[7]Folding intermediates studied by circular dichroism

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more slowly equilibrating, native-like states, having vastly different activity but only slightly different structure, i.e., slow

Active.

" inactive

In at least some cases where such subtle equilibria are known to be important in regulating biological activity [i.e., for concanavalin A , 7 prothrombin fragment 1,8 and superoxide dismutase 3° among proteins, and for the peptide hormone(s) angiotensin II and its analogs 31] there is already indirect evidence to suggest that peptide bond isomerization may be the rate-limiting process. If nature has generally taken advantage of the high activation barrier of isomerization for providing kinetic control of activity-regulating equilibria, then the ISP method should prove to be quite valuable in the study of such phenomena. 3o j. S. Valentine and M. W. Pantoliano, Met. lons Biol. 3, 291 (1981). 31 H. E. Bleich, R. J. Freer, S. S. Stafford, and R. E. Galardy, Proc. Natl. Acad. Sci. U.S.A. 75, 3630 (1980).

[7] F o l d i n g I n t e r m e d i a t e s S t u d i e d b y C i r c u l a r D i c h r o i s m

By A. M. LABHARDT In protein folding investigations two kinds of information are of special interest: (1) the character of stable intermediate states of partial folding, and (2) the kinetics of the folding pathway. (1) Local folding is presumed to seed the folding pathway. 1-4 The seeding structure has been termed the kernel if it is stable by itself and the nucleus if it is n o t ) Various suggestions have been made regarding the local structures (hydrophobic cluster, 6-8 a-helix,9-1t /3-hair-pins,12-14 or t S. Tanaka and H. A. Scheraga, Proc. Natl. Acad. Sci. U.S.A. 72, 3802 (1975). 2 M. Karplus and D. L. Weaver, Nature (London) 260, 404 (1976). 3 M. I. Kanehisa and T. Y. Tsong, J. Mol. Biol. 124, 177 (1978). 4 N. Go, Annu. Rev. Biophys. Bioeng. 12, 183 (1983). 5 p. S. Kim and R. L. Baldwin, Annu. Rev. Biochem. 51, 459 (1982). 6 W. Kauzmann, Adv. Protein Chem. 14, 1 (1959). 7 R. R. Matheson, Jr. and H. A. Scheraga, Macromolecules 11, 819 (1978).

METHODS IN ENZYMOLOGY, VOL. 131

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reverse turns 7) involved in kernels or nuclei of folding. Kernels have been searched for with circular dichroism (CD) by seeking particularly stable elements within the overall structure in unfolding conditions, ~5-17 in the reduced protein in conditions that favor folding ~8-23 and in proteolytic fragments or synthetic peptides, z4-26 Folding intermediates have been characterized by CD at enthalpic minima in nonnative conditions 17,27,2s or preceding a high activation barrier (either an intrinsic barrier of the folding reaction 29,30 or an artificial barrier produced by blocking the disulfide bond formation during reoxidation of reduced protein23,31-34; for a review see, e.g., Ref. 35) or using fragments of the native protein. 36-45 s H. A. Scheraga, in "Protein Folding" (R. Jaenicke, ed.), p. 261. Elsevier, Amsterdam, 1980. 9 D. Kotelchuck and H. A. Scheraga, Proc. Natl. Acad. Sci. U.S.A. 62, 14 (1969). 10 V. I. Lim, FEBS Lett. 89, 10 (1978). n V. 1. Lira, in "Protein Folding" (R. Jaenicke, ed.), p. 149. Elsevier, Amsterdam, 1980. 12 O. B. Ptitsyn and A. V. Finkelstein, in "Protein Folding" (R. Jaenicke, ed.), p. 101. Elsevier, Amsterdam, 1980. ~30. B. Ptitsyn and A. V. Finkelstein, Q. Rev. Biophys. 13, 339 (1980). 14 O. B. Ptitsyn, A. V. Finkelstein, and P. Falk, FEBS Lett. 101, 1 (1979). 15 K. Kuwajima, K. Nitta, M. Yoneyama, and S. Sugai, J. Mol. Biol. 106, 359 (1976). 16 K. Kuwajima, Y. Ogawa, and S. Sugai, J. Biochem. 89, 759 (1981). 17 R. W. Henkens, B. B. Kitchell, S. C. Lottich, P. J. Stein, and T. J. Williams, Biochemistry 21, 5918 (1982). ~s C. B. Anfinsen, E. Haber, M. Sela, and F. H. White, Proc. Natl. Acad. Sci. U.S.A. 47, 1309 (1961). ~9A. Fontana, C. Vita, and D. Dalzoppo, Proc. Int. Symp. Biomol. Struct. Interactions, Suppl. J. Biosci. 8, 57 (1985). 2o A. M. Tamburro, E. Boccu, and L. Celotti, Int. J. Pept. Protein Res. 2, 157 (1970). 2~ S. W. Schaffer, Int. J. Pept. Protein Res. 7, 179 (1975). 22 S. Takahashi, T. Kontani, M. Yoneda, and T. Ooi, J. Biochem. 82, 1127 (1977). 23 M. Hollecker, T. E. Creighton, and M. Gabriel, Biochimie 63, 835 (1981). 24 j. E. Brown and W. A. Klee, Biochemistry 10, 470 (1971). 25 A. Bierzynski, P. S. Kim, and R. L. Baldwin, Proc. Natl. Acad. Sci. U.S.A. 79, 2470 (1982). 26 p. S. Kim, A. Bierzynski, and R. L. Baldwin, J. Mol. Biol. 162, 187 (1982). 27 M. Desmadril and J. M. Yon, Biochemistry 23, 11 (1984). 2s S. Era, H. Ashida, S. Nagaoka, H. Inouye, and M. Sogami, Int. J. Pept. Protein Res. 22, 333 (1983). 29 L. F. McCoy, E. S. Rowe, and K.-P. Wong, Biochemistry 19, 4738 (1980). 3o S. Gerard, D. Puett, and W. M. Mitchell, Biochemistry 20, 1857 (1981). 3~ A. Galat, T. E. Creighton, R. C. Lord, and E. R. Blout, Biochemistry 20, 594 (1981). 32 A. M6nez, F. Bouet, W. Guschlbauer, and P. Fromageot, Biochemistry 19, 4166 (1980). 33 p. A. Kosen, T. E. Creighton, and E. R. Blout, Biochemistry 20, 5744 (1981). 34 p. A. Kosen, T. E. Creighton, and E. R. Blout, Biochemistry 22, 2433 (1983). 35 T. E. Creighton, Prog. Biophys. Mol. Biol. 33, 231 (1978). 36 R. G. Reed, R. C. Feldhoff, O. L. Clute, and T. Peters, Jr., Biochemistry 14, 4578 (1975).

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(2) Analyzing the kinetics of folding by CD gives information on the structure built up during the rate-limiting step(s). Two possibilities exist: Formation of the structure itself is rate limiting or else a transient intermediate impedes folding. For example, the folding of S-peptide of ribonuclease S when added to folded S-protein is a rapid reaction44; however, during folding of ribonuclease S the a-helix 3-13 of S-peptide is formed in a slow reaction 45 although combination between S-peptide and refolding S-protein occurs rapidly. 46 Evidently a transient intermediate is detected in this way. Alternatively the absence of CD detected kinetics has been used to make inferences on secondary structure formation preceding the ratelimiting step(s). 29,41,45,47

Depending on the perspective, equilibrium or kinetic (stopped flow) CD has been used, in connection with various chemical and biochemical modifications of the proteins, to search for intermediates. The CD of equilibrium folding intermediates has been treated in some c a s e s 17,22,23,28,43,48 by methods established for folded proteins 49-55 of extracting the secondary structure composition from CD spectra. Implicit assumptions and potential pitfalls are discussed in the section below. The use of near-UV CD is not reviewed here. For fast kinetic measurements, home built or modified CD spectrome-

37 E. A. Carrey and R. H. Pain, Biochem Soc. Trans. 5, 689 (1977). 38 A. Hogberg-Raibaud and M. E. Goldberg, Biochemistry 16, 4014 (1977). 39 A. M. Labhardt, in "Protein Folding" (R. Jaenicke, ed.), p. 401. Elsevier, Amsterdam, 1980. 4o B. Adams, R. J. Burgess, E. A. Carrey, I. R. Mackintosh, C. Mitchinson, R. M. Thomas, and R. H. Pain, in "Protein Folding" (R. Jaenicke, ed.), p. 447. Elsevier, Amsterdam, 1980. 4~ K. O. Johanson, D. B. Wetlaufer, R. G. Reed, and Th. Peters, Jr., J. Biol. Chem. 256, 445 (1981). 42 A. M. Labhardt, Biopolymers 20, 1459 (1981). 43 A. M. Labhardt, J. Mol. Biol. 157, 331 (1982). A. M. Labhardt, J. Mol. Biol. 157, 357 (1982). 45 A. M. Labhardt, Proc. Natl. Acad. Sci. U.S.A. 81, 7674 (1984). 46 A. M. Labhardt, J. A. Ridge, R. N. Lindquist, and R. L. Baldwin, Biochemistry 22, 321 (1983). 47 M. Erard, E. Burggraf, and J. Pouyet, FEBS Lett. 149, 55 (1982). 48 A. J. Hillquist Damon and G. C. Kresheck, Biopolymers 21, 895 (1982). 49 N. Greenfield and G. D. Fasman, Biochemistry 8, 4108 (1969). ~0 V. P. Saxena and D. B. Wetlaufer, Proc. Natl. Acad. Sci. U.S.A. 68, 969 (1971). 5, y . H. Chen, 3. T. Yang, and H. M. Martinez, Biochemistry 11, 4120 (1972).

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ters with mechanical stopped-flow devices 47,56,57 have been used. For a recent review of fast CD instrumentation, see Bayley) 8

Characterization of Folding Kernels By definition, only a folding kernel can be observed and characterized by equilibrium spectroscopy. A nucleus will decay or rapidly induce further folding and hence will not accumulate. Such presumed kernels have been investigated with CD by unfolding whole proteins by extremes of pH, 28'44'45'59by the action of heat 39,42,43 or denaturants such as GuHC1 or urea, 15-17,27,29,6° by the reduction of disulfide bonds, 2°-23,32-34,61,62 or by characterizing the conformation of protein fragments. 24-26,37,41 The interpretation of the spectral properties of the unfolded state is difficult. It has been suggested on the basis of CD and ORD data that pH and heat unfolded proteins 15,16,39,42,43,62-65 as well as reduced proteins 2°-23 retain structure capable of being disrupted in concentrated solutions of GuHCI. CD and optical rotatory dispersion data taken with solutions of denaturants must be interpreted with caution since anomalous optical effects have been proposed to arise from the interaction of proteins with denaturants, such as GuHCI, urea, and sodium dodecyl sulfate (SDS). 64,66-68 For arguments for the contrary, see Ref. 48 and references cited therein. E n h a n c e m e n t s of aromatic bands upon denaturation of pro-

52y. H. Chen, J. T. Yang, and K. H. Chau, Biochemistry 13, 3350 (1974). 53C. T. Chang, C.-S. C. Wu, and J. T. Yang, Anal. Biochem. 91, 13 (1978). 54S. Brahms and J. Brahms, J. Mol. Biol. 138, 149 (1980). 55j. p. Hennessey and W. C. Johnson, Biochemistry 20, 1085 (1981). 56j. Luchins and S. Beychok, Science 199, 425 (1978). 57H.-P. B/ichinger, H.-P. Eggenberger, and G. H~inisch,Rev. Sci. Instrum. 511,1367(1979). 58p. M. Bayley, Prog. Biophys. Mol. Biol. 37, 149 (1981). 59K. Nitta, T. Segawa, K. Kuwajima, and S. Sugai, Biopolymers 16, 703 (1977). 6oF. X. Schmid, FEBS Lett. 139, 190 (1982). 61 N. Okabe, E. Fujita, and K.-I. Tomita, Biochim. Biophys. Acta 700, 165 (1982). 62Ch. Tanford and K. C. Aune, Biochemistry 9, 206 (1970). 63K. C. Aune, A. Salahuddin, M. H. Zarlengo, and Ch. Tanford, J. Biol. Chem. 242, 4486 (1967). Ch. Tanford, Adv. Protein Chem. 23, 121 (1968). 65Ch. Tanford, Adv. Protein Chem. 24, 1 (1970). 66B. Jirgensons and S. Capetillo, Biochim. Biophys. Acta 214, 1 (1970). 67M. L. Tiffany and S. Krimm, Biopolymers 12, 575 (1973). D. Balasubramanian, Biopolymers 13, 407 (1974).

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teins with alkali have been observed. 69-73 To assess the contributions of aromatic residues or disulfide bonds to the weak amide region of unfolded proteins the disulfide bonds can be reduced 2° or the tyrosine residues can be titrated. ~5,~6,71 N o n r a n d o m CD spectra in the unfolded state when generated by residual structure m a y be protease sensitive. 24,44 Characterization of Equilibrium Folding Intermediates by CD CD is becoming increasingly popular as a spectral probe to monitor equilibrium transitions. In single w a v e length experiments, the strong ahelix band is usually selected, generally at 222 nm. 27,29,37,4s,74 Recently attempts have been made to analyze and quantitate the secondary structure of folding intermediates by amide CD measured at multiple wavelengths.17,19,22,23,28,39,43,48 Spectral decompositions have been carried out using the methods of P r o v e n c h e r and Gl6ckner 75 and Chen e t al. 51,52 or modifications thereof. 39,43 These procedures use the CD spectra of proteins of k n o w n conformation as a reference base. All procedures are reported to be successful in estimating the correct a-helix and/3-pleated sheet contents. In general, varying success is reported when such additional information is extracted as the average length of helical segments, 52 the fraction of fl-turns, 53,54,76 or differentiation between parallel and antiparallel fl-sheets. 77 F o r this reason it seems inevitable to limit the CD analysis of folding intermediates to monitoring the changes of a-helix and /3-sheet contents. The following p r o b l e m s and potential pitfalls associated with spectral decomposition must be clearly realized. I. Strong aromatic contributions to the amide region generally cannot be excluded. F o r example, the CD of native B P T I is considered abnormal 33,34,78 and an incorrect analysis for secondary structure is obtained from CD data. 23 A c o m p a r a t i v e study of the homologous proteins toxins I and K from black m a m b a has led to the suggestion that the unusual CD of 69 B. Jirgensons, Biochim. Biophys. Acta 317, 131 (1973).

70y . . y . T . Su and B. Jirgensons, MacromoL Chem. 180, 367 (1979). vl B. Jirgensons, Biochim. Biophys. Acta 625, 193 (1980). 72 E. H. Strickland, CRC Crit. Rev. Biochem. 2, 113 (1974). 73y. Tamura and B. Jirgensons, Arch. Biochem. Biophys. 199, 413 (1980). 74j. L. Barbero, L. Franco, F. Montero, and F. Moran, Biochemistry 19, 4080 (1980). 75 S. W. Provencher and J. Glfckner, Biochemistry 20, 33 (1981). 76S. Brahms and J. G. Brahms, J. Chim. Phys. 76, 841 (1979). 77I. A. Bolotina, V. O. Chekhov, and V. Y. Lugauskas, Int. J. Quantum Chem. 16, 819 (1979). 7s H. Rosenkranz, in "Protein Inhibitors" (H. Fritz, H. Tschesche, L. J. Greene, and E. Truscheit, eds.), p. 458. Springer-Verlag, Berlin, 1974.

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BPTI and toxin K results from Tyr-35. 23 In BPTI this residue is immobilized. 79 Toxin I but not K lacks Tyr-35. A strong and broad positive band around 226 nm is thought to be generated by tyrosine residues interacting with peptide b o n d s . 72,73,8°,81 Likewise the far-UV CD of RNase A may be influenced by broad positive contributions from tyrosines 82 which overlap with the more narrow amide bands. As a result, the region of cancellation around 235 nm may be particularly sensitive to changes in CD which remain undetected at shorter wavelengths. 83 For bovine carbonate dehydratase, Henkens e t al. 17 report slight but systematic deviations due to aromatic contributions when fitting the amide CD by the method of Chen e t a l . , 51 and the estimates for helix and sheet fractions remain lower than the crystal structure values. 84 In particular, single wavelength measurements made in the amide region, but outside the strong a-helix bands, or with proteins that lack significant fractions of helix do not necessarily report secondary structure changes. 2. When using reference spectra determined from native proteins to analyze folding intermediates, the following additional problems exist. (1) During unfolding new conformations may arise and dominate the CD spectrum. Tests must be made for the presence of CD active conformations in the unfolded protein (compare section on kernels). (2) By using "native" basis spectra for a-helix and /3-sheet, without correction for size, exposure to solvent, or involvement in supersecondary structure, one implicitly assumes that these factors are unimportant. Evidence to the contrary has been given. Chen e t al. 52 have shown that the a-helical CD spectrum depends on helix length. Manavalan and Johnson 85 summarize evidence for the dependence of the CD spectra on the supersecondary structure and on the conformational class (all-a, a +/3, a//3, all/3).86 The CD spectra of some all-/3 proteins resemble the spectra of models for the random coil. 85 Analysis of the CD spectrum of reduced toxin I predicts 50%/3-sheet. 23 As pointed out by the authors, this is probably incorrect. Similarly, the calculated /3-pleated sheet content of reduced and 79 G. H. Snyder, R. Rowan, M. Karplus, and B. D. Sykers, Biochemistry 14, 3765 (1975). 8o M. Baba, K. Hamaguchi, and T. Ikenaka, J. Biochem. 65, 113 (1969). 81 R. W. Woody, Biopolymers 17, 1451 (1978). 82 C. R. Cantor and S. N. Timasheff, "The Proteins" (H. Neurath and R. L. Hill, eds.), Vol. 5, 3rd Ed., p. 145 (1982). 83 M. N. Pflumm and S. Beychok, J. Biol. Chem. 244, 3973 (1969). K. K. Kaunan, A. Liljas, I. Waara, P.-C. Bergsten, S. Lovgren, B. Standberg, U. Bengtsson, U. Carbom, K. Fidborg, L. Jarup, and M. Petef, Cold Spring Harbor Syrup. Quant. Biol. 136, 221 (1972). 85 p. Manavalan and W. C. Johnson, Jr., Nature (London) 305, 831 (1983). 86 M. Levitt and C. Chothia, Nature (London) 261, 552 (1976).

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carboxymethylated RNase A, has a value between 0 and 25% depending on the reference spectra used. 22 (3) The CD of the nonperiodic structure in the native protein (referred to as irregular structure, excluding the a-helix and fl-sheet regions) may be significantly different from that of the unfolded "random coil" protein. Fitting the CD spectra of RNase A or S either in the thermally denatured state (pH 1.7, 60°), or in the presence of 5 M GuHC1, or after tryptic digestion by the method of Chen e t al., 51 would incorrectly indicate that one-third to one-half of the native fl-sheet content is retained. 87 This result has been traced to the inappropriate use of the irregular structure reference spectrum for the unfolded random coil state. Irregular structure reference spectra vary significantly, 51-53,77 depending on the types and number of reference proteins. They tend to deviate more or less systematically from CD spectra obtained from polypeptides that are considered to be models for the random coil state. It has been s u g g e s t e d 39,43 that the reference spectrum for the irregular structure in the native state should be taken from the measured random coil spectrum of the same protein, since inadequacies in the presence of the strong helix and sheet CD spectra become relatively smaller. 3. Alternatively one can depart completely from the use of pure-state reference spectra and instead decompose the spectral changes measured on unfolding into fractional changes based on sufficiently orthogonal spectra obtained from known mixed conformations. Using native RNase S and folded and thermally denatured S-protein as references, the spectra in the unfolding transitions of RNase S and A were decomposed in this way 43 and given a structural interpretation. As noted by Era et al., 28 extension of this approach to a basis set of five reference proteins yields a decreased signal-to-noise ratio and the conceptually equivalent regulator method of Provencher and Gl6ckner 75 should be used. Characterization of Transient Folding Intermediates by Stopped Flow CD CD as a kinetic technique has several practical difficulties. Before deciding to start such investigations, these problems should be carefully considered. 1. The sensitivity in the amide region is low when short response times are needed. 2. As a consequence, high protein concentrations (above 1 mg/ml) and multiple signal averaging are necessary to obtain reliable results. 87 A. M. Labhardt, unpublished results.

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3. The instrumentation needed for fast kinetics is currently still beyond the reach of most laboratories. 56-58 The following points have been emphasized as advantages of the CD stopped-flow technique for investigations of folding. It is a multiparameter approach. Differences in the kinetics can be monitored by aromatic absorbance, aromatic CD, and amide CD. This allows one to order, respectively, the burial of the aromatic groups in the interior of the protein, the fixation of aromatic groups in a folded environment, and the generation of the backbone conformation. Intrinsic fluorescence from tyrosine residues has also been used to monitor hydrogen bonding of these residues during folding. The kinetics of heme-induced refolding of human a-globin has been investigated 88 using the far-UV instrument described by Luchins and Beychok. 56 The kinetics were observed by CD at 222 nm and compared to the kinetics measured by absorbance in the Soret band and by quenching of tryptophan fluorescence. The instrument was calibrated at 290 nm against d-10-camphorsulfonic acid and at 222 nm against a solution of methemoglobin. The signal-to-noise ratio at 222 nm was optimized by testing samples of various concentrations with absorbance in the range 0.15 to 0 . 5 0 D . Data acquisition used a Nicolet Explorer 1090A digital oscilloscope. Based on the measured rates, the following interpretation of events was given. (1) First the heme enters the pocket with a half-time of 10 msec at 2.4 /zM concentration, 4°. (2) Subsequently the pocket assumes its final conformation (halftime 40 sec). (3) The induced growth of the a-helices is slow and biphasic with the major component having a halftime of about 160 sec. Slow helix formation has also been observed for the S-peptide helix 313 of RNase $45: In pH-jumps from low to neutral pH this helix forms in the terminal phase of folding with the same time constant measured for the burial of the tyrosines in the S-protein moiety (42 -+ 6 sec). The regain of affinity for 2'-CMP occurs in the same terminal phase of folding of the S-peptide:S-protein complex. In this work the far-UV instrument of B/ichinger et al. 57 w a s used. Up to 30 single shots, each one consuming 0.4 ml of solution, were averaged to improve the signal-to-noise ratio in the millisecond time range. Data collection used a Datalab transient recorder model DL 905 interfaced to an LSI 11/23 computer. A significant improvement of the signal-to-noise performance was achieved by executing a sliding average (integration) over 5 to 10 subsequent points of the digital transients. In order to avoid distortion of the progress curves, care had to be taken that the time interval corresponding to the length of the ss y . Leutzinger and S. Beychok, Proc. Natl. Acad. Sci. U.S.A. 78, 780 (1981).

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sliding average did not exceed 0. l/rain, where tmin is the fastest relaxation time. An alternative smoothing procedure 89 has been used by Hasumi in kinetic CD studies of the alkaline isomerization of ferricytochrome c 9° and spinach ferredoxin. 91 Terminal secondary structure formation may be the exception rather than the rule. In refolding of bovine carbonate dehydratase B from 6 M GuHCI, 29 secondary structure forms (as monitored by CD22z) before the burial of the chromophores (as monitored by absorbance at 291.5 nm) and clustering of the aromatic residues (as measured by CD270). The kinetic CD measurements were made by rapid manual mixing, with a dead time of 20 sec. Data were collected either at single wavelength settings, o r - because of the very slow kinetics--by a 8.8 min scan of the wavelength region 240 to 205 nm on a Jasco J 20 spectropolarimeter. Goto and Hamaguchi 92 report the refolding kinetics of the constant fragment of the immunoglobin light chain. Of the backbone CD 70% returns in the dead time of the experiment (1 min). Refolding was monitored by manually diluting the 7 mg/ml protein solution from 4 M GuHCI to 0.1 M GuHCI. The CD signal was recorded with a Jasco J-20 photometer with CD attachment at 17 wavelengths between 235 and 208 nm. The refolding of swine pepsinogen was investigated in a similar fashion. 93 The protein was unfolded at pH 8 or 11.5 by 6 M urea. Dilution to 0.29 M urea produces within the manual mixing dead time a folding intermediate. Its UV-CD spectrum was recorded at 13 wavelengths between 240 and 210 nm. It closely resembles the CD spectrum of native pepsinogen. Spectral decomposition was attempted as described by White. 94 Refolding monitored by other probes appears slow. Kuwajima e t al. 95 have introduced a rapid dilution device consisting of a conventional cuvette and a magnetic stirrer. The dead-time is decreased to 3 sec. They have investigated the refolding of a-lactalbumin and lysozyme by a 20-fold dilution of the protein solutions containing 6 M GuHC1. In the aromatic region almost the full changes expected from equilibrium studies are detected kinetically with time constants larger than 10 and 15 sec, respectively. In contrast most of the ellipticity changes in the peptide region occur in the dead-time. They extrapolate the CD kinetics to the dilution time point to get the CD spectra of the early folding intermedi89 A. Savitzki and M. J. E. Golay, Anal. Chem. 36, 1627 (1964). 9o H. Hasumi, Biochim. Biophys. Acta 626, 265 (1980). 9z H. Hasumi, J. Biochem. 92, 1049 (1982). 92 y . Goto and K. Hamaguchi, J. Mol. Biol. 156, 891 (1982). 93 p. McPhie, Biochemistry 21, 5509 (1982). 94 F. H. White, Biochemistry 15, 2906 (1976). 95 K. Kuwajima, Y. Hiraoka, M. Ikeguchi, and S. Sugai, Biochemistry 24, 874 (1985),

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ates. In the case of a-lactalbumin this spectrum is compatible with the spectrum of the A state. The latter is an equilibrium state of intermediate folding observed in 2 M GuHC1 below pH 515 and above pH 8.5.16 Its spectral characteristics are the absence of aromatic CD and the presence of most of the peptide CD. It had been observed quite early by CD that secondary structure formation precedes the regain of activity during reoxidation of reduced R N a s e A . 18,21,22 This is consistent with the common opinion that local structural preferences drive the selection of disulfide bond formation (e.g., Ref. 35). Note that the time range for these experiments is hours instead of seconds for refolding with the disulfide bonds intact. The unfolding of apomyoglobin 96 and myoglobin97,98 after a pH jump from neutral to low pH has been reported by Kihara et al. using stopped flow measurements of CD at 222 nm. A Union Giken CD-1002 stopped flow spectrophotometer with 2 msec dead-time and an electronic response time of 1/13 msec was used. In such experiments, the helical structure of apomyoglobin breaks down too rapidly to be followed kinetically. In the case of myoglobin, however, part of the helical CD disappears in the second time range. The unfolding of the RNase S-peptide helix 3-13 when lowering the pH from 6.6 to 1.7 could be monitored at 13° by CD at 225 nm. 45,57 This reaction has a time constant of 513 -+ 150 msec and represents the first conformational change detected in the unfolding of RNase S. At pH 3 the tetrameric lectin concanavalin A (Con A) dissociates into dimers with no gross conformational change of the subunits. 99 The kinetics of unfolding of dimeric Con A by 4 to 8 M urea was investigated. 1°° The CD signal was measured after manual mixing (30 sec) at 218,225, and 283 nm with a Jasco 500 dichrometer. The traces were digitized and smoothed by the Savitzky-Golay procedure, s9 The denaturation of TMV coat protein by urea or by GuHCI has been reported using stopped-flow measurements of CD at 222 nm. TM An entirely commercial setup with a dead time of 20 msec and averaging capabilities of up to 64 transients was used.

H. Kihara and E. Takahashi, Biochem. Biophys. Res. Commun. 95, 1687 (1980). 97 H. Kihara, E. Takahashi, K. Yamamura, and I. Tabushi, Biochim. Biophys. Acta "/02, 249 (1982). H. Kihara, E. Takahashi-Ushijima, and S. Saigo, Jiehi Med. J. 6, 143 (1983). H. E. Auer and T. Schilz, Int. J. Pept. Protein Res. 24, 462 (1984). ~ H. E. Auer and T. Schilz, Int. J. Pept. Protein Res. 24, 569 (1984). ~0~y. Sano and H. Inoue, Chem. Lett. 1087 (1979).