Essential Cell Biology 5th edition
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Essential Cell Biology 5th edition

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158 CHAPTER 4 Protein Structure and Functionprotein scaffoldsamyloidproduct(A)RNA scaffolds(B) (C) (D)2 µm 1 µm 1 µmFigure 4−54 Intracellular condensatescan form biochemical subcompartmentsin cells. These large aggregates form as aresult of multiple weak binding interactionsbetween scaffolds and other macromolecules.When these macromolecule–macromoleculeinteractions become sufficiently strong, a“phase separation” occurs. This creates twodistinct aqueous compartments, in one ofwhich the interacting molecules are denselyaggregated. Such intracellular condensatesconcentrate a select set of macromolecules,thereby producing regions with a specialbiochemistry without the use of anencapsulating membrane.(A) Schematic illustration of a phaseseparatedintracellular condensate. Thesecondensates can create a factory thatcatalyzes the formation of a specific type ofproduct, or they can serve to store importantentities, such as specific mRNA molecules,for later use. As shown, reversible amyloidstructures often help to create theseaggregates. These β-sheet structures formbetween regions of unstructured amino acidsequence within the larger protein scaffolds.(B–D) Three examples that illustrate howintracellular condensates (colorized regions)are thought to be used by cells. (B) Inside theinterphase nucleus, the nucleolus is a largefactory that produces ribosomes. In addition,many scattered RNA production factoriesconcentrate the protein machines thattranscribe the genome. (C) In the cytoplasm,a matrix forms the centrosome that nucleatesthe assembly of microtubules. (D) In a patchunderlying the plasma membrane at thesynapse where communicating nerve cellstouch, multiple interacting scaffolds producelarge protein assemblies; these create a localbiochemistry that makes possible memoryformation and storage in the nerve cellnetwork. (B, courtesy of E.G. Jordan andJ. McGovern; C, from M. McGill,D.P. Highfield, T.M. Monahan, andB.R. Brinkley, J. Ultrastruct. Res. 57:43–53,1976. With permission from Elsevier;D, courtesy of Cedric Raine.)“hydrogel” that pulls other molecules into the condensate (Figure 4−54).Amyloid-forming proteins thus have functional roles in cells. But for ahandful of these amyloid-forming proteins, mutation or perturbation canlead to neurological disease, which is how some of them were initiallydiscovered.HOW PROTEINS ARE STUDIEDECB5 04.54Understanding how a particular protein functions calls for detailed structuraland biochemical analyses—both of which require large amounts ofpure protein. But isolating a single type of protein from the thousandsof other proteins present in a cell is a formidable task. For many years,proteins had to be purified directly from the source—the tissues in whichthey are most plentiful. That approach was inconvenient, entailing, forexample, early-morning trips to the slaughterhouse. More importantly,the complexity of intact tissues and organs is a major disadvantage whentrying to purify particular molecules, because a long series of chromatographysteps is generally required. These procedures not only take weeksto perform, but they also yield only a few milligrams of pure protein.Nowadays, proteins are more often isolated from cells that are grown ina laboratory (see, for example, Figure 1−39). Often these cells have been“tricked” into making large quantities of a given protein using the geneticengineering techniques discussed in Chapter 10. Such engineered cellsfrequently allow large amounts of pure protein to be obtained in only afew days.In this section, we outline how proteins are extracted and purified fromcultured cells and other sources. We describe how these proteins areanalyzed to determine their amino acid sequence and their three-dimensionalstructure. Finally, we discuss how technical advances are allowingproteins to be analyzed, cataloged, manipulated, and even designed fromscratch.Proteins Can Be Purified from Cells or TissuesWhether starting with a piece of liver or a vat of bacteria, yeast, or animalcells that have been engineered to produce a protein of interest, thefirst step in any purification procedure involves breaking open the cellsto release their contents. The resulting slurry is called a cell homogenateor extract. This physical disruption is followed by an initial fractionationprocedure to separate out the class of molecules of interest—for example,all the soluble proteins in the cell (Panel 4−3, pp. 164–165).With this collection of proteins in hand, the job is then to isolate thedesired protein. The standard approach involves purifying the proteinthrough a series of chromatography steps, which use different materialsto separate the individual components of a complex mixture into

How Proteins Are Studied159portions, or fractions, based on the properties of the protein—such assize, shape, or electrical charge. After each separation step, the resultingfractions are examined to determine which ones contain the proteinof interest. These fractions are then pooled and subjected to additionalchromatography steps until the desired protein is obtained in pure form.matrix ofaffinitycolumnprotein X covalentlyattached tocolumn matrixThe most efficient forms of protein chromatography separate polypeptideson the basis of their ability to bind to a particular molecule—a processcalled affinity chromatography (Panel 4−4, p. 166). If large amounts ofantibodies that recognize the protein are available, for example, they canbe attached to the matrix of a chromatography column and used to helpextract the protein from a mixture (see Panel 4−2, pp. 140–141).Affinity chromatography can also be used to isolate proteins that interactphysically with a protein being studied. In this case, the purified proteinof interest is attached tightly to the column matrix; the proteins that bindto it will remain in the column and can then be removed by changing thecomposition of the washing solution (Figure 4−55).Proteins can also be separated by electrophoresis. In this technique, amixture of proteins is loaded onto a polymer gel and subjected to anelectric field; the polypeptides will then migrate through the gel at differentspeeds depending on their size and net charge (Panel 4−5, p. 167). Iftoo many proteins are present in the sample, or if the proteins are verysimilar in their migration rate, they can be resolved further using twodimensionalgel electrophoresis (see Panel 4−5). These electrophoreticapproaches yield a number of bands or spots that can be visualized bystaining; each band or spot contains a different protein. Chromatographyand electrophoresis—both developed more than 70 years ago but greatlyimproved since—continue to be instrumental in building an understandingof what proteins look like and how they behave. These and otherhistorical breakthroughs are described in Table 4−2.Once a protein has been obtained in pure form, it can be used in biochemicalassays to study the details of its activity. It can also be subjectedto techniques that reveal its amino acid sequence and, ultimately, its precisethree-dimensional structure.Determining a Protein’s Structure Begins withDetermining Its Amino Acid SequenceThe task of determining a protein’s primary structure—its amino acidsequence—can be accomplished in several ways. For many years,sequencing a protein was done by directly analyzing the amino acidsin the purified protein. First, the protein was broken down into smallerpieces using a selective protease; the enzyme trypsin, for example,cleaves polypeptide chains on the carboxyl side of a lysine or an arginine.Then the identities of the amino acids in each fragment were determinedchemically. The first protein sequenced in this way was the hormoneinsulin in 1955.A much faster way to determine the amino acid sequence of proteins thathave been isolated from organisms for which the full genome sequenceis known is a method called mass spectrometry. This technique determinesthe exact mass of every peptide fragment in a purified protein,which then allows the protein to be identified from a database that containsa list of every protein thought to be encoded by the genome of therelevant organism. Such lists are computed by taking the organism’sgenome sequence and applying the genetic code (discussed in Chapter 7).To perform mass spectrometry, the peptides derived from digestion withtrypsin are blasted with a laser. This treatment heats the peptides, causingthem to become electrically charged (ionized) and ejected in theELUTION WITHHIGH SALTOR A CHANGEIN pHMIXTURE OFPROTEINSAPPLIEDTO COLUMNproteins thatbind to protein Xadhere to columnpurified X-binding proteinsmost proteins passthrough the columnFigure 4−55 Affinity chromatography canbe used to isolate the binding partners ofa protein of interest. The purified proteinof interest (protein X) is covalently attachedto the matrix of a chromatography column.An extract containing a mixture of proteinsis then loaded onto the column. Thoseproteins that ECB5 associate 04.55 with protein X insidethe cell will usually bind to it on the column.Proteins not bound to the column pass rightthrough, and the proteins that are boundtightly to protein X can then be released bychanging the pH or ionic composition of thewashing solution.

How Proteins Are Studied

159

portions, or fractions, based on the properties of the protein—such as

size, shape, or electrical charge. After each separation step, the resulting

fractions are examined to determine which ones contain the protein

of interest. These fractions are then pooled and subjected to additional

chromatography steps until the desired protein is obtained in pure form.

matrix of

affinity

column

protein X covalently

attached to

column matrix

The most efficient forms of protein chromatography separate polypeptides

on the basis of their ability to bind to a particular molecule—a process

called affinity chromatography (Panel 4−4, p. 166). If large amounts of

antibodies that recognize the protein are available, for example, they can

be attached to the matrix of a chromatography column and used to help

extract the protein from a mixture (see Panel 4−2, pp. 140–141).

Affinity chromatography can also be used to isolate proteins that interact

physically with a protein being studied. In this case, the purified protein

of interest is attached tightly to the column matrix; the proteins that bind

to it will remain in the column and can then be removed by changing the

composition of the washing solution (Figure 4−55).

Proteins can also be separated by electrophoresis. In this technique, a

mixture of proteins is loaded onto a polymer gel and subjected to an

electric field; the polypeptides will then migrate through the gel at different

speeds depending on their size and net charge (Panel 4−5, p. 167). If

too many proteins are present in the sample, or if the proteins are very

similar in their migration rate, they can be resolved further using twodimensional

gel electrophoresis (see Panel 4−5). These electrophoretic

approaches yield a number of bands or spots that can be visualized by

staining; each band or spot contains a different protein. Chromatography

and electrophoresis—both developed more than 70 years ago but greatly

improved since—continue to be instrumental in building an understanding

of what proteins look like and how they behave. These and other

historical breakthroughs are described in Table 4−2.

Once a protein has been obtained in pure form, it can be used in biochemical

assays to study the details of its activity. It can also be subjected

to techniques that reveal its amino acid sequence and, ultimately, its precise

three-dimensional structure.

Determining a Protein’s Structure Begins with

Determining Its Amino Acid Sequence

The task of determining a protein’s primary structure—its amino acid

sequence—can be accomplished in several ways. For many years,

sequencing a protein was done by directly analyzing the amino acids

in the purified protein. First, the protein was broken down into smaller

pieces using a selective protease; the enzyme trypsin, for example,

cleaves polypeptide chains on the carboxyl side of a lysine or an arginine.

Then the identities of the amino acids in each fragment were determined

chemically. The first protein sequenced in this way was the hormone

insulin in 1955.

A much faster way to determine the amino acid sequence of proteins that

have been isolated from organisms for which the full genome sequence

is known is a method called mass spectrometry. This technique determines

the exact mass of every peptide fragment in a purified protein,

which then allows the protein to be identified from a database that contains

a list of every protein thought to be encoded by the genome of the

relevant organism. Such lists are computed by taking the organism’s

genome sequence and applying the genetic code (discussed in Chapter 7).

To perform mass spectrometry, the peptides derived from digestion with

trypsin are blasted with a laser. This treatment heats the peptides, causing

them to become electrically charged (ionized) and ejected in the

ELUTION WITH

HIGH SALT

OR A CHANGE

IN pH

MIXTURE OF

PROTEINS

APPLIED

TO COLUMN

proteins that

bind to protein X

adhere to column

purified X-binding proteins

most proteins pass

through the column

Figure 4−55 Affinity chromatography can

be used to isolate the binding partners of

a protein of interest. The purified protein

of interest (protein X) is covalently attached

to the matrix of a chromatography column.

An extract containing a mixture of proteins

is then loaded onto the column. Those

proteins that ECB5 associate 04.55 with protein X inside

the cell will usually bind to it on the column.

Proteins not bound to the column pass right

through, and the proteins that are bound

tightly to protein X can then be released by

changing the pH or ionic composition of the

washing solution.

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