboxyl groupsof the side chains during the coupling steps, a protecting(blocking) group is attached to the side chains. Without theseprotecting groups, branched peptides would be generated. Inthe last steps of synthesis, the side chain–protecting groupsare removed and the peptide is cleaved from the resin onwhich synthesis takes place.Protein Conformation Is Determinedby Sophisticated Physical MethodsIn this chapter, we have emphasized that protein function isdependent on protein structure.
Thus, to figure out how aprotein works, its three-dimensional structure must beknown. Determining a protein’s conformation requires sophisticated physical methods and complex analyses of the experimental data. We briefly describe three methods used togenerate three-dimensional models of proteins.X-Ray Crystallography The use of x-ray crystallography todetermine the three-dimensional structures of proteins waspioneered by Max Perutz and John Kendrew in the 1950s.
Inthis technique, beams of x-rays are passed through a proteincrystal in which millions of protein molecules are preciselyaligned with one another in a rigid array characteristic of theprotein. The wavelengths of x-rays are about 0.1–0.2 nm,short enough to resolve the atoms in the protein crystal.Atoms in the crystal scatter the x-rays, which produce a diffraction pattern of discrete spots when they are interceptedby photographic film (Figure 3-38). Such patterns are extremely complex—composed of as many as 25,000 diffraction spots for a small protein. Elaborate calculations andmodifications of the protein (such as the binding of heavymetals) must be made to interpret the diffraction pattern andto solve the structure of the protein. The process is analogousto reconstructing the precise shape of a rock from the ripples that it creates in a pond.
To date, the detailed threedimensional structures of more than 10,000 proteins havebeen established by x-ray crystallography.Cryoelectron Microscopy Although some proteins readilycrystallize, obtaining crystals of others—particularly largemultisubunit proteins—requires a time-consuming trial-and-96CHAPTER 3 • Protein Structure and Functionof electrons to prevent radiation-induced damage to the structure. Sophisticated computer programs analyze the imagesand reconstruct the protein’s structure in three dimensions.Recent advances in cryoelectron microscopy permit researchers to generate molecular models that compare withthose derived from x-ray crystallography.
The use of cryoelectron microscopy and other types of electron microscopyfor visualizing cell structures are discussed in Chapter 5.(a)X-raysourceX-raybeamCrystalDetector(e.g., film)DiffractedbeamsNMR Spectroscopy The three-dimensional structures ofsmall proteins containing about as many as 200 amino acidscan be studied with nuclear magnetic resonance (NMR)spectroscopy. In this technique, a concentrated protein solution is placed in a magnetic field and the effects of differentradio frequencies on the spin of different atoms are measured. The behavior of any atom is influenced by neighboring atoms in adjacent residues, with closely spaced residuesbeing more perturbed than distant residues.
From the magnitude of the effect, the distances between residues can becalculated; these distances are then used to generate a modelof the three-dimensional structure of the protein.Although NMR does not require the crystallization of aprotein, a definite advantage, this technique is limited to proteins smaller than about 20 kDa.
However, NMR analysiscan also be applied to protein domains, which tend to besmall enough for this technique and can often be obtainedas stable structures.KEY CONCEPTS OF SECTION 3.6Purifying, Detecting, and Characterizing ProteinsProteins can be separated from other cell componentsand from one another on the basis of differences in theirphysical and chemical properties.■Centrifugation separates proteins on the basis of theirrates of sedimentation, which are influenced by theirmasses and shapes.■▲ EXPERIMENTAL FIGURE 3-38 X-ray crystallographyprovides diffraction data from which the three-dimensionalstructure of a protein can be determined. (a) Basiccomponents of an x-ray crystallographic determination.
When anarrow beam of x-rays strikes a crystal, part of it passes straightthrough and the rest is scattered (diffracted) in various directions.The intensity of the diffracted waves is recorded on an x-ray filmor with a solid-state electronic detector. (b) X-ray diffractionpattern for a topoisomerase crystal collected on a solid-statedetector. From complex analyses of patterns like this one, thelocation of every atom in a protein can be determined. [Part (a)adapted from L. Stryer, 1995, Biochemistry, 4th ed., W. H.
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