Proa Nati. Acad. Sci. USAVol. 80, pp. 7060-7064, December 1983Biochemistry

Genetic analysis of the folding pathway for the tail spike proteinof phage P22

(protein folding/temperature-sensitive mutant/subunit assembly/structural protein)

DAVID P. GOLDENBERG*, DONNA H. SMITH, AND JONATHAN KINGDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139

Communicated by William B. Wood, June 20, 1983

ABSTRACT Temperature-sensitive mutations in the gene en-coding the trimeric tail spike protein of phage P22 interfere withprotein maturation at 39C. We show here that temperature-sen-sitive mutations at many sites block the folding pathway prior toaccumulation of the partially folded protrimer intermediate.Temperature-shift experiments indicate that at least some of themutants accumulate an earlier intermediate in the folding path-way. Immunoprecipitation experiments suggest that the confor-mation of the isolated temperature-sensitive polypeptide chains iscloser to that of the unfolded chain than to that of the mature spikeformed at permissive temperature. The sites of these mutationsprobably represent amino acid sequences that play key roles dur-ing the folding of the tail spike polypeptide chain but are not im-portant in the mature protein.

Though numerous proteins can be refolded from a fully dena-tured state back to the native form (1, 2), relatively little is knownof the detailed pathways polypeptide chains follow in achievingtheir native configuration. Studies of bovine pancreatic trypsininhibitor (3), ribonuclease (4), and collagen (5), among others,have identified kinetic intermediates in refolding, showing thatthe native proteins are formed via multistep pathways. In thecase of the trypsin inhibitor, the intermediates contain disul-fide bonds, and presumably local conformations, not found inthe mature protein (6). Analysis of chemically modified formsof trypsin inhibitor protein reveals that some residues have rolesin the folding intermediates different from their role in the na-tive conformation (6, 7). Such intermediate structures could nothave been predicted from investigation of the native structure.The half-life of intermediates in the in vitro refolding of small

proteins is quite short, and they have proved difficult to bothidentify and characterize. This has limited experimental in-vestigation of the role of local amino acid sequences in deter-mining critical kinetic steps in chain folding. As a result, it isstill not possible to accurately predict native conformation fromsequence -data alone (8, 9).We have taken a genetic approach to the related problems

of identifying kinetic intermediates during in vivo folding andsubunit association and of identifying those sequences that playa critical role in directing the polypeptide chain-folding path-way. This has involved the isolation and characterization of con-ditional lethal mutants defective in the folding and subunit as-sembly pathway of a bacteriophage structural protein, the tailspike endorhamnosidase of phage P22 (10-12). The native tailspike is composed of three polypeptide chains of 71,600 daltonseach, which are encoded by gene 9 of P22 (13). The tail spikes-function in the attachment of the phage to the bacterial surface(14); the distal end possesses an endorhamnosidase activity thatdigests the Salmonella 0 antigen during adsorption (15). The

mature tail spike is exceptionally resistant to heat denaturation,to denaturation by NaDodSO4, and to proteolytic digestion (13,16). The native trimer is formed via an intermediate composedof three partially folded chains, the protrimer, which can betrapped at low temperature and is separable from the nativetrimer by gel electrophoresis (17). Though its maturation path-way is multistep, there is no evidence for any covalent modi-fication of the tail spike (13, 16-19).

Temperature-sensitive (ts) mutations have been isolated atmore than 30 sites in gene 9, as shown in Fig. 1 (10). Fifteenof the ts mutant tail spike proteins have been characterized (11,12). At the restrictive temperature, 390C, the mutants syn-thesize polypeptide chains that possess none of the activities ofthe native tail spike (12). However, at the permissive temper-ature, 30'C, the mutants produce native tail spikes that are fullyactive. These remain so even when heated to elevated tem-perature (11). Thus, the mutations act not by altering or desta-bilizing the mature protein but by blocking the maturationpathway.

In the present study, we have attempted to identify the stageof the folding and subunit assembly pathway at which the tsmutants are blocked.

MATERIALS AND METHODS

Bacteria and Bacteriophage. Salmonella typhimuriumDB7000 (suppressor-) was used as the host for all experiments.The ts mutations in gene 9 have been described (10-12). The9- amber mutant amN110 produces only a small fragment (13,900daltons) of the tail spike polypeptide chain (10). The phage strainsused here carried, in addition to the gene 9 mutation of in-terest, three additional alleles, in gene 5 (amN114), gene 13(amH101), and the cl gene (cl-7). Gene 5 encodes the phagecapsid protein (18, 19). In its absence, no head-related struc-tures are formed and the tail spike trimer remains soluble (16).In the absence of the gene 13 product, infected cells do not lyseand phage protein synthesis continues past the normal lysis time.Late after infection in cells in which capsid assembly, andtherefore DNA packaging, is blocked, the tail spike protein be-comes one of the major proteins of P22-infected cells.

Gel Electrophoresis. Gel electrophoresis in the presence ofNaDodSO4 was carried out in the discontinuous buffer systemof Laemmli (21, 22). The samples were not warmed above roomtemperature, except as noted in the figure legends. Nondena-turing gel electrophoresis was carried out in the discontinuousbuffer system of Davis (23) and Ornstein (24). Electrophoresiswas carried out at 40C at a constant current of 20 mA (200-400V) for =z2 hr.

Abbreviation: ts, temperature sensitive.* Present address: Medical Research Council, Laboratory of MolecularBiology, Hills Road, Cambridge, CB2 2QH, England.

7060

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 8Q (1983) 7061

tsU34

aamNI10

tsUlI1sH300 tsRH tsU18 ts9IItsV tsItsU9 tSN48 IJsHKtsH3OI

I U 9?p ?IT T TI T

'6 06amHI291 amHI2006

am H840

tsN42ts U389?I

6arnEI017

aamNI08 amH1014

FIG. 1. Map of ts mutations in gene 9. Markers above the gene denote sites of ts mutations (10). The labeled mutations are those used in theexperiments described here. The markers below the gene represent sites of amber mutations. One of the mutants included in this study, tsH305,has not been precisely located but maps to the left of all those shown (unpublished results). The complete sequence of gene 9 has been determinedby Sauer et al. (20).

Immunodiffusion. Antisera were prepared in rabbits againstpurified native tail spikes and against a guanidine hydrochlo-ride (6 M)-denatured portion of the same preparation. Samplesof the final sera were absorbed by incubation with the proteinsample used to make-the other serum. The infected cells wereprepared and labeled as described above, except that the cellswere concentrated 5-fold by centrifugation prior to lysis byfreezing and thawing. The ts proteins do not reactivate once thecells are broken in the cold (12).

Wells of standard Ouchterlony immunodiffusion slides wereprepared containing 5 ,ul of lysate each. The antibody-antigenreactions were allowed to proceed overnight at room temper-ature in a humidity chamber. The Ouchterlony slides werewashed extensively with 1% NaCl, rinsed with distilled water,air-dried, and then applied to x-ray films to produce the au-toradiograms shown.

RESULTSThe ts Mutations Prevent Formation of the NaDodSO4-Re-

sistant Native Trimer. The mature tail spike protein is resistantto denaturation by NaDodSO4 at room temperature (13). Todetermine whether the ts mutations prevent the formation ofthe NaDodSO4-resistant trimer, we examined the NaDodSO4resistance of mutant polypeptide chains synthesized at restric-tive temperatures. Bacteria were infected with the ts mutantphage, and the cultures were split and incubated with 14C-la-beled amino acids to label the newly synthesized polypeptidechains at both restrictive and permissive temperatures. Sam-ples of the labeled cells were lysed 3 and 20 min after the la-beling period. These samples were mixed with NaDodSO4without heating and electrophoresed through a NaDodSO4/polyacrylamide gel to fractionate the native spikes from the in-completely folded chains.

The proteins from lysates labeled at 30'C are shown in Fig.2a. The first two lanes display the proteins synthesized by thecontrol 9+-infected cells. The native trimer does not bindNaDodSO4 and migrates more slowly than the NaDodSO4-polypeptide chain complex, which migrates with a mobility cor-responding to its molecular weight (13, 18). These NaDodSO4-denatured chains represent incompletely or incorrectly foldedchains present in the cells at the time of lysis (13). By 20 minafter the labeling period, most of the wild-type chains had formedthe NaDodSO4-resistant trimer. Both the NaDodSO4-polypep-tide complex and the native trimer were absent from the lysatesof cells infected with phage carrying a nonsense mutation ingene 9, shown in the adjacent two lanes.

At 300C, all of the cultures infected with one of the sevents mutants produced significant levels of the NaDodSO4-re-sistant trimer. However, the mutants varied in their efficien-cies of maturation. tsUl8 was close to wild type, whereas tsU38is partially defective even at permissive temperatures. The tsH304mutation alters the mobility of the trimer, probably due to theintroduction of a charge change (13). tsH305 appears to differfrom the other mutants in that the synthesis of the polypeptidechain itself is altered.The parallel lysates labeled at the restrictive temperature are

shown in Fig. 2b. At 39TC, a fraction of the wild-type chainsmatured into native trimers, though the efficiency of foldingwas only -25% of that at 30TC, as described previously (11, 13).In six of the seven mutant-infected cells, the gene 9 polypep-tide chains accumulated in a NaDodSO4-sensitive state and failedto form NaDodSO4-resistant trimers. The other eight ts mu-tants shown on the map in Fig. 1 showed the same behavior.Thus, in general, the mutations block the folding or subunitassociation prior to formation of the NaDodSO4-resistant com-

a 300C9-AM TSUII TSU38 TSH304

9 TSH301 TSUI8 TSU34 TSH305TI2MEAFTER 2O3'20320

NATIVETRIMER -

POLYPEPTIDECHAIN --% _ ___ _

L ' LI L. LJ 4 I4 l4 L II4

b 39°C9-AM TSUII TSU38 TSU304

g9+ T5H301 TSUIS TSU34 TSH305TIMEAFTER__LABELING- 13, 24OII3'3d1 03'

NATIVETRIMER I-I

POLYPEPTIDECHAIN

A aSMM Ji WO an -~ -M.

~~~ ~ ~

FIG. 2. ts mutant polypeptide chains do not form NaDodSO4-re-sistant trimers at restrictive temperature. The native trimer migratesmore slowly in NaDodSO,-containing gels than the denatured NaDod-S04-polypeptide complex (13). The latter populations include inter-mediates notyet native atthe time oflysis. Bacteria (su-) were infectedwith phage strains carrying the mutations of interest. Portions of theinfected cultures were incubated at the permissive temperature, 3000(a), and at the restrictive temperature, 3900 (b). At 3 and 20 min afterthe labeling period, samples were withdrawn and lysed by freezing andthawing. Portions of the lysates were mixed with NaDodSO4 samplebuffer(withoutheating) andelectrophoresed through NaDodSO4/poly-acrylamide gels. All the strains carry, in addition to the alleles indi-cated, an amber mutation in gene 5 (amN114) to prevent capsid assem-bly, an amber mutation in gene 13 (amffl0l) to extend the lysis period,and a clear plaque allele (cl-7) to ensure entry into the lytic cycle.

4. .: -1%.F .. '. -1 .; ; -,- ., '." _, --'. '. -N. '... '..- :: Z. -. ': tifit-t-I .. .:. -.1 J-11 -t :1:- .7.! Z-, '--- -,I I I

Biochemistry: Goldenberg et al.

7062 Biochemistry: Goldenberg et al.

plex. For tsH305, the level of gene 9 polypeptide chain syn-thesis sharply decreased, making it difficult to determine whethera folding or assembly block is also present.The ts Mutations Interfere with Protrimer Formation or Sta-

bility. The native NaDodSO4-resistant tail spike is formed invivo via a partially folded trimeric intermediate, the protrimer.This intermediate can be trapped at low temperature and iden-tified by electrophoresis through a nondenaturing gel (no Na-DodSO4) (17). The ts mutations could block the folding and as-sembly pathway before or after the formation of this inter-mediate. To locate the defect, we pulse-labeled ts mutant-in-fected cells, then rapidly chilled the cells to trap any protrimerintermediates. The cells were then lysed and samples wereelectrophoresed through a native gel at 40C.

Fig. 3a shows an autoradiograph of the gels displaying lysateproteins labeled at 30'C. The first two lanes show cells infectedwith control gene 9+ phage sampled 3 and 20 min after label-ing. The most heavily labeled band is the native trimer. Theprotrimer, which Migrates more slowly than the native trimer,appears early after labeling and is then converted to the maturetail spike. Neither the native trimer nor the protrimer bandsare present in the adjacent lanes containing the gene 9- amberlysates.The nondenaturing gels confirmed that, at 30'C, the mutant

polypeptide chains formed native trimers, though with variousefficiencies. The protrimer intermediate was also present inmutant-infected cells lysed early after labeling. Those mutantswith reduced yields of the native trimer, tsH301, tsUll, andtsU18, also displayed lower levels of protrimer intermediate.

Fig. 3b shows the proteins from lysates labeled at the re-strictive temperature. The native trimer is absent from all ofthe ts mutant lysates. However, the ts polypeptide chains arenot found accumulated as protrimers. Rather both species areabsent from the mutant lysates. Since the mutant polypeptidechains, with the possible exception of tsH305, were synthe-sized and present in the lysates (Fig. 2), the ts amino acid sub-stitutions must either destabilize the protrimer itself or inter-fere with the folding pathway at a stage prior to protrimerformation. Similar results were obtained with the other mu-tants shown on the map in Fig. 1. The 39TC mutant chains werenot resolved by the nondenaturing gel electrophoresis, prob-ably as a result of aggregation after cell lysis into a complex toolarge to enter the gel (unpublished experiments).

Pathway of Chain Maturation After Shift to Permissive Tem-perature. Smith and King (12) have shown that for many of themutants the inactive polypeptide chains that accumulate at 39°(can form active tail spikes within infected cells on lowering thetemperature. If the ts mutants are blocked at the protrimer orearlier stage of the pathway, then the prttrimner should appeartransiently when the temperature of incubation of the mutant-infected cells is lowered.

Bacteria were infected at the restrictive temperature withphage carrying tsUIl, and with gene 9+ and 97 amber controls,and were labeled for 4 min with '4C-labeled amino acids. Chlor-amphenicol was then added to inhibit further protein synthesis.The culture was then shifted to 24°C and, at various times, sam-ples were withdrawn and quickly frozen. These samples weresubsequently thawed at0OC and then electrophoresed througha nondenaturing gel to resolve the protrimer. The bands of in-terest were quantitated with a microdensitometer (Fig. 4a).

At the time of the shift down, neither- native trimer nor pro-trimer species were labeled. After shift down, labeled nativetrimer appeared in the tsUll-infected cells with a half-time of_5 min. As the native trimer accumulated, labeled protrimerappeared and disappeared, reaching a maximum-4 min afterthe shift. The absence of these bands in gene 9- amber-in-

a 300C9YAM TSUII TSU38 TSh304

9+ TSH301i TSU18 TSU34 'TSH305

3-729 2 220532'34,2i 2O3--201.9@@@R@RAR-I!~ ~ ~ WFW"|EI1I|I

PROTRIMER

NATIVETRIMER -

b 3 9"C9-AM TSUII TSU38 TSU304

9+ TSH30I TSU34 TSH305

PROTRIMERN A T VETRIMER - -R

FIG. 3. The ts mutant-infected cells do not accumulate protrimersat restrictive temperature. Samples of the labeled lysates described inthe legend to Fig. 2 were electrophoresed through polyacrylamide gelsunder nondenaturing conditions at 40C. The protrimer (which is dena-tured and thus not resolved in the NaDodSO4 gels of Fig. 2) migratesmore slowly than the native trimer. At the restrictive temperature, themature trimer does not form nor does the protrimer accumulate.

fected cells confirmed the identifications. The data are con-sistent with a simple kinetic scheme in which the protrimerspecies is formed after the shift and converts to product (Fig.4b). These results confirm that the tsUll mutation leads to theaccumulation of tail spike polypeptide chains at a step prior tothe protrimer.

Similar results were obtained with tsU18. A third mutant,which was studied in detail, tsU24, gave ambiguous results.The Conformation of to Mutant Polypeptide Chains at 390C

Resembles an Unfolded State. Previous studies of the ts mu-tant polypeptide chains synthesized at restrictive temperatureshowed that they did not block neutralizing antibody directedagainst the distal end of native tail spikes (12). To further com-pare the conformations of the inactive mutant chains with thatof the native tail spike, we carried out precipitation reactionssensitive to antibody-antigen reactions over the entire protein.In addition to antiserum raised against the native tail spike, wealso used an antiserum raised against guanidine hydrochloride-denatured tail spikes.

Fig. 5a shows an Ouchterlony diffusion plate in which anti-serum against the native tail spike was placed in the center well.The outer well at the upper center contained a gene 9+ controllysate. The native tail spikes in the lysate formed a strong pre-cipitin band with the antibodies. The identity of this band isconfirmed by its absence from the zone in front of the upperleft-hand well, which contained a lysate of cells infected witha deletion of gene 9. The other wells contained ts mutant ly-sates prepared at the restrictive temperature. These lysates,which contained normal levels of the tail spike polypeptide chain,showed only a very weak reaction with the antisera. When serumthat had been absorbed with denatured protein to remove an-tibody species against denatured chains was used, only a traceof precipitation was observed.

The Ouchterlony plate shown in Fig. 5b contained anti-serum raised against denatured tail spikes. The control-9 ly-sate contained two antigen species, presumably the native tail

Proc. Natl. Acad. Sci. USA 80 (1983)

.a

Proc. Natl. Acad. Sci. USA 80 (1983) 7063

I

a0

00

0

4

FIG. 4. Temperature shiftdown with tsUll. Cells were infected withtsUMM and control gene 9+ and 9- amber phage at 390C. At 60 min, theinfected cells were incubated with '4C-labeled amino acids for 4 min.At 65 min, chloramphenicol was added to stop protein synthesis and,2 min later, the culture was shifted to 24TC. Samples were removed atthe times shown and quickly frozen. After thawing in the cold, the sam-ples were electrophoresed through anondenating gel at 40C. The bandsof interest were quantitated with aJoyce-Loebl microdensitometer. (a)Curvesfittothe experimental points; o, protrimer band; *, mature spike.(b) Theoretical curves assuming that, after the shift, the accumulatedhigh-temperature intermediate of the protrimer intermediate speciesforms the protrimer band, which then converts directly to the native

kltail spike. The kinetic expressions used were as follows: pp -+ pTk2-* N, wherepp = concentration ofnewly synt edpolypeptide chains,pT = concentration of protrimer, andN = concentration of native tailspikes. The startingconcentrationofpolypeptide chains wascalculatedfrom the addition of all species at late times, ppo = pp + pT + N. Ex-pressingpp = ppOexp(-k t), we find that

[T=poklexp(-kit) klexp-k2t)1pT= ppo [ mk2 - kl k2 - Ji

and

N =ppo - k2exp(-kit) + kep(-k2t)]L k2 - kI h2 - ki J'

Forthe curves shown,ppo = 0.36,kl = 0.30 min-1, and k2 = 0.19 minfat 240C.

4- b

l

spike and non-native forms, which reacted with different an-tibodies in the serum. When this serum was absorbed with thenative protein, one of the precipitin bands disappeared, as shownin the right-hand wells. We conclude that this band representsnative tail spikes and that the band remaining after absorptionof the serum contained non-native forms of the gene 9 poly-peptide chain from the 9+ lysate. This population correspondsto the NaDodSO4-sensitive wild-type chains shown in Fig. 2and is probably a mixture of incompletely and incorrectly foldedchains.The ts mutant lysates formed only one precipitin band with

the antidenatured serum, and this band was not affected whenthe serum was absorbed with native protein. The amber frag-ment from the 9- amber lysate was also precipitated by serumagainst denatured chains, suggesting that these antibodies rec-ognize the amino-terminal region of the polypeptide chain. Sincethis serum was prepared with guanidine hydrochloride-dena-tured protein, the inactive mutant polypeptide chains resembleunfolded chains rather than native protein. The other ts mutantlysates also showed reactions of identity with one another andwith the non-native wild-type chains.

DISCUSSIONts Mutations and Protein Folding. We have shown previ-

ously (11) that ts mutations in gene 9 do not affect the functionor thermal stability of the mutant proteins once matured at per-missive temperature. They therefore must define sites in thepolypeptide chain that can be thought of as specialized for di-recting the chain-folding or association pathway. The ts mu-tations could interfere with the early stages of chain folding, orthey could interfere with specific polypeptide chain interac-tions needed for association into the protrimer or stabilizationof the protrimer. However, it seems to us unlikely that singleamino acid substitutions, at 14 different sites in the polypeptidechain, would all specifically destabilize interchain interactionswithin the protrimer at high temperature. Some, if not manyof the mutants, probably act by preventing the polypeptide chainfrom achieving the conformation needed for subunit recogni-tion and association.The maturation of the P22 wild-type tail spike is itself tem-

perature sensitive, with the yield of native trimer from poly-peptide chain decreasing from 90% at 30TC to <20% at 40TC

a ANTI -NATIVE b ANTI -DENATURED

Anti-native, Absorbedwith denatwed VS

Anti-dweatued Anti-denotured, Absorbedwith native p9

3 k1=

4.

en

- .30

> .26

- .22

< .18

- .14

z .lowZ .06

z< .02m

0m 8 12 16 20 0 4 8 12 16 20TIME AFTER SHIFTDOWN (MINUTES)

Anti-native

9+ 9+9 0° tOsN42 A9 0 Ad

am9 O 0° tsH300 om9 0O0) 0 tstsRH O tsH301 ts 00ts

ts U56 ts

9+ 9+

69 04Uts t9 0_0 tsam9 ct~pts am9 Q g)Ots

ts 03 ts ts O OtsIts ts

FIG. 5. Immunological properties ofts mutant polypeptide chains synthesized atthe restrictive temperature. Thecentral wellscontain antibody.The lysates were prepared at the restrictive temperature and then rapidly chilled and lysed. The lysate samples are the same for the four sera Astrain with gene 9 deleted serves as the negative control. Similar results were found with all the ta mutants shown on the map ofFig. 1. The amberfragments crossreat with the antidenatured serum, suggesting that the serum is directed against NH2-terminal determinants.

Biochemistry: Goldenberg et al.

3.

3

P. .

i

7064 Biochemistry: Goldenberg et al.

(11, 13). This decrease in yield is not associated with increasedaccumulation of the protrimer, as shown by the control samplesin Fig. 3. Thus, the temperature sensitivity of the wild-typepathway is probably due to the thermolability of the protrimeritself or of intermediates prior to protrimer formation. The tsmutations probably act by further destabilizing these thermola-bile intermediates or interactions already present in the wild-type pathway.None of the ts mutants we have studied decreases the sta-

bility of the mature mutant protein. Such phenotypes are com-mon with ts mutants affecting other proteins, such as RNApolymerase or lysozyme (25, 26). This probably reflects the highthermostability of the tail spike; single amino acid substitutionsare not likely to alter the melting temperature of the proteinby 400C-from >80TC to s40TC. The efficient isolation of mu-tations specifically affecting the maturation pathway probablyreflects both the nature of the pathway itself and the stabilityof the native protein.The High-Temperature State of the Mutant Polypeptide

Chains. For many of the ts mutants, the inactive chains ac-cumulating at high temperature can be converted to native tailspikes on shift to permissive temperature (12). The high tem-perature mutant chains within infected cells must thereforerepresent either intermediates in protein folding or states re-versibly related to them. Since shiftdown of tsUll and tsU18results in the appearance of the protrimer, these chains mustaccumulate at 39TC at an earlier stage of the pathway.The lack of reaction between any of the ts high temperature

chains and antibody to native tail spikes indicates that the tschains are in a conformation very different from that of the na-tive tail spikes. The positive reaction with antibody against de-natured chains implies that at least some part of the 390C chainsare in a conformation resembling partially folded chains or oth-erwise different from the native spike.We do not know whether the high-temperature polypeptide

chains are partially folded monomers, dimers, or higher ag-gregates. They were not detected as discrete bands during na-tive gel electrophoresis, though we have found candidates forsuch species among spike precursors from wild-type-infectedcells (17). Efforts to isolate incompletely folded chains from tsmutant-infected cells revealed that a large fraction of the chainsin the lysate were in a rapidly sedimenting complex. These largeaggregates would not enter the native gel. Such off pathwayaggregates have been described by Jaenicke and Rudolph (27)in the refolding of oligomeric proteins.

It is possible that some of the mutations act not by blockingthe productive pathway but by increasing the rate of an off-pathway folding reaction. A ts mutant of RNA polymerase hasrecently been described that aggregates at restrictive temper-ature, though the mutant chains do appear to achieve close totheir native conformation (28).

Folding Pathway for Thermostabile Oligomers. The remark-able stability of the native tail spike to heat, detergent, and pro-teases may involve extensive wrapping of the three chains aroundeach other. This would require a precursor to the association

reaction that was only partially folded and might be quite un-stable (29, 30). Because three subunits must be incorporated,the efficiency of the formation of the protrimer would be quitesensitive to the accumulation of partially folded monomers.

In such a model, the gene 9 temperature-sensitive mutationswould mark local sequences or residues that stabilize the in-termediates along the pathway to the final stable, but difficultto achieve, conformation.We thank Edward Loechler for help with the kinetic model and Peter

Berget and Robert Sauer for communication of unpublished results.This research was supported by National Science Foundation GrantPCM77-15017 and National Institutes of Health Grant GM17,980.

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Proc. Natl. Acad. Sci. USA 80 (1983)