View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Research Paper 19

Discrete intermediates versus molten globule models for folding: characterization of partially folded intermediates of apomyoglobin Anthony L Fink, Keith A Oberg and Sangita Seshadri

Background: Although small may fold in an apparent two-state Address: Department of Chemistry and manner, most studies of reveal transient intermediates. The Biochemistry, The University of California, Santa Cruz, CA 95064, USA. ‘molten globule’ has been proposed to be a general intermediate in protein folding. Relatively little is known about the structure of partially folded Correspondence: Anthony L Fink intermediates, however. E-mail: [email protected]

Results: Three different partially folded intermediates of apomyoglobin, having Key words: A states, apomyoglobin, molten globule, partially folded intermediates, protein 35%, 50% and 60% helix, were characterized at low pH in the presence of folding different anions. It was found that increasing helical structure correlated with decreasing size and increasing stability to urea. Similar intermediates have been Received: 01 September 1997 observed transiently during the folding of apomyoglobin. Revisions requested: 06 October 1997 Revisions received: 20 October 1997 Accepted: 28 October 1997 Conclusions: The results are consistent with a model for folding in which structural units coalesce to form a core of relatively native-like structure, the Published: 27 November 1997 remainder of the protein being relatively disordered. For a given protein there http://biomednet.com/elecref/1359027800300019 will be certain partially folded conformations of particularly low free energy that Folding & Design 27 November 1997, 3:19–25 are preferentially populated under both equilibrium and transient folding conditions. The conformation and topology of the intermediates will be specific © Current Biology Ltd ISSN 1359-0278 to a given protein, so there are no ‘general’ intermediates, such as the molten globule, in folding.

Introduction molten globule (e.g. the ‘pre-molten globule’ and the Several models for the mechanism of protein folding ‘highly ordered molten globule’; [1,13,17–19]). The orig- have been presented and there is considerable contro- inal definition of the molten globule was a compact inter- versy regarding these competing hypotheses. Recently, mediate state with native-like secondary structure but the emphasis has been on ensembles of conformational fluctuating tertiary structure [11,20]. In fact, the ten- (sub)states rather than on traditional kinetic pathways dency has been to call any partially folded intermediate a [1–3]. Current sources of contention include whether molten globule regardless of its properties. A key feature there are on-pathway intermediates in folding and, if so, of the molten globule model is that the intermediates the nature of such partially folded intermediates. There formed from different proteins will have similar struc- is now substantial evidence to indicate that small pro- tural features. The data presented here suggest that the teins may, under appropriate conditions, fold to the molten globule model is incorrect and that each protein within a few milliseconds with no detectable will potentially have partially folded intermediates intermediates [2,4–6]. Such folding is consistent with (ensembles of substates) with regions of unique struc- smooth-funnel energy landscape models [3,7]. On the ture. This is a reflection of the fact that the amino acid other hand, for many proteins there is substantial experi- sequence determines the fold and that each protein has a mental data for transient intermediates [8–10], including unique sequence, and thus potentially a unique folding the molten globule [11–14]. pathway. Several observations suggest that for at least some proteins the observed intermediate species are on The ‘molten globule’ has been proposed as a common the productive folding pathway, rather than there being (general) intermediate in all folding pathways [12,15] misfolded, off-pathway species [1,21]. Several reports and experimental support for intermediates with several have described the apomyoglobin molten globule (e.g. properties common to molten globules have now been [19,22–28]). Some evidence for defined structure in other reported for many proteins [13,16]. A number of observa- partially folded intermediates, such as intermediates of tions have been made, however, that are inconsistent α-lactalbumin, has been reported [29–31]. with the molten globule model as originally conceived and these observations have led to proposed modifica- Here, we describe evidence for the existence of three dis- tions, such as the existence of more than one form of crete, relatively stable, partially folded conformations of 20 Folding & Design Vol 3 No 1

Figure 1

Far UV CD spectra of apomyoglobin in the

native (pH 7.0; spectrum 1), A3 (30 mM TCA, 10 pH 2; spectrum 2), A2 (100 mM TFA, pH 2; spectrum 3), A1 (500 mM KCl, pH 2; spectrum 4) and acid-unfolded (UA; pH 2.0; spectrum 5) states. Protein concentrations

/dmol) 0 were 0.3 mg/ml, determined using a 1 mm 2 5 pathlength cell at 22°C. deg cm

–3 –10 4 (x 10 3 222 ] θ [ –20 2 1

200 210 220 230 240 250

Wavelength (nm) Folding & Design

apomyoglobin. We believe that these conformations are TCA, A3) to complete the transition from UA to the particularly stable and are populated under appropriate A state [24]. Apomyoglobin is an all-helix protein [33]: transient and equilibrium folding conditions. Previously, the amounts of helix, determined by CD and FTIR, for we have shown that conditions of low pH and moderate the native, UA and three A states are shown in Table 1. salt can be used to stabilize partially folded intermediates The amount of helix in each A state corresponds well in many proteins, including apomyoglobin [16,22–24,32]. with that observed in transient intermediates during the These intermediates are known as A states (note that the refolding of apomyoglobin using amide-protection NMR term state is not used in its strict thermodynamic sense experiments [34]. here). Anions differ in their effectiveness in ‘neutralizing’ the net positive charges [24]. We find that different The size of the different A states was measured using anions also result in different amounts of secondary struc- dynamic light scattering, small-angle X-ray scattering and, ture, as determined by CD spectroscopy upon completion where possible, size exclusion HPLC. The Stokes radius of the anion titration of acid-unfolded (UA) apomyoglobin (Rs) and radius of gyration (Rg) for the different conforma- (Figure 1). Interestingly, for all the 10 anions we have tions are shown in Table 1. These experiments revealed examined [23] the end-point of the anion titration curve that at suitably low protein concentrations the solutions of θ ≈ always had one of three values: [ ]222 –12,000, –15,000 each A state were monodisperse, indicating the absence of or –19,000 deg cm2/dmol, indicating that three different native or unfolded states, as well as showing that each intermediates are formed. These species will be referred A state is a discrete species. The relationship between Rs to as A1, A2 and A3, respectively. For the native state, and Rg is quite sensitive to the shape and compactness of θ ≈ 2 [ ]222 –21,000 deg cm /dmol. The A states arising from the protein. The data indicate that the less structured A1 three monovalent anions (chloride, A1 state; trifluoro- state has a significantly non-globular shape, whereas the acetate [TFA], A2 state; and trichloroacetate [TCA], A3 two more structured A states are relatively globular. state) were chosen for more detailed study. We will show by several criteria that the least structured intermediate, The stability of the A states was examined in the pres- ∆ A1, is ~50% folded (relative to the native state) and A2 ence of urea: both the stability ( G) and the cooperativity and A3 have correspondingly more ordered structure and (m) of the unfolding transition decreased as the amount compactness. The A states are separated from each other, of structure in the intermediate decreased (Table 1, θ and from the unfolded state, by significant free energy Figure 2). The thermal unfolding (monitored by 222) of differences, as determined by urea-induced unfolding the A states was increasingly non-cooperative for the less transitions. structured intermediates (Figure 3). For A3, a clear three- state transition was observed in which the ellipticity at the

Results and discussion completion of the first transition was similar to that of A2. Properties of apomyoglobin A states The three different A states were made by adding a suf- The near-UV CD spectra (Figure 4) for the A states ficient quantity of the anions (chloride, A1; TFA, A2; and were essentially identical and of lower intensity than the Research Paper Protein folding intermediates Fink et al. 21

Table 1 Figure 2

Properties of different conformational states of apomyoglobin.

Property N A3 A2 A1 UA

% Helix from: CD* 71 60 49 38 6 0 FTIR† 74 53 49 34 Calculated‡ 68 63 51 39 –5 § Rs 20 27 31 37 43

¶ /dmol) Rg 18.7 22.4 25.9 27.2 34 2 # Relative volume 1.0 2.8 4.3 7.4 11.6 –10 ¥ Cm (M) 2.5 2.4 1.9 1.8 deg cm ∆ –3 G0 (kcal/mol)** 4.4 2.5 1.8 1.5 m†† 1.6 1.0 0.92 0.85 (x 10 –15 222 Propensity to aggregate‡‡ 0 0 0.32 0.74 ] θ [

A1, A2 and A3 are three different partially folded conformations of –20 apomyoglobin; UA, acid-unfolded state; Rs, the Stokes radius; Rg, radius of gyration; Cm, the midpoint of the urea denaturation transition; ∆G , free energy indicating the stability of the unfolding transition; and 0 –25 m, the cooperativity of the unfolding transition. *Using the method of Chen et al. [40]. Error is ± 5%. †Transmission FTIR data, using FSD 02468 Urea concentration (M) and second derivative deconvolution. Estimated error is ± 6–7%. ‡ A1 (KCl) From the X-ray structure of myoglobin, assuming the presence of: A1, A2 (TFA) only the core A, G, and H helices; A2, the core and helix B or E; A3, the core and helix B and E. §From dynamic light scattering measurements. A3 (TCA) Native apomyoglobin, pH 7.5 Radii in Å. Error is ± 5%. ¶From small-angle X-ray scattering Folding & Design measurements. Radii in Å. Error is ± 5–6%. #Relative volume of a sphere with radius R with native apomyoglobin = 1.0. ¥Midpoint of the s Urea-induced unfolding transitions of different conformational states of major urea-induced unfolding transition (see Figure 2). **Extrapolated apomyoglobin as measured by far-UV CD at 20°C. Experimental free energy of the major urea-induced unfolding transition to zero urea conditions were as noted in Figure 1. concentration. ††Slope of the plot of ∆G versus concentration of urea for the major transition. ‡‡From dynamic light scattering measurements: slope of R versus protein concentration. s shows that the propensity for aggregation decreases with increasing structure (Table 1). spectrum for the native state, but the spectra for the A states show the presence of some tertiary interactions. These data indicate that under mildly destabilizing con- Detailed analysis suggested that the tyrosine contribu- ditions apomyoglobin can form three discrete partially tions were absent from the spectra of the A states (indi- folded intermediates, differing in their degree of folding. cating the absence of the native CFGH helix interface) The observation of urea-induced transitions between the and that the tryptophan residues (in the AGH interface intermediates indicates that there are significant energy in the native conformation) were involved in specific barriers between them, and that the intermediates are λ interactions (Figure 4; [35]). The max values of the discrete states and not different populations of a single intrinsic tryptophan fluorescence for all three A states state. Differential destabilization of the native state rela- λ were similar to max for the native state (333 nm), tive to the partially folded intermediates is required for whereas the fluorescence intensity of the A states, population of the intermediate states. except that in TCA, was 11% higher than that of the native protein. Correction for the quenching effect of Structure of the A states TCA, using the Stern–Volmer relation, yielded a trypto- All three A states of apomyoglobin contain a core of the A, phan fluorescence intensity similar to that of the other G and H helices, as do the early kinetic intermediate A states. [34,36] and the intermediate at pH 4 [25,37]. The pres- ence of the core in the A states is supported by several The A states did not bind heme, based on the lack of lines of evidence, including: the helix content; the similar- absorbance in the Soret region when hemin was added. ity in tryptophan fluorescence and the near-UV CD Thus, a stable tight-binding heme pocket is absent even spectra in the three A states and the native state; and, in the most structured intermediate. Information concern- especially, fluorescence energy transfer experiments using ing exposed hydrophobic surfaces comes from dynamic nitrotyrosine-modified apomyoglobin, which shows similar light scattering measurements of the propensity of the distances between the A and H helices in the three A states to aggregate at higher protein concentrations, and A states and the native state (unpublished observations). 22 Folding & Design Vol 3 No 1

Figure 3 disordered (Figure 5). A2 and A3, the more structured inter- mediates, arise by the coalescence of additional structural

–2 units onto this core. For example, the A2 intermediate has an additional helix (B or E) present and in A3 an additional –4 helix (or helices) docks to the core [19,34]. Increasing structure in the intermediates correlates with decreased –6 size, decreased hydrophobic surface area (reduced aggrega- –8 tion), and little change in the core tryptophan environ- ment. A linear relationship was found between the amount /dmol) 2 –10 θ of helix (i.e. 222) and the volume of the different A states, –12 indicating that the amount of structure in the partially

deg cm folded intermediate and its degree of compactness are inti- –3 –14 mately related. This strongly supports the notion that col-

(x 10 lapse (and hence compactness) and secondary structure –16 222 ] formation are concurrent. It is useful to consider the θ [ –18 progress of folding as shown in Figure 6, in which the three A states are shown as a function of the ‘degree of folding’, –20 which can be a measure of either the amount of secondary –22 structure or the compactness, based on their correlation; 0 20406080100this is equivalent to a measure of the reaction progress or Temperature (ºC) reaction coordinate. That these observations may be rela-

UA apomyoglobin tively general is supported by similar findings for staphylo- A1 (KCl) coccal nuclease (unpublished observations). In particular, A (TFA) 2 the same anions as in apomyoglobin result in comparable A3 (TCA) Native apomyoglobin, pH 7.5 A1, A2 and A3 states, with the amounts of native-like struc- Folding & Design ture in the range 50–95%, based on ellipticity. Importantly, three-state urea-induced transitions were observed for A Thermal unfolding transitions of different conformational states of 3 apomyoglobin as measured by far-UV CD. Except for the temperature, and A2. the experimental conditions were as noted in Figure 1. Protein folding model The results are consistent with the following model for

Thus, A1, the least compact and least structured interme- protein folding. Placing an unfolded protein under native- diate, consists of a core of relatively native-like structure like conditions leads to a rapid collapse driven by burial of (the AGH helices), with the rest of the polypeptide chain hydrophobic surface area, with concomitant formation of

Figure 4

Near-UV CD spectra of apomyoglobin in the

native (spectrum 1), A3 (TCA; spectrum 2), A2 (TFA; spectrum 3), A1 (KCl; spectrum 4) and 600 1 UA (spectrum 5) states. Protein concentrations were 1 mg/ml in a 1 cm

) 400 pathlength cell. Spectra are offset for clarity.

–1 The scale on the y axis is for the native state. 200

/dmol 2 2 0 3

(deg cm 200 4 222 ] θ

[ –400 5 –600

255 265 275 285 295 305 315

Wavelength (nm) Folding & Design Research Paper Protein folding intermediates Fink et al. 23

Figure 5 Figure 6

N UA state A states Native state A1 A2 A3

G

C H 0 0.5 1.0 Degree of folding Folding & Design

A The relative degree of folding for the partially folded intermediate A states of apomyoglobin.

Folding & Design In summary, we propose two key points: at least some proteins fold via a hierarchical path in which additional A schematic model for the structure of the A1 intermediate of apomyoglobin. The A, G and H helices form a native-like core, structural units coalesce to an initially formed core with connected by an essentially disordered polypeptide. Successive native-like structure, and the nature of the intermediates intermediates have more core and less disordered polypeptide chain. will be specific to a given protein (i.e. there are no ‘general’ or common intermediates in folding; in fact, each protein provides a unique situation). For a given protein, there will some secondary structure. The nature of this collapsed also be certain partially folded conformations that corre- state will vary depending on the conditions and the partic- spond to particularly low free energies, which are thus pref- ular protein, but in general it will consist of a very large erentially populated under both equilibrium and transient number of substates. Further condensation will lead to folding conditions. one or more particularly stable intermediate(s) and, again depending on the particular protein, the intermediate(s) Materials and methods will have regions of unique structure, especially in terms Preparation of apomyoglobin of the compactness, amount of secondary structure and Horse apomyoglobin was prepared using the heme extraction method topology. It is very likely that in most cases these interme- of Teale [38] with a pH 2 dialysis immediately following 2-butanone extraction. Protein concentration was determined using an ε of diates will consist of a core of native-like structure with 280 15,700 M–1 cm–1. the remainder of the protein in varying degrees of disor- der. Regions of the non-ordered chain are probably flick- Generation of A states ering in and out of their native-like secondary structure Conditions used to induce the A states were chosen at a point where → conformation. Thus, the intermediate will be an ensemble the UA A state transitions were nearly complete [24]. Anion solutions of many substates having in common a core of relatively were prepared by dissolving the appropriate acid or sodium salt in native-like structure. This picture fits nicely with the 10 mM HCl, then adjusting the pH to 2.0. Anion concentrations were as follows: TCA 15 mM, 30 mM or 50 mM (we found that high protein funnel type of folding model, but in this case the funnel concentrations required 30–50 mM TCA to ensure completion of the shape leads to a directed pathway of folding involving the transition, although the transition from UA to A3 is complete by 20 mM intermediate cores. If more than one intermediate is TCA at low protein concentrations); 100 mM TFA; 500 mM chloride. formed, each successive one will have more native-like Any undissolved protein was removed by centrifugation. Typically, A state solutions were used within 0.5 h after mixing. For native than its predecessor. This model also readily the conditions were 50 mM sodium phosphate, pH 7.0. UA was made allows for parallel pathways, for example those in which by dissolving salt-free apomyoglobin in 10 mM HCl (pH 2). Solutions of either two different cores are formed of similar stability or apomyoglobin are stable at pH 2 and room temperature for several subsequent regions of ordered structure adding to the core hours after mixing. lead to more than one species of equivalent stability. The Dynamic light scattering existence of a limited number of especially stable partially Dynamic light scattering (DLS) measurements were made with a folded conformations (i.e. significant energy minima) sug- Brookhaven model BI2030 instrument with a tunable 2 W laser at gests that these species would be detectable under both 514 nm, running at 0.4–0.8 W. Samples were collected in random transient (kinetic) folding conditions and selected equilib- order. Protein solutions were filtered directly into clean, dust-free µ rium conditions. It is the presence of the native-like core cuvettes through 0.22–0.45 m filters. DLS measurements were taken of 1.5–3 ml samples at 20°C. Instrumental parameters were as follows: that leads to the enhanced stability and population of the detector slit width, 200–800 nm; sample time, 0.3–1.5 µs; 2–7 × 107 intermediates. readings collected for each run, 7–10 runs for each sample. Actual 24 Folding & Design Vol 3 No 1

protein concentrations were determined after DLS measurements by 4. Sosnick T.R., Mayne, L., Hiller, R. & Englander S.W. (1994). The ⋅× barriers in protein folding. Nat. Struct. Biol. 1, 149-156. denaturing the protein in 6 M guanidinium HCl and measuring A280. 5. Huang, G.S. & Oas, T.G. (1995). Submillisecond folding of monomeric lambda repressor. Proc. Natl Acad. Sci. USA 92, Fluorescence 6878-6882. Fluorescence measurements were made on a Perkin Elmer MPF 4 6. Schindler, T., Herrler, M., Marahiel, M.A. & Schmid, F.X. (1995). instrument using ratio mode with a 4 nm excitation slit, and 10 nm emis- Extremely rapid protein folding in the absence of intermediates. Nat. sion slit. Protein concentrations were ~0.1 mg/ml. Intrinsic (tryptophan) Struct. Biol. 2, 663-673. fluorescence was measured using excitation at 280 nm. 7. Onuchic J.N., Wolynes P.G., Luthey-Schulten, Z. & Socci N.D. (1995). Toward an outline of the topography of a realistic protein-. Proc. Natl Acad. Sci. USA 92, 3626-3630. FTIR 8. Varley, P., Gronenborn A.M., Christensen, H., Wingfield P.T., Pain R.H. FTIR interferograms were collected on a Nicolet 800SX instrument & Clore G.M. (1993). Kinetics of folding of the all-beta sheet protein with an MCT detector. 4000 scans were co-added at 4 wavenumber interleukin-1 beta. Science 260, 1110-1113. 1 resolution. All samples were prepared with H2O. Transmission spectra 9. Bai, Y., Milne J.S., Mayne, L. & Englander S.W. (1994). Protein were collected with protein concentrations in the range 10–40 mg/ml stability parameters measured by hydrogen exchange. Proteins 20, µ 4-14. in a demountable cell with BaF2 windows and a 15 m Teflon spacer. Open beam spectra were collected for use as backgrounds. All data 10. Radford S.E. & Dobson C.M. (1995). Insights into protein folding processing and analysis was performed with GRAMS/386 and Lab using physical techniques: studies of lysozyme and alpha-lactalbumin. 348 Calc (Galactic) as previously described [39]. Phil. Trans. R. Soc. Lond. Ser. B. , 17-25. 11. Ptitsyn, O.B. (1987). Protein folding: hypothesis and experiment. J. Protein Chem. 6, 273-293. CD 12. Ptitsyn, O.B., Pain, R.H., Semisotnov, G.V., Zerovnik, E. & Razgulyaev, CD data were collected on an Aviv 60DS CD spectrometer. Far-UV O.I. (1990). Evidence for a molten globule state as a general spectra from 185–260 nm were collected with 5 s/point signal averag- intermediate in protein folding. FEBS Lett. 262, 20-24. θ 13. Ptitsyn O.B. (1995). Molten globule and protein folding. Adv. Protein ing; 222 measurements were taken in kinetics mode with the signal averaged over 120 s. For apomyoglobin concentrations < 0.6 mg/ml a Chem. 47, 83-229. 0.1 cm fixed pathlength cell was used. For 0.6–1 mg/ml and > 1 mg/ml 14. Kuwajima, K. (1989). The molten globule state as a clue for understanding the folding and cooperativity of globular-protein samples 0.05 cm and 0.001 cm sandwich cells were used, respectively. structure. Proteins 6, 87-103. The pathlength of the 0.001 cm cell was checked using the log of 15. Ptitsyn, O.B. (1992). In Protein Folding. (Creighton, T.E., ed.). detector dynode voltage at 222 nm and a calibration curve generated pp. 243-300. W.H. Freeman and Company, New York. for native apomyoglobin in a 0.1 cm cell. α Helix content was estimated 16. Fink, A.L. (1995). Compact intermediate states in protein folding. using the method of Chen et al. [40]. For near-UV CD, 10 scans at Annu. Rev. Biophys. Biomolec. Struct. 24, 495-522. 5 s/point were collected using 1 mg/ml apomyoglobin in a 1 cm path- 17. Uversky V.N. & Ptitsyn O.B. (1994). ‘Partly folded’ state, a new length circular cell. Soret spectra were collected with holomyoglobin equilibrium state of protein molecules: four-state guanidinium chloride- under the same conditions used for apomyoglobin. induced unfolding of beta-lactamase at low temperature. Biochemistry 33, 2782-2791. 18. Uversky V.N. & Ptitsyn O.B. (1996). Further evidence on the Stability of the A states equilibrium ‘pre-molten globule state’: four-state guanidinium chloride- Urea-induced unfolding transitions were monitored by the changes in induced unfolding of carbonic anhydrase B at low temperature J. Mol. ellipticity at 222 nm. Stock solutions of 9 M urea were made in a buffer Biol. 255, 215-228. containing the respective anion and the pH adjusted to 2. The concen- 19. Loh S.N., Kay M.S. & Baldwin R.L. (1995). Structure and stability of a tration of the urea was checked using the refractive index. Thermal second molten globule intermediate in the apomyoglobin folding denaturation was monitored by changes in the ellipticity at 222 nm. The pathway. Proc. Natl Acad. Sci. USA 92, 5446-5450. 20. Ohgushi, M. & Wada, A. (1983). ‘Molten-globule state’: a compact temperatures were raised from 0–100°C at a rate of > 0.3°C/min. form of globular proteins with mobile side-chains. FEBS Lett. 164, 21-24. Small-angle X-ray scattering 21. Miranker A.D. & Dobson C.M. (1996). Collapse and cooperativity in Solution X-ray scattering experiments were carried out at the Stanford protein folding. Curr. Opin. Struct. Biol. 6, 31-42. Synchrotron Radiation Laboratory (SSRL) on Beam Line 4–2. A flow- 22. Goto, Y., Calciano, L.J. & Fink, A.L. (1990). Acid-induced folding of cell with 10 µm thick mica windows, a pathlength of 1.3 mm and a proteins. Proc. Natl Acad. Sci. USA 87, 573-577. sample capacity of 45 µm was used to reduce extended exposure of the 23. Goto, Y. & Fink, A.L. (1990). Phase diagram for acidic conformational states of apomyoglobin. J. Mol. Biol. 214, 803-805. sample to radiation. The sample cell was thermostatted and maintained 24. Goto, Y., Takahashi, N. & Fink, A.L. (1990). Mechanism of acid- at 20°C. Protein concentrations were varied from 1–5 mg/ml for the induced folding of proteins. Biochemistry 29, 3480-3488. native state, 1–2 mg/ml for the TCA-induced A state and 0.25 mg/ml for 25. Barrick, D. & Baldwin R.L. (1993). Stein and Moore award address. the acid-denatured, KCl-induced and TFA-induced A states. These con- The molten globule intermediate of apomyoglobin and the process of centrations were chosen based on the Raleigh light scattering data protein folding. Protein Sci. 2, 869-876. which showed the presence of aggregation in the solutions of UA, TFA 26. Gast, K., Damaschun, H., Misselwitz, R., Muller-Frohne, M., Zirwer, D. and KCl A states at concentrations 0.3 mg/ml. & Damaschun, G. (1994). Compactness of protein molten globules: temperature-induced structural changes of the apomyoglobin folding intermediate. Eur. Biophys. J. 23, 297-305. Acknowledgements 27. Nishii, I., Kataoka, M. & Goto, Y. (1995). Thermodynamic stability of We thank Dan Segel and Seb Doniach for assistance with the small-angle the molten globule states of apomyoglobin. J. Mol. Biol. 250, X-ray scattering experiments, and Alice Gast and Linda DeYoung for assis- 223-238. tance with the dynamic light scattering experiments. This research was sup- 28. Kiefhaber, T. & Baldwin R.L. (1995). Intrinsic stability of individual ported by grants from the National Science Foundation and beamtime was alpha helices modulates structure and stability of the apomyoglobin from SSRL. molten globule form J. Mol. Biol. 252, 122-132. 29. Schulman B.A., Redfield, C., Peng Z.Y., Dobson C.M. & Kim P.S. References (1995). Different subdomains are most protected from hydrogen 1. Baldwin R.L. (1995). The nature of protein folding pathways: the exchange in the molten globule and native states of human alpha- classical versus the new view. J. Biomolec. NMR 5, 103-109. lactalbumin. J. Mol. Biol. 253, 651-657. 2. Privalov, P.L. (1996). Intermediate states in protein folding. J. Mol. 30. Chyan C.L., Wormald, C., Dobson C.M., Evans P.A. & Baum, J. Biol. 258, 707-725. (1993). Structure and stability of the molten globule state of guinea- 3. Dill, K.A. & Chan, H.S. (1997). From Levinthal to pathways to funnels. pig alpha-lactalbumin: a hydrogen exchange study. Biochemistry 32, Nat. Struct. Biol. 4, 10-19. 5681-5691. Research Paper Protein folding intermediates Fink et al. 25

31. Alexandrescu, A.T., Evans, P.A., Pitkeathly, M., Baum, J. & Dobson, C.M. (1993). Structure and dynamics of the acid-denatured molten globule state of alpha-lactalbumin: a two-dimensional NMR study. Biochemistry 32, 1707-1718. 32. Fink, A.L., Calciano, L.J., Goto, Y., Kurotsu, T. & Palleros, D.R. (1994). Classification of acid denaturation of proteins - intermediates and unfolded states. Biochemistry 33, 12504-12511. 33. Eliezer, D. & Wright, P.E. (1996). Is apomyoglobin a molten globule? Structural characterization by NMR. J. Mol. Biol. 263, 531-538. 34. Jennings, P.A. & Wright, P.E. (1993). Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin Science 262, 892-896. 35. Irace, G., Bismuto, E., Savy, F. & Colonna, G. (1986). Unfolding pathway of myoglobin: molecular properties of intermediate. Arch. Biochem. Biophys. 244, 459-469. 36. Ballew, R.M., Sabelko, J. & Gruebele, M. (1996). Direct observation of fast protein folding: the initial collapse of apomyoglobin. Proc. Natl Acad. Sci. USA 93, 5759-5764. 37. Hughson, F.M., Wright, P.E. & Baldwin, R.L. (1990). Structural characterization of a partly folded apomyoglobin intermediate. Science 249, 1544-1548. 38. Teale, F.W.J. (1959). Cleavage of the haem-protein link by acid methylethylketone. Biochim. Biophys. Acta 35, 543. 39. Oberg, K., Chrunyk, B.A., Wetzel, R. & Fink, A.L. (1994). Native-like secondary structure in interleukin-1 beta inclusion bodies by attenuated total reflectance FTIR. Biochemistry 33, 2628-2634. 40. Chen, Y.H., Yang, J.T. & Martinez, H.M. (1972). Determination of the secondary structures of proteins by and optical rotatory dispersion. Biochemistry 11, 4120-4131.

Because Folding & Design operates a ‘Continuous Publication System’ for Research Papers, this paper has been published on the internet before being printed. The paper can be accessed from http://biomednet.com/cbiology/fad.htm — for further information, see the explanation on the contents pages.