unraveling the mysteries of protein folding

8/7/2019 Unraveling the mysteries of protein folding

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Alzheimers disease. Cystic fibro-sis. Mad Cow disease. An inher-ited form of emphysema. Evenmany cancers. Recent discoveriesshow that all these apparentlyunrelated diseases result from

protein folding gone wrong. Asthough that werent enough, manyof the unexpected difficultiesbiotechnology companies encoun-ter when trying to produce humanproteins in bacteria also resultfrom something amiss whenproteins fold.

What exactly is this phenom-enon? We all learned that pro-teins are fundamental compo-

nents of all living cells: our own,the bacteria that infect us, theplants and animals we eat. Thehemoglobin that carries oxygen toour tissues, the insulin that sig-nals our bodies to store excesssugar, the antibodies that fightinfection, the actin and myosin

that allow our muscles to con-tract, and the collagen that makesup our tendons and ligaments(and even much of our bones)allare proteins.

To make proteins, machinesknown as ribosomes string to-

gether amino acids into long,linear chains. Like shoelaces,these chains loop about each otherin a variety of ways (i.e., theyfold). But, as with a shoelace,only one of these many waysallows the protein to functionproperly. Yet lack of function isnot always the worst scenario.For just as a hopelessly knottedshoelace could be worse than one

that wont stay tied, too much of amisfolded protein could be worsethan too little of a normally foldedone. This is because a misfoldedprotein can actually poison thecells around it.

Early Studies

The importance of protein foldinghas been recognized for manyyears. Almost a half-century ago,Linus Pauling discovered two quite

Unraveling the Mystery of Protein Folding

A series of articles for general audiences

This series of essays wasdeveloped as part of FASEBsefforts to educate the generalpublic, and the legislators whomit elects, about the benefits offundamental biomedicalresearchparticularly how

investment in such researchleads to scientific progress,improved health, and economicwell-being.

W. A. (Bill) Thomasson, Ph.D., is a science

and medical writer based in Oak Park, IL.

Jonathan A. King served as science writer.

This series is available on FASEBs Public

Policy Home Page at

http://www.faseb.org/opa/ or as reprints

from FASEBs Office of Public Affairs, 9650

Rockville Pike, Bethesda, MD 20814.

by W. A. (Bill) Thomasson


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simple, regular arrangements ofamino acidsthe -helix and the-sheet (see the box, FundamentalPatterns of Protein Structure) that are found in almost everyprotein. And in the early 1960s,Christian Anfinsen showed that

the proteins actually tie them-selves: If proteins become un-folded, they fold back into propershape of their own accord; noshaper or folder is needed.

Of course, neither Pauling norAnfinsen nor the committees thatawarded them their respectiveNobel prizes knew at the timethat these discoveries would be soimportant for understanding

Alzheimers disease or cysticfibrosis. And when Pauling, atleast, was doing his breakthroughstudies, he could hardly haveimagined the enormity of todaysbiotechnology industry. Whatscientists did know is that anyprocess that was so fundamentalto life as protein folding wouldhave to be of the utmost practicalimportance.

But research did not stop withPauling and Anfinsen. Indeed, wenow know that Anfinsens conclu-sions needed expansion: Some-times a protein will fold into awrong shape. And some proteins,aptly named chaperones, keeptheir target proteins from gettingoff the right folding path (see thebox, Molecular Chaperones).These two small but important

additions to Anfinsens theoryhold the keys to protein foldingdiseases.

Weve known since antiquity(but didnt know we knew) thatprotein folding can go wrong.When we boil an egg, the proteinsin the white unfold. But when the

egg cools, the proteins dont re-turn to their original shapes.Instead, they form a solid, in-soluble (but tasty) mass. This ismisfolding. Similarly, biochemistshave always cursed the tendencyof some proteins to form the in-

soluble lumps in the bottom oftheir test tubes. We now knowthat these, too, were proteinsfolded into the wrong shapes.

Until recently, biochemistslacked the tools to study theseinsoluble lumps. Nor did theyexpect such masses would beparticularly interesting. Theprevailing view at the time wasthat the lumps were just hope-

lessly tangled and completelyamorphous masses of proteinfibers (aggregation). Researcherseventually discovered that theseaggregates of incorrect foldingcould be highly structured, butbefore this crucial insight andbefore proper investigative toolswere developed, biochemistssimply threw their fouled testtubes away.

Gunking Up Tissues

As far back as the start of thiscentury, physicians have beennoticing that certain diseases arecharacterized by extensive pro-tein deposits in certain tissues.Most of these diseases are rare,but Alzheimers is not. It wasAlois Alzheimer himself whonoted the presence of neurofibril-

lary tangles and neuritic plaquein certain regions of his patientsbrain. Tangles are more or lesscommon in diseases that featureextensive nerve cell death; plaque,however, is specific to Alzheimers.The major question, which hasonly recently been answered, is


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whether plaque causesAlzheimers or, like tangles, is aconsequence of it.

Further investigation showedthat neuritic plaque (unrelated tothe plaque that clogs atheroscle-rotic blood vessels and causes

heart attacks) is composed almostentirely of a single protein. De-posits of large amounts of asingle, insoluble protein aroundthe degenerating nerve cells ofAlzheimers disease eventuallyprovided a key to understandingthe disorder.

It was development of the bio-technology industry that unex-pectedly spurred interest in in-

soluble protein gunk. This indus-try can produce proteins (oftenotherwise difficult-to-obtain hu-man proteins) quickly and eco-nomically in bacteria. To theirsurprise, however, scientists whoworked for biotech companiesoften found two things: proteinthat was supposed to be solubleinstead precipitated as insolubleinclusion bodies within the bacte-

ria and proteins that were sup-posed to be secreted into thesurrounding medium instead gotstuck at the bacterial cell wall.

This puzzling activity led scien-tists, almost for the first time, toseriously study just what goeswrong during protein folding.

Further Studies

In the decades after Anfinsens

work, the National Institutes ofHealth and the National ScienceFoundation continued to financeresearch in several laboratories.Working in relative obscurity,these protein biochemists tried todiscover how a completely un-folded protein, with hundreds of

millions of potential folded statesto choose from, consistently foundthe correct oneand did so withinseconds to minutes.

Could there be specific, criticalintermediates (partially foldedchains) in the folding process?

This turned out to be a difficultquestion to answer. Partiallyfolded chains dont stay that wayvery long; they become fullyfolded chains in a fraction of asecond. Nevertheless, by theearly 1980s researchers had notonly found clear evidence for theexistence ofpartially folded pro-teins, but also realized the keyrole these played in the folding

process.One study involved the diffi-

culty in getting bovine growthhormone to fold properly. Al-though the unfolded proteins werenot sticky, and the fully foldedproteins were not sticky, thepartially folded molecules stuck toeach othera first clue as to theorigins of misfolded lumps (atleast for purified proteins in test

tubes). It still remained unclearwhy misfolding occurred in cellsunder certain circumstances butnot under others.

Temperature Sensitivity

The early 1980s also saw one ofthe first serious investigations ofprotein misfolding. These stud-ies focused on temperature-sensitive mutations (mutations

allowing growth at 75_F but notat 100_F) in the tailspike proteinof bacteriophage P22. Neitherbacteriophage P22, a virus thatinfects certain bacteria, nor itstailspike protein has any practi-cal importance in themselves.Faced with thorny problems,


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The molecular surface of the immunodominant heat-shock chaperonin-

10 of mycobacterium leprae shows the multiple subunits of the protein

complex forming one doughnut layer (Protein data bank entry 1lep).

The second complex would stack together with this complex having the

chaperoned protein inside the open hole. Each subunit is colored differ-

ently. (Courtesy: Stanley Krystek, Bristol-Myers Squibb, Pharmaceuti-

cal Research Institute)

Molecular Chaperones

The beauty and the frustration of science are that they are constantly producing surprises. Almostthree decades after Christian Anfinsen had won the Nobel Prize for demonstrating that protein foldingis governed solely by the protein itself, other scientists discovered that some proteins have helped inthe process. This help consists of proteins called chaperones (or chaperonins) that are associated withthe target protein during part of its folding process. However, once folding is complete (or even before)the chaperone will leave its current protein molecule and go on to support the folding of another.

Proper folding of some proteins appears to call for not just one chaperone, but several. Especiallyclear evidence for such multi-step chaperoning is provided by test-tube experiments on a protein knownas rhodanese. Proper folding of this pro-tein, the experiments show, requires fivedifferent chaperone-type proteins acting attwo distinct steps in the operation. Earlyin the folding process, rhodanese binds toa chaperone known as DnaK; the complexthat binds a further chaperone: DnaJ.Somewhat later, a protein known as GrpEcatalyzes transfer of the partially foldedrhodanese to another chaperone, GroEL,

and its partner, GroES. These latter twoproteins then see rhodanese all the waythrough to its properly folded state.

Several lines of evidence suggest thatchaperones primary function may be toprevent aggregation. For example, a chap-erone found in the power plant organellesof mammalian cells (but otherwise simi-lar to GroEL) has been shown to consist of14 protein chains arranged as two dough-nuts stacked on top of each other (see fig-ure). The chaperoned protein sits inside

the two doughnut holes, safely sequesteredfrom other molecules with which it mightaggregate.

A role for chaperones in preventing ag-gregation is also suggested by what hap-pens to mammalian proteins produced inbacteria. Although bacteria have chaper-ones, they are not the same as those inmammals. It is thus easy to imagine thatthey may be relatively ineffective towardmammalian proteins, and that this re-sults in the aggregation so often seen.Indeed, there has been one case in which bacteria engineered to overproduce their own chaperonessuccessfully produced a mammalian protein that otherwise irretrievably aggregated. Unfortunately,this approach has failed in other cases. And no one has yet reported introduction of mammalianchaperones into bacteria to help produce soluble mammalian proteins. Yet this, along with the intro-duction of mutations that block the aggregation pathway and the discovery of small molecules thatprevent aggregation, is one of the most promising ways to overcome the roadblocks that biotechnologycompanies have so often encountered.


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however, scientists often look forexperimental systems that willallow them to get a foothold orfind a way around them. In thiscase, they thought that a largeprotein, whose folding passesthrough multiple stages, would bea good system for looking at fold-

ing pathways within cells. Manytemperature-sensitive mutationshad already been isolated inbacteriophages, but never exam-ined for their effect on folding.

Their hopes were realized: Themajority of the temperature-sensitive mutations they found,despite having only one aminoacid altered, caused the tailspikeprotein to end up as insoluble

gunk at high temperatures. Sincethese folding failures were occur-ring in bacterial cells that weregrowing in the laboratory, it wasnow possible to analyze whatwent wrong in a proteins foldingprocess.

The obvious guess at the time

was that the mutant proteinswere less stable. After all, thetemperature scale is fundamen-tally defined by how much atomic-scale shaking or motion is goingon; in other words, the higher thetemperature, the more shakingthere is. This implies that a less

stable protein is more likely to fallapart at elevated temperaturesand might therefore be morelikely to end up (like cooked eggs)as insoluble gunk.

But this turned out not to bethe case. If the mutant chainswere allowed to fold up at lowtemperature, and were thenheated, they were as stable aswild-type. It turned out to be a

partially folded intermediate, onthe route from the random shoe-lace to the correctly folded pro-tein, that was sensitive to tem-perature. At higher temperaturesthese intermediates would stick tothemselves and be unable to reachthe properly folded state.

Partially folded intermedi-ates at the junction be-tween productive and off-pathway folding. General-ized pathways showing an

inclusion body derivedfrom an intermediate onthe folding pathway. Thisillustration shows aspeculative intermediatein the formation of an /protein in which a helicaldomain is docking againsta sheet. In the inclusionbody pathway, the sameinteraction proceedsbetween intermediates,resulting in a polymericaggregate (3, 62). [Re-

drawn from FASEB J. 10,58 (1997)]


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This turned out to be a generalproblem in the folding of manyproteins: They have to passthrough partially folded states inwhich they are delicately poisedbetween folding all the way to thecorrect state or becoming seri-

ously stuck as a result of prema-ture entanglement with othermolecules. Recognizing that itwas the intermediates and not thefully folded protein that were introuble opened the way to under-standing some aspect of a range ofdiseases.

Familial AmyloidoticPolyneuropathy

Over the past several years, col-laborators have conducted similarstudies in connection with a hu-man disease. The minor differ-ences between their results andothers are very revealing.

In the hereditary disease famil-ial amyloidotic polyneuropathy(FAP), peripheral nerves andother organs are damaged bydeposits of amyloid-type protein.

Although the disorder is quiterare, extensive genetic studieshave shown that the diseaseresults from mutations in theprotein transthyretin. As with theP22 tailspike protein,transthyretin contains largeamounts of-sheet structure andnormally consists of several iden-tical amino acid chains (four inthis case) associated into a single,

three-dimensional structure.FAP results from any of more

than 50 distinct mutations withinthe transthyretin protein, eachaltering a single amino acid.After studying several of these,scientists found that their four-chain structure is less stable

under mildly acid conditions than

is the wild-type structure. Thiscontrasts with the P22 tailspikemutations, which fold slowly butare stable once folded. It alsoappears that transthyretin aggre-gation takes place from a mono-meric unfolding intermediate,rather than the folding intermedi-ate involved in P22 tailspikeaggregation (the pathway may ormay not be the same in both

directions).In both cases, however, the

single-chain intermediates havestructures that nature has de-signed for association with otherchains of the same type. It appar-ently takes only a very smallchange in the shape of these

A ribbon diagram showing two molecules of the protein transthyretin

docked together. The spiral coils of ribbon represent a-helix, while the flat

arrows running alongside each other represent b-sheet. (Generated with

Molscript by Scott Peterson, Texas A&M University)


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intermediates to alter their nor-mal linkage with two or threeother chains into an endless seriesof linkages that creates insolublegunk.

There is yet another contrastbetween the P22 tailspike muta-

tions and those in transthyretin:From the P22 viruss view, theproblem with the tailspike muta-tions is that not enough normalprotein is made. People withtransthyretin mutations, on theother hand, have all the normaltransthyretin they need to carryout its usual function (transport-ing the thyroid hormone). Theproblem is that, as the protein is

being broken down, it forms in-soluble gunk, and the insolublegunk poisons the tissues where itis deposited.

Alzheimers Disease

FAP is a rare disease; not soAlzheimers, which afflicts 10percent of those over 65 years oldand perhaps half of those over 85.Every year Alzheimers not only

kills 100,000 Americans, but alsocosts society $82.7 billion to carefor its victims.

In 1991, several different re-search groups found that indi-viduals with specific mutations intheir amyloid precursor proteindeveloped Alzheimers disease asearly as age 40. The body pro-cesses amyloid precursor proteininto a soluble peptide (small

protein) known as A; undercertain circumstances, A thenaggregates into long filamentsthat cannot be cleared by thebodys usual scavenger mecha-nisms. These aggregates thenform the -amyloid, which makeup the neuritic plaque in

Alzheimer patients. So the con-sistent association of amyloidprecursor protein mutations withearly-onset Alzheimers hasfinally answered a long-debatedquestion: the deposition of neu-ritic plaque is part of the pathway

leading to the disease, not a lateconsequence of it.

To help understand the Aaggregation process, researcherschemically synthesized fragmentsof the 40-amino-acid-long peptide.By using these fragments, theyshowed that the key step is get-ting started. Specifically, theprecursor fragments have to forma specific nucleus, which then

grows into the amyloid process.Possibly the slowness of this firststep is why Alzheimers disease isalmost entirely limited to olderpeople, and it could be that themutations in amyloid precursorprotein that lead to early-onsetAlzheimers are the ones thatmake it progress more quicklyand easily.

Even so, Aremains soluble inmost people. Most individualswho develop Alzheimers diseasehave the normal form of amyloidprecursor protein, indistinguish-able from that in people whonever acquire the disorder. Whythe same form of Aaggregates insome peoples brain but not inothers remains a mystery, al-though a recent discovery hassuggested an intriguing possibility.

We know that people withdifferent genetic variants of theprotein apolipoprotein E (apoE)have quite different risks of devel-oping Alzheimers disease. Com-pared to those with the mostcommon variant, known as apoE3,those with the apoE4 variant are


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significantly more likely to de-velop the disease. Some studiessuggest that those with the apoE2variant may be at lower risk,although other studies disagree.

These findings are particularlysurprising because apoE is best

known as part of the complex thattransports cholesterol and otherfatty materials in the blood-stream. What could a fat-trans-porting protein have to do withAlzheimers disease? It may besignificant that small amounts ofthis protein are associated withneuritic plaque and that apoEbinds to A in the test tube. Theresults of this binding are in

dispute, however.Researchers report that adding

apoE to a test-tube solution ofsoluble A causes rapid formationof plaque-type -amyloid fibersand that apoE4 does so morerapidly than apoE3. Others,however, have obtained oppositeresults: apoE prevents fibril for-mation. Thus, whereas somesuggest that apoE acts as a patho-

logical chaperone, one that actu-ally promotes misfolding, otherresearchers believe that it exertsa normal chaperones protectiveeffect. In either case, apoEsinfluence on the folding of Ab mayplay a major role in developmentof Alzheimers disease.

Mad Cow and Other Species

Perhaps the most interesting

example of a protein folding disor-der is Mad Cow disease and itshuman equivalent, Creutzfeldt-Jacob disease. These diseases,along with the sheep versionknown as scrapie, have had thescientific community in an uproarfor years. They are infectious

diseases transmitted by prions, or

protein particles. Prions seem tobe pure protein; they containneither DNA nor RNA. Yet aninfectious agent is necessarilyself-replicating. How, scientistsasked themselves, could a pureprotein replicate itself?

The answer now starting toemerge may be viewed as a varia-tion on the concept of the patho-logical chaperone, only in this

case the protein serves as its ownchaperone.

The protein whose aggregationdamages nerve cells in Mad Cowdisease is constantly being pro-duced by the body. Normally,though, it folds properly, remainssoluble, and is disposed of withoutproblem. But suppose that some-how a small amount misfolds in aparticular way so as to become a

scrapie prion. If this scrapie prionbumps into a normal-foldingintermediate, it shifts the foldingprocess in the scrapie directionand the protein, despite its per-fectly normal amino acid se-quence, ends up as more scrapieprion. And the process continues:

Courtesy: National Institute on Aging, Bethesda, MD.


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So long as the body keeps producingthe normal protein, a little bit ofscrapie prion can keep on creat-ing more and then more. Ineffect, the prion is replicatingitself without needing anynucleic acid of its own.

What old-school scientists findeven more strange is that theprocess resembles somethingakin to genetics. Differentstrains of these diseases, withsomewhat different clinicalsymptoms, breed true as theyare transmitted from one animalor human to another. Moreover,these strain differences areassociated with slight differ-

ences in the protein depositsthat apparently cause the dis-ease. (Scientists have recentlyused these strain differences toshow that a few Britons trulyhave Mad Cow disease, the formseen in cattle, rather than theusual human form ofCreutzfeldt-Jacob disease.)

Just as replication can occurwithout DNA or RNA, other

experiments have shown howgenetics is possible withoutnucleic acids. Thus, when re-searchers mix seed quantities oftwo different scrapie prionstrains in separate test tubeswith large amounts of normalprotein, each test tube producesmore of the specific scrapie prionstrain that was added. That is,each strain induces the normal

protein to fold in exactly the sameway as the original seed. Thestrain breeds true in the testtube, just as it does in the body.Odd as it may seem, genetics with-out nucleic acid is truly possible in

the world of protein folding.

Too Little, Too Late

Despite the examples of FAP,Alzheimers disease, and MadCow disease, in which the prob-lem derives from accumulation oftoxic, insoluble gunk, many hu-

man diseases arise from proteinmisfolding leaving too little of thenormal protein to do its job properly.The most common hereditarydisease of this type is cystic fibrosis.

Recent research has clearlyshown that the many, previouslymysterious symptoms of thisdisorder all derive from lack of aprotein that regulates the trans-port of the chloride ion across the

cell membrane. More recentlyscientists have shown that by farthe most common mutation un-derlying cystic fibrosis hinders thedissociation of the transport-regulator protein from one of itschaperones. Thus, the final stepsin normal folding cannot occur,and normal amounts of activeprotein are not produced.

A hereditary form of emphy-

sema shows an even greateranalogy to the mutations studiesin P22 tailspike protein. Investi-gators have found that one of themost common mutations produc-ing this disorder greatly slows thenormal folding process, just as theP22 temperature-sensitive muta-tions do. As with the tailspikemutations, the resulting buildupof a crucial folding intermediate

leads to aggregation, which de-prives affected individuals ofenough circulating

1-antitrypsin

to protect their lungs. Emphysemais the result.

As intriguing as these examplesmay be, there is a far more com-


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appears to be a promising treat-ment for FAP.

Developing small-moleculetherapies is quite straightforwardfor proteins like transthyretinthat naturally bind small mol-ecules, but these therapies are

more difficult to apply to proteinsthat do not have a small-moleculebinding site.

One of the few other groupscurrently publishing their re-search on small-molecule struc-ture stabilizers is working tostabilize p53, an acknowledgeddifficult target. In fact, onelaboratory has obtained encourag-ing results by using two different

approaches.Treatments based on our grow-

ing knowledge and contined re-search of protein folding are onthe way. When they arrive, thesaga that began with Paulingsfundamental studies of proteinstructure and Anfinsens investi-gation of what some call thesecond genetic code will reach itspractical fruition.

Suggested Readings

For a thorough discussion of protein

folding, read a thematic issue on the

topic in FASEB J. (1996) Protein Folding

10.

A very short review of protein folding

with superb illustration is: Jonathan

King (1993) The Unfolding Puzzle ofProtein Folding, Technology Review, 58-

61.

For an overview of the role of protein

folding in human disease see Gary

Taubes (1996) Misfolding the Way to

Disease, Science271, 1493-1495.

For a short, comprehensive review of

diseases due to protein misfolding, read

Philip J. Thomas, Bao-He Qu, and Peter

L. Pedersen (1995) Defective Protein

Folding as a Basis of Human Disease,

TIBS, 20, 456-459.

A good discussion of amyloid precursor

protein and its role in Alzheimers

disease is: Celia Hooper (1991) An

Exciting If in Alzheimers, The Journal

of NIH Research, 3 (April) 65-70.

For coverage of recent research on p53

and efforts to stabilize it, read: Rebecca

L. Rawls (1997) Keeping Cancer in

Check with p53, Chemical & Engineer-

ing News, 75 (February 18) 39- 41.

For everything you might want to knowabout prion disease, read Richard

Rhodes (1997) Deadly Feasts, Simon &

Schuster, New York, and a review by

Neil Stahl and Stanley B. Prusiner

(1991) Prions and prion proteins, FASEB

J. 5, 2799-2807.

unraveling the mysteries of protein folding

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