Preliminary circular dichroism study of the conformations of intermediates trapped during protein folding

B I O C H I M I E , 1981, 63, 835-839.

Preliminary circular dichroism study of the conformations of intermediates trapped during protein folding. M. HOLLECKER *, T. E. CREIGHTON * and M. GABRIEL ¢.

* MRC Laboratory o[ Molecular Biology, Hills Road, Cambridge CB2 2QH, U.K. <>ISIN, Universit~ Nancy I, Parc R. Bentz, 54500 Vandwuvre, France.

Summary.

two such intermediates has suggested an early appearance of helical con]urinations, but that of ~-structure is not yet clear.

The pathway of protein folding is being studied by CD analysis o/ the conformational properties of trapped intermediates. Preliminary analysis of

Introduction. Globular proteins fold spontaneously from random-coil states to their complex, three-dimensional, native conformations via a finite number of partially-folded intermediates, ,in a self-assembly process directed by the amino acid sequence [1-3].

Key-words : circular dichroism, protein folding.

folding involves disulphide bond formation between Cys residues, intermediates may be trapped in a stable form by quenching disulphide formation, breakage and rearrangement; the trapped intermediates may then be isolated and characterized. The pathway of unfolding and refolding of BPTI has been determined in this way [2, 4].

1 5 i0 15 BPTI Arg-Pro-Asp-Phe-CYS-Leu-GIu-PRO-Pro-Tyr-Thr-GLY-PRO-CYS-LYS-AI a-Ar g- ILEI G1 n-Pro-Leu-Arg-LYS-Leu-CYS- I Ie-LEU-Hi s-Arg-Asn-Pro-GLY-Arg-CYS-Tyr-GIn-LYS- ILEK A1 a-Al a-LYS-Tyr -CYS-Lys-LEU-PRO-Leu-Arg- I Ie-GLY-PRO-CYS-LYS-Arg-LYS- ILE2O 25 3O 35 I I e -Arg-Tyr -Phe-TYR-ASN-AI a-LYS-ALA-GIy-Leu-CYS-GIn-Thr-PHE-VaI-TYR-GIy-GLY-CYSPRO-A1 a-PHE-TYR-TYR-ASN-GIn-LYS-Lys-LYS-GLN-CYS-GI u-GIy-PHE-Thr -Trp- SER-GLY-CYSPRo-Ser-PHE-TYR-TYR-Lys-Trp-LYS-ALA-LYS-GLN-CYS-Leu-Pr•-PHE-Asp-TYR-SER-GLY-CYS40 45 50 55 Arg-AIa-Lys-Arg-ASN-Asn-PHE-LYS-Ser-AI a-GLU-Asp-CYS-Met-ARG-THR-CYS-GIy-GIy-AI a GLY-GLY-ASN-Ser -ASN-ARG-PHE -LYS -THR- ILE -GLU-GLU-CYS-ARG-ARG-THR-CYS - Iie -Arg-Lys GLY-GLY-ASN-AI a-ASN-ARG-PHE-LYS-THR- ILE-GLU-GLU-CYS-ARG-ARG-THR-CYS-VaI-GIy

FIG. 1. - -

The amino acid sequences of BPTI and o] black mamba toxins 1 and K [12], using the standard threeletter abbreviations. A m i n o acid identities are indicated by capital letters.

Experimental elucidation of the folding pathway is difficult, as the partially-folded states are unstable and normally may be detected only as transient kinetic intermediates. However, when Abbreviations : CD, circular dichroism ; BPTI, bovine pancreatic trypsin inhibitor, <> Address correspondence t o ~ M. Hollecker & T. E. Creighton.

The conformational forces which direct the non-random, non-sequential formation of the three disulphide bonds of BPTI is not clear. It should be apparent from the conformational properties of the trapped intermediates [5-9] and from the refolding of related proteins with different amino acid sequences [10-11]. Therefore, we are studying the folding pathway of two proteins from the venom of black mamba snakes (Dendroaspis polylepis polylepis) ~ toxins I and K [12] - - which

836

M . Hollecker and coll.

are clearly h o m o l o g o u s to B P T I (fig. 1). T h e y each differ f r o m B P T I at a b o u t 2 / 3 of their a m i n o acid residues a n d from each other at a b o u t 1 / 3 . However, all of the i n t e r n a l side-chains a p p a r e n t l y i m p o r t a n t for the folded c o n f o r m a t i o n of B P T I are conserved [13], so it is virtually certain that both have the B P T I - l i k e c o n f o r m a t i o n of the polypeptide b a c k b o n e (fig. 2). T h e conformations of the folded and u n f o l d e d proteins, a n d of their t r a p p e d folding intermediates, are being studied b y C D spectroscopy, one of the most powerful methods for characterizing flexible protein c o n f o r m a t i o n s in solution, being especially sensitive to secondary structure. W e report here our initial o b s e r v a t i o n s with toxin I.

14

method of Strydom [14]. Unfolding and refolding were followed with the same techniques developed for BPTI to accumulate, trap, and purify the intermediates [2, 4]. CD spectra were measured at room temperature with the Jobin-Yvon CD III dichrograph of the (>, which was calibrated with a solution of isoandrosterone in freshly distilled dioxane (As = 3310 cm -1 1 mole -1 at 304 nm). Proteins were dissolved in 6.0 mM PIPES (piperazine-N,N'-bis(2-ethanesulphonic acid)) buffer, pH 6.8, then filtered, to a Concentration which gave a maximum absorbance of 0.7 to 1.4 ; the cell path length was 0.1 to 10 mm. The spectra data were measured every nm, digitized, and analyzed directly with a Tektronix 4052 computer [15]. Each point of the curve was the mean value of ten successive measurements, and three such spectra were averaged and smoothed.

38

Results. R e f o l d i n g a n d disulphide f o r m a t i o n i n unfolded, reduced toxin I were followed by t r a p p i n g the species present after various times, rapidly reacting all thiol groups with iodoacetate. T h e t r a p p e d species were separated b y ion-exchange c h r o m a t o graphy o n the basis of the differences i n charge between p r o t e i n molecules with different n u m b e r s 1 disulphides

2 disulphides

(30 511 t

/

58 Fro. 2. - - Ribbon diagram o] the backbone o/BPT1 as determined crystallographically [28], with the three disulphide bonds linking Cys residues 5 to 55, 14 to 38, and 30 to 51. Residues 2 to 7 and 47 to 55 are considered to be in a helical conformation, 14 to 25, 28 to 37, and 43 to 46 in 413-strands, 8, 9, 12, 13, 26, 27, 38, 39 and 56 in reverse turns, and 1, 10, 11, 40 to 42, 57 and 58 in irregular conformations [29].

(30-51) (5-551

FIG. 3. - - Chromatographic isolation o] the species of black mamba toxin 1 trapped during re]olding. The elution profile from a carboxymethyl cellulose column is shown as a tracing of UV absorbance at 280 nm as a function of time ; the upper absorbance scale is contracted 5-fold. The intermediates had been trapped by iodoacetate after refolding of 30 ,~M reduced toxin I for 5 minutes in the presence of 0.15 mM oxidized glutathione [2, 4, 17, 18]. R represents residual reduced protein, whereas N is refolded, with three disulphides. The elution positions of intermediates (30-51) and (30-51, 5-55) studied here are indicated.

Materials and Methods. Dessicated black mamba snake venom was obtained from D. Muller, Professional Snake Catcher, Johannesburg, South Africa. Toxins I and K were isolated by the BIOCH1M1E, 1981, 63, n ° 11-12.

of disulphide b o n d s [2, 4]. Substantial quantities of one- and two-disulphide intermediates a c c u m u lated transiently (fig. 3).

837

Conformations of protein folding intermediates.

The disulphide bonds present in two of the intermediates have been identified by diagonal electrophoresis ,[16-18]. The diagonal m a p of the two-disulphide intermediate is shown in figure 4 ; the two pairs of peptides demonstrate unambi-

duced upon trapping. The absence of other nondiagonal peptide spots and of carboxymethyl derivatives of Cys 5, 30, 51, and 55 demonstrate that this species was homogeneous and that the disulphide bonds were intramolecular ; it is designated as (30-51, 5-55). The corresponding intermediate in B P T I is a crucial productive intermediate which forms directly the third, 14-38, disulphide. The major single-disulphide intermediate was similarly identified as (30-51). This is also the major, productive one-disulphide intermediate in refolding of B P T I . Diagonal maps of native and refolded toxin I and of native toxin K showed that they have the same disulphide bonds as B P T I .

S ()38CM

/30

FIo. 4. - - Diagonal map of toxin I intermediate (30-51, 5-55). After digestion with thermolysin, the peptides were subjected to electrophoresis at pH 3.5 in the first, horizontal dimension, starting at the origin, with the cathode on the right. The paper was then exposed to performic acid vapour, to cleave the disulphides and oxidize the carboxymethyl Cys residues to the sull~hones. Electrophoresis was then repeated under the same conditions as, but perpendicular to, the first dimension, with the cathode at the top. Peptides unaltered by the performic acid migrate identically in both dimensions and define a diagonal, which is indicated by crosshatching [1648]. Peptides containing Cys residues are generally more acidic after performic acid treatment and usually lie below the diagonal ; they were identified from their amino acid compositions. Peptides 30 and 51 had the same mobility in the first dimension and contained cysteic acid residues, indicating that they were originally linked by a disulphide bond, as were peptides 5 and 55. Peptides 14CM and 38 CM had carboxymethyl Cys sulphone residues. Peptides 5, 14 CM, 30, 38 CM, 51, and 55 consisted of residues 4 to 5, 7 to 14, 29 to 32, 35 to 41, 48 to 53, and 54 to 55, respectively.

The far-UV C D spectra of native, of reduced, and of the two intermediates of toxin I are illustrated in figure 5. That of the reduced protein is characteristic of an unfolded protein, with a minim u m at 197.5 nm, negative ellipticity below 200 nm, and littie or no shoulder in the region of 220 nm. Both intermediates have their spectra shifted to higher wavelengths, with minima at 203.5 nm, maxima at about 187 nm, and pronotmced shoulders at 220 nm. The spectrum of (30-51, 5-55) is closer to that of the native and refolded proteins, which have minima at 206.5 nm and the most pronounced shoulders at 220 rim. X

10 -6

0.4 0.2 0.0

1

200

215

230

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d

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-0.6 -(28

FIo. 5. - - CD spectra o] the trapped species o! toxin 1 : fully reduced, - - - - - - ; intermediate (30-51), - . . . . . ; intermediate (30-51, 5-55). . . . . . . ; and native, - The molar ellipticity, 0 = 3300 Ae, is plotted in units 05 degrees cm2 mole-1.

Discussion.

gnously the original presence of the 30-51 and 555 disulphide bonds (using the B P T I numbering system of fig. 1), whereas Cys 14 and 38 were present only in the carboxymethylated form proBIOCH1MIE, 1981, 63, n ° 11-12.

The CD spectra of the initial, intermediate, and final conformational states of toxin I (figure 5) clearly show the progressive acquisition of non-

838

M. Hollecker and coll.

random conformation during refolding. As such spectra are sensitive to secondary structure, they should resolve the question of when such conformations appear during refolding. The early predo-

XlO 6 0.4 02 I! /'

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I

I

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-0.8I FIG. 6. - - Example of experimental (Q) and calculated (- - -)CD spectra of native toxin I. The spectra of the other protein species of table I were fit just as closely.

minance of the (30-51) intermediate in both to,xin I and BPTI is consistent with interactions between t3-structure around Cys 30 and ~-helix around Cys 51, as occurs in the final folded conformation

was considered to be unusual, not consistent with its known structure, as was also concluded by Rosenkranz [19]. Using the methods of Greenfield and Fasman [20] ,and of Chen et al. [21] to deconvolute the far-UV CD spectrum, very incorrect values of secondary structure content were obtained [8]. We have found the spectrum of toxin K to be similar to that of BPTI, whereas that of native toxin I is significantly different, even though t h e amino acid sequences and identical disulphide bonds of the three proteins make it extremely likely that they have very similar backbone conformations. The anomalous spectrum of BPTt was considered to be due to contributions by the aromatic side-chains [8, 22], so a similar spectrum with toxin K, but not I, suggests that it is due to the immobile Tyr 35 [23], as this residue is Trp in toxin I (figure 1). These observations prompted us to pursue interpretation of these spectra, and we have found the procedure of Provencher and G1/Sckner [24] to give intriguing results. First, the method was able to fit all the spectra to within their experimental uncertainty (e.g. fig. 6). Secondly, the indicated values of helix, ~-strands, reverse turns, and irregular conformations indicated for BPTI are much closer to the observed values than other analyses

TABLE I. Percent content oJ secondary structure estimated ]rom CD spectra. CD Spectra 09 Secondary structure

BPTI crystal structure (a)

Toxin I BPTI

N (9 (30-51,5-55) (30-51) R (9 Helix ,~-strand Revetse turns Irregular

26 (d) 45 16 14

10 58 4 28

19 42 13 25

16 48 12 25

15 31 26 27

1 49 32 19

(a) F r o m the anMysis by Levitt & G r e e r [29] of the crystal structure by Deisenhofer & Steigemann [281. (b) Estimated by the computer program of Provencher ~: G16ckner [241. (c) N : native ; R : reduced. (d) Only 16 per cent of the residues are in a regular ~-helical conformation, the other 10 per cent in a different helical conformation•

(figure 2), being important at this stage [17], but the secondary structures should then be present in the trapped intermediate. A thorough CD study of all the BPTI species was carried out b y Kosen et al. [6, 8] to answer this question, but the CD spectrum of native BPTI BIOCH1M1E, 1981, 63, n ° 11-12.

of its CD spectrum (table I). There are still significant discrepancies, but it must be kept in mind that of the 15 residues of BPTI considered to be hefical (26 per cent of the chain), 6 are in an irregular helical conformation of residues 2 through 7 ; only 16 per cent of the chain is an e-helix (figure 2).

Conformations

o f p r o t e i n f o l d i n g intermediates.

839

In contrast, the secondary structure indicated from the C D spectrum of toxin I is remarkably close to that of the B P T I crystal structure, which makes this protein the most favourable for CD analysis.

obtained thus far demonstrate the potential of CD analysis of chemically trapped intermediates in protein folding.

The C D spectrum of reduced toxin I is like that of reduced B P T I [8] and ribonuclease [9] and similar to that expected for a fully unfolded chain. Accordingly, analysis of the spectrum (table I) indicates absence of helices, but does indicate presence of ~-strands and reverse turns. Presumably, this implies that :an unfolded polypeptide, which is rapidly interconverting between very m a n y conformations of similar energy, is different from the irregul~ar, but fixed, portions of folded proteins, i3-sheets and reverse turns are not as reliably measured as helices by CD in this wavelength range [24-27] ; spectra at shorter wavelengths are required for more accurate measurements [26, 27]. Nevertheless, it is virtually certain that these reduced protvins do not have t-sheets of hydrogen-bonded t-strands.

We thank S. W. Provencher for providing a copy o] his computer program ; M. Hollecker was supported by the Max Perutz Fund.

Acknowledgements.

REFERENCES.

Intermediate (30-51, 5-55) has a CD spectrum approaching that of native toxin I (figure 5), although not as closely similar as with the corresponding two forms of BPTI, where the two-disulphide species has a conformation very close to native BPTI. In the case of toxin I, (30-51, 5-55) m a y have significant conformational differences from native, but its helical content is similar to that of the earlier intermediate (30-51), with an increased content of extended [3-strands.

1. Baldwin, R. L. (1975) Ann. Rev. Biochem., 44, 453475. 2. Creighton, T. E. (1978) Prog. Biophys. Mol. Biol., 33, 231-297. 3. Baldwin, R. L. & Creighton, T. E. (1980) in <~Protebz Folding >>(Jaenicke, R., ed.), pp. 217-260, Elsevier/ North Holland, Amsterdam. 4. Creighton, T. E. (1980) J. Mol. Biol., 144, 521-550. 5. Creighton, T. E., Kalef, E. & Arnon, R. (1978) J. Mol. Biol., 123, 129-147. 6. Kosen, P. A. (1978) Ph.D. Dissertation. Harvard University, U.S.A. 7. Kosen, P. A., Creighton, T. E. & Blout, E. R. (1980) Biochemistry, 19, 4936-4944. 8. Kosen, P. A., Creighton, T. E. & Blout, E. R. (1981) Biochemistry, 20, 5744-5754. 9. Galat, A., Creighton, T. E., Lord, R. C. & Blout, E. R. (1981) Biochemistry, 20, 594-601. 10. Creighton, T. E., Dyckes, D. F. & Sheppard, R. C. (1978) 1. Mol. Biol., 119, 507-518. 11. Creighton, T. E. & Dyckes, D. F. (1981) J. Mol. Biol., 1,16, 375-381. 12. Strydom, D. J. (1973) Nature N e w Biol., 243, 88-89. 13. Creighton, T. E. (1975) Nature (London), 255, 743745. 14. Strydom, D. J. (1972) 1. Biol. Chem., 247, 4029-4042. 15. Ernst, C. (1979) Th~se de Docteur Ingtnieur. Universit6 de Nancy I, France. 16. Brown, J. R. ~ Hartley, B. S. (1966) Biochem. I., 101, 214-228. 17. Creighton, T. E. (1974) J. Mol. Biol., 87, 603-624. 18. Creighton, T. E. (1975) 1. Mol. Biol., 95, 167-199. 19. Rosenkranz, H. (1974) in <~Proteinase lnhibitors ~ (Fritz, H., Tschesche, H., Greene, L. J. & Truscheit, E., eds.), pp. 458-462, Springer-Verlag, Berlin. 20. Greenfield, N. ~ Fasman, G. D. (1969) Biochemistry, 8, 4108-4116. 21. Chen, Y.-H., Yang, J. T. & Chau, K. H. (1974) Biochemistry, 13, 3350-3359. 22. Woody, R. W. (1978) Biopolymers, 17, 1451-1467. 23. Snyder, G. H., Rowan, R., Karplus, S. ~ Sykes, B. D. (1976) Biochemistry, 14, 3765-3777. 24. Provencher, S. W. & G16ckner, J. (1981) Biochemistry, 20, 33-37. 25. Baker, C. C. & Isenberg, I. (1976) Biochemistry, 15, 629-634. 26. Brahms, S. s: Brahms, J. (1980) 1. Mol. Biol., 138,

Clearly, this report is i n c o m p l e t e , the identities, roles in unfolding and refolding, relative energies, and conformational properties of the other intermediates of toxin I (figure 3), and those of toxin K, are being determined. Nevertheless, the results

27. Hennessey, J. P. ~ Johnson, W. C. (1981) Biochemistry, 20, 1085-1094. 28. Deisenhofer, J. ,~ Steigemann, W. (1975) Acta Cryst., B 31, 238-250. 29. Levitt, M. & Greer, J. (1977) 1. Mol. Biol., 114, 181239 (1977).

The CD spectrum of toxin I intermediate (3051) indicates the presence of nearly as much helical conformation as does the spectrum of the native protein (table I), so the s-helix around Cys 51 m a y be stable in this intermediate. CD spectra are most sensitive to helix content [24-27], and this is the first direct experimental indication of the presence of secondary structure at this early stage of folding. On the other hand, the content of ~-strands is lower than in the reduced protein, which suggests that the ~-sheet around Cys 30 in the final conformation may not be present at this stage of folding. However, interpretation of these figures is very uncertain, as explained above for the reduced protein.

BIOCHIMIE, 1981, 68, n ° 11-12.

149-178.