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1 School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom
2 Oxford Centre of the Molecular Sciences, University of Oxford, New Chemistry Laboratory, Oxford OX1 3QT, United Kingdom
3 Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom
(RECEIVED December 20, 2000; FINAL REVISION March 13, 2001; ACCEPTED March 20, 2001)
Article and publication are at www.proteinscience.org/cgi/doi/10.1110/ps.52801
4 Reprint requests to: Sheena E. Radford, School of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK; e-mail: s.e.radford{at}leeds.ac.uk; fax: 44-0113-233-3167. 
5 Present address: Departments of Chemistry and Molecular Biology, and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
6 Present address: Department of Nuclear Magnetic Resonance Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
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Abstract |
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90 kJ/mole and is catalyzed by cyclophilin A, indicating that folding is limited by trans-cis proline isomerization, presumably around the Xaa-Pro 20 bond that is in the cis isomer in the native state. Before proline isomerization, an intermediate accumulates during folding. This species has a substantial signal in the far-UV CD, a nonnative signal in the near-UV CD, exposed hydrophobic surfaces (judged by 1-anilino naphthalenesulphonate binding), a noncooperative denaturation transition, and a dynamic structure (revealed by line broadening on the nuclear magnetic resonance time scale). We compare the properties of this intermediate with partially folded states of other proteins and discuss its role in folding of this complex Greek key protein. Keywords: Pseudoazurin; proline isomerization; ß-sheet; protein folding
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Introduction |
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/ß counterparts (Capaldi and Radford 1998). For example, cold shock protein B (Csp B), SH3 domains, and tendamistat are known to fold rapidly to the native state without populating detectable intermediates (Viguera et al. 1994; Schindler et al. 1995; Schönbunner et al. 1997; Plaxco et al. 1998). In contrast, the folding of more complex ß-sheet structures such as interleukin-1ß (Heidary et al. 1997) and some, but not all, immunoglobulin domains and Greek key proteins (Clarke et al. 1997; Parker et al. 1997; Capaldi et al. 1999; Cota and Clarke 2000) involve the formation of populated intermediates, which have been suggested to be essential for productive folding to the native state. In the case of CD2, an intermediate has been shown to form its native topology within the dead time of stopped flow experiments, leaving open the question of how the Greek key fold is initiated (Parker et al. 1997). A variety of folding models have been proposed in attempts to rationalize how the complex Greek keys and jelly roll motifs might form, focusing on early hydrogen bonding (Richardson 1977), hydrophobic zippers (Dill et al. 1993), ß-zippers (Hazes and Hol 1992), and tyrosine corners (Hemmingsen et al. 1994), although a single such folding mechanism is unlikely to encompass the vast array of Greek key structures (Hutchinson and Thornton 1993).
The cupredoxins are copper-containing proteins that are found in a variety of organisms including bacteria, plants, and algae, in which they function as electron transport proteins in several redox reactions (Adman 1991). The cupredoxins share a common Greek key fold that consists of two ß-sheets arranged as a ß-sandwich. Although retaining a common architecture involving two overlapping Greek key motifs, each family member has unique structural details. For example, the pseudoazurins contain two -helices toward the C terminus of the polypeptide chain (Fig. 1
), whereas the azurins have a single -helix interdigitated within the ß-sandwich structure (Adman 1991). How the cupredoxins fold presents a challenging problem, because the double wound Greek key motif involves an array of long-range interactions between ß-strands (Fig. 1). In addition, as a consequence of their biological location in the bacterial periplasm and in chloroplasts, folding in vivo must be coupled with translocation across one or more biological membranes, and it has been suggested that cis/trans proline isomerization could play an important role in tailoring the rate of folding of these proteins such that membrane traversal is facilitated (Koide et al. 1993).
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; Williams et al. 1995). Like the other cupredoxins, the structure of pseudoazurin remains largely unchanged upon reduction or removal of the copper (Petratos et al. 1995; Thompson et al. 2000). The protein is 123 residues in length, has no disulphide bonds or tryptophan residues and eight proline residues, seven of which are located in interstrand loops in the native protein and are in the trans isomer (Fig. 1). The remaining proline (Pro 20) is in the cis conformation in the native state. This residue kinks strand 2 and allows the connection of this strand to ß-sheets on each side of the ß-sandwich (Fig. 1). Interestingly, this feature is highly conserved amongst all cupredoxins, and in the pseudoazurins and plastocyanins involves a cis proline at this site (Murphy et al. 1997). Here, we investigate the role of proline isomerization in apo-pseudoazurin folding by using several kinetic probes and discuss the role of proline isomerization in the folding of this, and other, proteins. |
Results |
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GH2O = 29 kJ/mole), folding is fully reversible, and the refolding properties can be compared directly with those obtained previously using sequential mixing stopped flow methods (Capaldi et al. 1999). The refolding kinetics of apo-pseudoazurin in 0.75 M urea, 0.5 M sodium sulphate, pH 7.0, measured by far-ultraviolet circular dichroism (UV CD) are shown in Figure 2a. Immediately after the dilution of denaturant (within the 15-sec dead time of these manual mixing experiments), a rapid increase in the negative ellipticity at 220 nm is observed. After this event (defined hereafter as the early folding phase), the native protein forms in a single exponential process with a rate constant of 0.04 ± 2.4 x 10-4 min-1. The refolding kinetics also were measured by near-UV CD at 261 nm (Fig. 2b). At this wavelength, the formation of the native protein occurs in a single exponential process with a rate constant of 0.03 ± 4.1 x 10-4 min-1. Interestingly, there is no significant change in ellipticity within the first 15 sec of initiating refolding at this wavelength, suggesting that the early folding phase involves the formation of one or more intermediates that possess significant secondary structure but lack persistent tertiary interactions (most probably involving phenylalanine residues). Control experiments in which apo-pseudoazurin was refolded in the absence of sodium sulphate also showed the rapid formation of an early folding intermediate as judged by far UV CD, indicating that an intermediate is stably populated during folding even in the absence of the stabilizing salt. Under these conditions, the rate constant of the proline-limited folding phase was 0.01 min-1 (A.P. Capaldi and S.E. Radford, unpubl.).
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). From these data, an activation enthalpy of 92 ± 9 kJ/mole was determined, a value that is typical of proline-limited refolding phases in other proteins (Brandts et al. 1975).
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) indicate that the rate of the slow phase of apo-pseudoazurin folding is increased dramatically in the presence of cyclophilin A (the rate is accelerated up to 12-fold at an enzyme : apo-pseudoazurin ratio of 1 : 450), demonstrating that proline isomerism is responsible for the slow folding phase. In accord with this view, the rate constant of the slow refolding phase does not change substantially when the concentration of urea in the refolding buffer is increased (the rate decreases approximately twofold between 0.5 and 3.5 M urea [data not shown]). Double jump refolding experiments also have demonstrated that denatured apo-pseudoazurin with all proline residues in their native isomeric state folds to the native state within 3 sec under identical refolding conditions to those used here (Capaldi et al. 1999).
Characterization of the kinetically trapped intermediate
To examine the nature of the intermediate populated rapidly during refolding in more detail, we studied the refolding kinetics by far-UV CD at various wavelengths, and the spectrum of the early folding intermediate was reconstructed from the amplitude of the early folding phase (obtained 15 sec after dilution of denaturant). The resulting spectrum (Fig. 4a) is consistent with that expected for a species containing a mixture of ß-sheet and random conformations (Yang et al. 1986), confirming that the intermediate possesses significant secondary structure. The spectrum of the protein calculated from the amplitude of the CD signal once folding was complete (after 3 h; see Materials and Methods) was indistinguishable from that of the native protein, confirming that folding is fully reversible under these conditions (not shown).
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. The spectrum is profoundly different from that of the unfolded and refolded states. The significant negative ellipticity of the intermediate at 270–280 nm suggests that this species possesses a significant content of fixed tertiary interactions involving one or more tyrosine residues, and that the packing of these residues differs substantially from that in the native state. In accord with these data, examination of the refolding kinetics of the protein by near-UV CD at 271 nm shows a large increase in negative ellipticity within 15 sec of the initiation of folding (Fig. 2c). Thereafter, the native state is formed in a single exponential process with a rate constant of 0.03 ± 5.1 x 10-4 min-1, consistent with the rate constant of this phase monitored by near-UV CD at 261 nm. The near-UV CD spectra of the early folding intermediate and unfolded states are similar at 261 nm (Fig. 4b), consistent with the observation that a significant dead-time phase is not observed in the refolding kinetics obtained at this wavelength (Fig. 2b). These data suggest that whereas one or more tyrosine residues are involved in fixed tertiary interactions in the folding intermediate trapped by proline isomerization, the intermediate lacks fixed interactions involving phenylalanine residues. The intermediate binds the hydrophobic dye, 1-anilino naphthalenesulphonate (ANS; Fig. 5), resulting in an increase in fluorescence intensity and a blue shift in the 
max of the dye, characteristic of ANS binding to partially folded states of other proteins (Ptitsyn 1995). After a refolding time of 3 h (in the absence of ANS), the dye then was added to the protein, and the emission spectrum was again monitored. This spectrum is very similar to that of the native protein in the presence of ANS (Fig. 5), consistent with the results from other spectroscopic methods that folding is complete by this time.
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. The data show that the intermediate unfolds in a broad transition, as expected for denaturation of an ensemble of partially folded molecules that bury only a small surface area from solvent (i.e., the intermediate[s] has a small m value [Myers et al. 1995]). This contrasts with the unfolding of the native protein that fits well to a two-state model with an m value of 7.2 ± 0.9 kJ/mole.M and a free energy of unfolding in water, GH2O, of 27.3 ± 3.3 kJ/mole. These values are similar to those obtained previously from equilibrium denaturation experiments (Capaldi et al. 1999), demonstrating that the refolded protein is native.
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. The spectrum is typical of that expected for a highly denatured protein: there is little chemical shift dispersion, the shifts of side chain resonances fit well with excepted values for disordered peptides (Merutka et al. 1995), and the resonances are sharp. In contrast, the spectrum of the refolded protein acquired 12 h after folding was initiated is identical to the spectrum of native apo-pseudoazurin (Thompson et al. 2000).
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. The spectrum is clearly very different from those of the native and denatured states, confirming that a distinct species is populated in the early folding phase. In contrast with the spectra of the native and denatured proteins, the spectrum of partially folded apo-pseudoazurin shows substantial line broadening, suggesting that the intermediate ensemble is a dynamic species in intermediate exchange on the millisecond time scale. These features are characteristic of partially folded states of other proteins (Balbach et al. 1995; Schulman et al. 1997). The line broadening unfortunately prevents the site-specific identification of aromatic residues involved in the fixed tertiary interaction(s) in this species visualised by near-UV CD (Fig. 4b).
Four well-dispersed resonances with very low intensity are seen in the upfield region (<0 ppm) of the NMR spectrum of the intermediate acquired 5.2 sec after the initiation of folding. The chemical shift of these resonances is identical to those of the 
CH3 groups of Val 76 and Ile 31, Val 68, and Val 23 in native pseudoazurin (Thompson et al. 2000) and indicate that a minor population of molecules fold rapidly to the native state. Assuming a ratio of cis : trans proline in the denatured ensemble of 1 : 5 (Kiefhaber 1995), 5% of molecules in the unfolded ensemble will have all eight proline residues in their native isomeric form (a ratio of 0.13 : 0.87 cis : trans proline was observed in an eight residue synthetic peptide from pseudoazurin that included Pro 20 in its sequence [data not shown]). Estimates of the population of fast-folding molecules from the peak heights of the CH3 groups of Val 76 and Ile 31, Val 68, and Val 23 5.2 sec after the initiation of folding accords well with these estimates.
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Discussion |
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5% of molecules fold to the native state within the 5.2-sec dead time of rapid mixing NMR experiments, and double jump refolding experiments have shown that apo-pseudoazurin containing only native proline isomers in the denatured state folds to the native state in under 3 sec under identical experimental conditions to those used here (Capaldi et al. 1999).
Individual proline residues have been shown to be responsible for slow proline-limited folding phases in several proteins (Evans et al. 1987; Koide et al. 1993; Mayr et al. 1993; Vanhove et al. 1996). For apo-pseudoazurin, a single peptidyl–proline bond involving Pro 20 is in the cis isomer in the native protein (Williams et al. 1995; Fig. 1). Trans–cis isomerism of Pro 20 thus is likely to make a significant contribution to the slow proline-limited refolding phase of apo-pseudoazurin. A synthetic octapeptide with the sequence equivalent to residues 17–24 in apo-pseudoazurin was shown to contain 87% trans and 13% cis Pro 20 by using NMR (G.S. Thompson and S.E. Radford, unpubl.), suggesting that isomerization of this residue will play a major role in the observed refolding kinetics, although isomerization of other residues and more complex kinetic scenarios including the rapid isomerization of prolines during folding (Kiefhaber et al. 1992; Plaxco et al. 1996) cannot be ruled out. Substitution of Pro 20 with either Ala or Gly or replacing the sequence Glu 19-Pro 20-Ala 21 of apo-pseudoazurin with the all-trans sequence, Asn-Thr-Asn, found in the equivalent location in the related protein, azurin, from Pseudomonas aeruginosa (Adman et al. 1982) failed to produce significant amounts of recombinant protein, despite exploring a range of expression conditions (Reader 1998). The data suggest that a cis proline at position 20 is important for the production of pseudoazurin in vivo, consistent with its conservation throughout known pseudoazurin and plastocyanin sequences (Adman 1991).
Interconversion of nonnative to native Xaa-Pro peptide bonds provides a significant barrier to folding, and as a consequence a partially folded state accumulates during the refolding of apo-pseudoazurin. In contrast with the rate of proline limited folding observed for other proteins (Jackson and Fersht 1991), proline-limited folding of apo-pseudoazurin is remarkably slow (0.04 min-1). In previous experiments, we have shown that the rate of proline isomerization in fully denatured apo-pseudoazurin under identical conditions to those used here is 0.24 min-1 (Capaldi et al. 1999). Hence, proline isomerization in fully unfolded molecules occurs significantly faster than the rate of acquisition of the native state observed here. These data suggest strongly that the rate determining step in apo-pseudoazurin refolding involves isomerization of proline residues in the partially folded intermediate species, although a contribution from isomerization of prolines in denatured molecules cannot be ruled out.
The intermediate of apo-pseudoazurin trapped by proline isomerization contains significant secondary structure, binds ANS, and displays significant line broadening in NMR spectra. It thus has properties akin to compact denatured states and molten globules in other proteins such as those observed for cytochrome c (Marmorino and Pielak 1995) and -lactalbumin (Balbach et al. 1997). Interestingly, however, the intermediate of apo-pseudoazurin unusually contains a significant signal in the near-UV CD at 270–280 nm, suggesting that it possesses significant fixed tertiary interactions involving one or more tyrosine residues. The absence of a significant signal in the near-UV CD 260 nm suggests, in contrast, that fixed tertiary interactions involving phenyalanine residues have not developed yet at this stage of folding.
A highly conserved feature of Greek key proteins is the tyrosine corner (Hemmingsen et al. 1994). In pseudoazurin, this feature involves the formation of a specific hydrogen bond between the side chain of Tyr 74 and the main chain carbonyl oxygen of Glu 70 (Williams et al. 1995). The tyrosine corner has been proposed to be important in the folding and/or stabilization of Greek keys and has been demonstrated to be important in the folding of at least some proteins containing this structure (Hamill et al. 2000). We have shown that a mutant of apo-pseudoazurin, Y74W, retains this feature in the native protein as well as in a partially folded intermediate formed at equilibrium in 4.3 M urea under conditions identical to those used here (Jones et al. 2000). This raises the possibility that the tyrosine corner is formed in the trapped intermediate of wild-type apo-pseudoazurin during proline-limited folding, and this feature could be responsible, at least in part, for the significant near-UV CD signal of this species. Further kinetic experiments will be required to determine if this is the case. Experiments in which apo-pseuodazurin was refolded from highly denaturing solutions containing 6 M urea, 3 M guanidinium chloride, or 6 M guanidinium chloride also showed the rapid formation of an intermediate species before slow, proline-limited formation of the native state, suggesting that residual structure in the denatured state is not a major contributing feature of intermediate formation in the early phase of folding (data not shown).
The data presented here show that isomerization of one or more proline residues is a prerequisite for apo-pseudoazurin to obtain a native-like fold. For other proteins, species resembling the native state may be obtained even in the presence of nonnative proline isomers (Evans et al. 1987; Schreiber and Fersht 1993; Killick et al. 1999). The finding that intermediates of proteins with complex folds such as the double wound Greek key of pseudoazurin can be trapped by proline isomerization provides opportunities to determine the structural hierarchy and role of intermediates in protein folding in vitro and the importance of these events in vivo where a host of cellular factors have evolved to facilitate slow folding events involving proline isomerization and protein translocation (Schmid et al. 1993; Economou 1999).
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Materials and methods |
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Expression, mutagenesis, and purification of pseudoazurin
Pseudoazurin was expressed in Escherichia coli strain XL1-Blue, transformed with the plasmid pJR2 containing the pazS gene (Leung et al. 1997) and purified as described by Jones et al. (2000). The sample was dialyzed extensively into H2O and lyophilized before use. The final protein concentration was estimated using 
590 = 1.36 mM-1 cm-1 (Moir et al. 1993) and Mr of pseudoazurin of 13,344. Copper was removed from the protein by incubation with DTT and EDTA as described by Jones et al. (2000). After this procedure, <5% of copper remained as judged by the absorbance at 590 nm after reoxidation with K3Fe(CN)6 and by electrospray ionization mass spectrometry under nondenaturing conditions. Mutagenesis of the pazS gene to introduce the changes P20A, P20G, and E19N-P20A-A21N was performed as described in Reader (1998).
Purification of cyclophilin A
Cyclophilin A was expressed using a BL21 strain of E. coli transformed with the plasmid pGEX-cycA (kindly provided by Dr. S.E. Jackson, University of Cambridge). The concentration of the purified cyclophilin was estimated using an extinction coefficient of 280 = 7920 M-1 cm-1. Purified cyclophilin A was assayed for peptidyl prolyl isomerase activity based on the method described by Harrison and Stein (1990).
Circular dichroism
CD measurements were performed on a Jasco J-715 spectropolarimeter using a Jasco PTC-348W peltier system for temperature control. Cuvettes with a pathlength of 1 mm were used for far-UV CD, and 10-mm pathlength cells were used for measurements in the near-UV CD. Protein concentrations ranged from 0.2 to 0.6 mg/mL for far-UV CD and were 3.0 mg/mL for near-UV CD. It was not possible to vary the concentration of protein further within an experimental technique because of the limitations of signal to noise. Consistent refolding kinetics were observed at various protein concentrations and by the several techniques used to monitor refolding, suggesting that transient protein association during refolding is unlikely but cannot be completely ruled out.
ANS binding
Fluorescence experiments were performed on a Perkin-Elmer LS 50B luminescence spectrometer using 10-mm pathlength quartz cuvettes. All solutions were filtered before use. The protein concentration was 0.45 µM (0.006 mg/mL), and a final ANS concentration of 0.25 mM was used. Temperature control was achieved using a circulating water bath attached to the cell holder. The excitation wavelength was 370 nm, and two scans were averaged.
Ultracentrifugation
Analytical ultracentrifugation was performed using a Beckman XLA ultracentrifuge equipped with absorption optics using a An60-Ti rotor with cells containing quartz windows and double-sector charcoal-filled epon centerpieces. Equilibrium measurements were performed at 30,000–40,000 rpm and were monitored using UV absorbance with variable optical pathlengths depending on the protein concentration used. Experiments were performed in 20 mM sodium phosphate buffer, pH 7.0, at 15°C containing 0.5 M sodium sulphate and various concentrations of urea and DTT as appropriate. Sedimentation equilibrium and velocity ultracentrifugation measurements demonstrated that under the refolding conditions used native apo-pseudoazurin and the refolded protein are both monomeric and monodisperse even at high protein concentrations (10 mg/mL).
Real-time refolding experiments
Apo-pseudoazurin was denatured in 20 mM sodium phosphate buffer, pH 7.0, containing 4 M urea and an excess DTT (higher than the protein concentration) for at least 1 h at 15°C. The unfolded protein then was refolded by manual dilution into refolding buffer (20 mM sodium phosphate, pH 7.0, containing a final concentration of urea of 0.75 M, 0.5 M sodium sulphate, and 1–2 mM DTT at 15°C). The dead time of these experiments was 15 sec. Data collected were processed and analyzed using the Kaleidagraph software package (Abelbeck). In all kinetic experiments, the data were fitted to single exponential functions. Although in some traces, minor deviations from an ideal single exponential are observed, in no case do the data fit adequately to double exponential functions.
NMR spectroscopy
Real-time refolding experiments by NMR were recorded using a 600-MHz Bruker spectrometer belonging to the Oxford Centre for Molecular Sciences. For these experiments, apo-pseudoazurin was denatured in 4 M deuterated urea as described above. Rapid introduction of the unfolded protein into the refolding buffer was achieved through injection of the protein through a Teflon transfer line into the NMR tube placed inside the NMR spectrometer as described in Balbach et al. (1995). The first spectrum was recorded within 5.22 sec after initiating folding, and the time increment of each spectrum was 4.23 sec. For refolding experiments at 15°C, the NMR spectrometer was sufficiently stable to make measurements without locking on the deuterium signal. The data were processed using FELIX (Hare Research).
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Acknowledgments |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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References |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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Adman, E.T., Canters, G.W., Hill, H.A., and Kitchen, N.A. 1982. The effect of pH and temperature on the structure of the active site of azurin from Pseudomonas aeruginosa. FEBS Lett. 143: 287–292.[CrossRef][Medline]
Balbach, J., Forge, V., van Nuland, N.A.J., Winder, S.L., Hore, P.J., and Dobson, C.M. 1995. Following protein folding in real time using NMR spectroscopy. Nat. Struct. Biol. 2: 865–870.[CrossRef][Medline]
Balbach, J., Forge, V., Lau, W.S., Jones, J.A., van Nuland, N.A., and Dobson, C.M. 1997. Detection of residue contacts in a protein folding intermediate. Proc. Natl. Acad. Sci. 94: 7182–7185.
Balbach, J., Steegborn, C., Schindler, T., and Schmid, F.X. 1999. A protein folding intermediate of ribonuclease T1 characterized at high resolution by 1D and 2D real-time NMR spectroscopy. J. Mol. Biol. 285: 829–842.[CrossRef][Medline]
Brandts, J.F., Halvorson, H.R., and Brennan, M. 1975. Consideration of the possibility that the slow step in protein denaturation reactions is due to cis-trans isomerism of proline residues. Biochemistry 14: 4953–4963.[CrossRef][Medline]
Capaldi, A.P. and Radford, S.E. 1998. Kinetic studies of ß-sheet protein folding. Curr. Opin. Struct. Biol. 8: 86–92.[CrossRef][Medline]
Capaldi, A.P., Ferguson, S.J., and Radford, S.E. 1999. The Greek key protein apo-pseudoazurin folds through an obligate on-pathway intermediate. J. Mol. Biol. 286: 1621–1632.[CrossRef][Medline]
Clarke, J., Hamill, S.J., and Johnson, C.M. 1997. Folding and stability of a fibronectin type III domain of human tenascin. J. Mol. Biol. 270: 771–778.[CrossRef][Medline]
Clarke, J., Cota, E., Fowler, S.B., and Hamill, S.J. 1999. Folding studies of immunoglobulin-like ß-sandwich proteins suggest that they share a common folding pathway. Structure Fold. Des. 7: 1145–1153.[Medline]
Cota, E. and Clarke, J. 2000. Folding of ß-sandwich proteins: Three-state transition of a fibronectin type III module. Protein Sci. 9: 112–120.[Abstract]
Dill, K.A., Fiebig, K.M., and Chan, H.S. 1993. Cooperativity in protein folding kinetics. Proc. Natl. Acad. Sci. 90: 1942–1946.
Dobson, C.M. 1999. Protein misfolding, evolution and disease. Trends Biochem. Sci. 24: 329–332.[CrossRef][Medline]
Economou, A. 1999. Following the leader: Bacterial protein export through the Sec pathway. Trends Microbiol. 7: 315–320.[CrossRef][Medline]
Evans, P.A., Dobson, C.M., Kautz, R.A., Hatfull, G., and Fox, R.O. 1987. Proline isomerism in staphylococcal nuclease characterized by NMR and site-directed mutagenesis. Nature 329: 266–268.[CrossRef][Medline]
Hamill, S.J., Cota, E., Chothia, C., and Clarke, J. 2000. Conservation of folding and stability within a protein family: The tyrosine corner as an evolutionary cul-de-sac. J. Mol. Biol. 295: 641–649.[CrossRef][Medline]
Harrison, R.K. and Stein, R.L. 1990. Substrate specificities of the peptidyl prolyl cis-trans isomerase activities of cyclophilin and FK-506 binding protein: Evidence for the existence of a family of distinct enzymes. Biochemistry 29: 3813–3816.[CrossRef][Medline]
Hazes, B. and Hol, W.G.J. 1992. Comparison of the hemocyanin ß-barrel with other Greek Key ß-barrels: Possible importance of the "ß-zipper" in protein structure and folding. Proteins Struct. Funct. Genet. 12: 278–298.[CrossRef][Medline]
Heidary, D.K., Gross, L.A., Roy, M., and Jennings, P.A. 1997. Evidence for an obligatory intermediate in the folding of interleukin-1ß. Nature Struct. Biol. 4: 725–731.[CrossRef][Medline]
Hemmingsen, J.M., Gernert, K.M., Richardson, J.S., and Richardson, D.C. 1994. The tyrosine corner: A feature of most Greek key ß-barrel proteins. Protein Sci. 3: 1927–1937.[Abstract]
Hoeltzli, S.D. and Frieden, C. 1996. Real-time refolding studies of 6–19F-tryptophan labeled Escherichia coli dihydrofolate reductase using stopped-flow NMR spectroscopy. Biochemistry 35: 16843–16851.[CrossRef][Medline]
Hutchinson, E.G. and Thornton, J.M. 1993. The Greek key motif: Extraction, classification and analysis. Protein Eng. 6: 233–245.
Ikeguchi, M., Kuwajima, K., Mitani, M., and Sugai, S. 1986. Evidence for identity between the equilibrium unfolding intermediate and a transient folding intermediate: A comparative study of the folding reactions of -lactalbumin and lysozyme. Biochemistry 25: 6965–6972.[CrossRef][Medline]
Jackson, S.E. and Fersht, A.R. 1991. Folding of chymotrypsin inhibitor 2. 2. Influence of proline isomerization on the folding kinetics and thermodynamic characterization of the transition state of folding. Biochemistry 30: 10436–10443.[CrossRef][Medline]
Jamin, M. and Baldwin, R.L. 1996. Refolding and unfolding kinetics of the equilibrium folding intermediate of apomyoglobin. Nat.e Struct. Biol. 3: 613–618.
Jeng, M.-F. and Englander, S.W. 1991. Stable submolecular folding units in a non-compact form of cytochrome c. J. Mol. Biol. 221: 1045–1061.[CrossRef][Medline]
Jennings, P.A. and Wright, P.E. 1993. Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin. Science 262: 892–895.
Jones, S., Reader, J.S., Healy, M., Capaldi, A.P., Ashcroft, A.E., Kalverda, A.P., Smith, D.A., and Radford, S.E. 2000. Unfolding of the ß-sheet protein Y74W apo-pseudoazurin: Evidence for an equilibrium intermediate species. Biochemistry 39: 5672–5682.[CrossRef][Medline]
Kay, M.S. and Baldwin, R.L. 1996. Packing interactions in the apomyoglobin folding intermediate. Nat. Struct. Biol. 3: 439–445.[CrossRef][Medline]
Kiefhaber, T. 1995. Protein folding kinetics. Methods Mol. Biol. 40: 313–341.[Medline]
Kiefhaber, T. and Schmid, F.X. 1992. Kinetic coupling between protein folding and prolyl isomerisation. II. Folding of ribonuclease A and ribonuclease T1. J. Mol. Biol. 224: 231–240.[CrossRef][Medline]
Kiefhaber, T., Kohler, H.-H., and Schmid, F.X. 1992. Kinetic coupling between protein folding and prolyl isomerization. I. Theoretical models. J. Mol. Biol. 224: 217–229.[CrossRef][Medline]
Killick, T.R., Freund, S.M., and Fersht, A.R. 1999. Real-time NMR studies on a transient folding intermediate of barstar. Protein Sci. 8: 1286–1291.[Abstract]
Koide, S., Dyson, H.J., and Wright, P.E. 1993. Characterization of a folding intermediate of apoplastocyanin trapped by proline isomerization. Biochemistry 32: 12299–12310.[CrossRef][Medline]
Kraulis, P.J. 1991. MOLSCRIPT: A program to produce both detailed and schematic plots of protein structures. J. Appl. Cryst. 24: 946–950.
Leung, Y.C., Chan, C., Reader, J.S., Willis, A.C., vanSpanning, R., Ferguson, S.J., and Radford, S.E. 1997. The pseudoazurin gene from Thiosphaera pantotropha: Analysis of upstream putative regulatory sequences and overexpression in Escherichia coli. Biochem. J. 321: 699–705.
Marmorino, J.L. and Pielak, G.J. 1995. A native tertiary interaction stabilizes the A state of cytochrome c. Biochemistry 34: 3140–3143.[CrossRef][Medline]
Mayr, L.M., Landt, O., Hahn, U., and Schmid, F.X. 1993. Stability and folding kinetics of ribonuclease T1 are strongly altered by the replacement of cis-proline 39 with alanine. J. Mol. Biol. 231: 897–912.[CrossRef][Medline]
Merutka, G., Dyson, H.J., and Wright, P.E. 1995. "Random coil" 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG. J. Biomol. NMR 5: 14–24.[CrossRef][Medline]
Moir, J.W.B., Baratta, D., Richardson, D.J., and Ferguson, S.J. 1993. The purification of a cd1 type nitrite reductase and the absence of a copper type nitrite reductase from the aerobic denitrifier Thiosphaera pantotropha; the role of pseudoazurin as an electron donor. Eur. J. Biochem. 212: 377–385.[Medline]
Murphy, E.P., Lindley, P.F., and Adman, E.T. 1997. Structural comparison of cupredoxin domains: Domain recycling to construct proteins with novel functions. Protein Sci. 6: 761–770.[Abstract]
Myers, J.K., Pace, C.N., and Scholtz, J.M. 1995. Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding. Protein Sci. 4: 2138–2148.[Abstract]
Parker, M.J., Dempsey, C.E., Lorch, M., and Clarke, A.R. 1997. Acquisition of native ß-strand topology during the rapid collapse phase of protein folding. Biochemistry 36: 13396–13405.[CrossRef][Medline]
Petratos, K., Papadovasilaki, M., and Dauter, Z. 1995. The crystal structure of apo-pseudoazurin from Alcaligenes faecalis S6. FEBS Lett. 368: 432–434.[CrossRef][Medline]
Plaxco, K.W., Spitzfaden, C., Campbell, I.D., and Dobson, C.M. 1996. Rapid refolding of a proline-rich all-ß-sheet fibronectin type III module. Proc. Natl. Acad. Sci. 93: 10703–10706.
Plaxco, K.W., Guijarro, J.I., Morton, C.J., Pitkeathly, M., Campbell, I.D., and Dobson, C.M. 1998. The folding kinetics and thermodynamics of the Fyn-SH3 domain. Biochemistry 37: 2529–2537.[CrossRef][Medline]
Ptitsyn, O.B. 1995. Molten globule and protein-folding. Adv. Protein Chem. 47: 83–229.[Medline]
Reader, J.S. (1998). Folding studies of the ß-sheet protein pseudoazurin. D. Phil. thesis, University of Oxford, UK.
Richardson, J.S. 1977. ß Sheet topology and the relatedness of proteins. Nature 268: 495–500.[CrossRef][Medline]
Schindler, T., Herrler, M., Marahiel, M.A., and Schmid, F.X. 1995. Extremely rapid protein folding in the absence of intermediates. Nat. Struct. Biol. 2: 663–673.[CrossRef][Medline]
Schmid, F.X., Mayer, L.M., Mucke, M., and Schnbrunner, E.R. 1993. Prolyl isomerases: Role on protein folding. Adv. Protein Chem. 44: 25–66.[Medline]
Schönbunner, N., Koller, K.-P., and Kiefhaber, T. 1997. Folding of the disulfide-bonded ß-sheet protein tendamistat: Rapid two-state folding without hydrophobic collapse. J. Mol. Biol. 268: 526–538.[CrossRef][Medline]
Schreiber, G. and Fersht, A.R. 1993. The refolding of cis- and trans-peptidylprolyl isomers of barstar. Biochemistry 32: 11195–11203.[CrossRef][Medline]
Schulman, B.A., Kim, P.S., Dobson, C.M., and Redfield, C. 1997. A residue-specific NMR view of the non-cooperative unfolding of a molten globule. Nat. Struct. Biol. 4: 630–634.[CrossRef][Medline]
Thompson, G.S., Leung, Y.C., Ferguson, S.J., Radford, S.E., and Redfield, C. 2000. The structure and dynamics in solution of Cu(I) pseudoazurin from Paracoccus pantotrophus. Protein Sci. 9: 846–858.[Abstract]
Vanhove, M., Raquet, X., Palzkill, T., Pain, R.H., and Frere, J.M. 1996. The rate-limiting step in the folding of the cis-pro167thr mutant of TEM-1 ß-lactamase is the trans to cis isomerization of a non-proline peptide-bond. Proteins Struct. Funct. Genet. 25: 104–111.[CrossRef][Medline]
Van Nuland, N., Forge, V., Balbach, J., and Dobson, C. 1998. Real-time NMR studies of protein folding. Acc. Chem. Res. 31: 773–780.[CrossRef]
Viguera, A.R., Martinez, J.C., Filimonov, V.V., Mateo, P.L., and Serrano, L. 1994. Thermodynamic and kinetic analysis of the SH3 domain of spectrin shows a two state folding transition. Biochemistry 33: 2142–2150.[CrossRef][Medline]
Williams, P.A., Fülöp, V., Leung, Y.-C., Chan, C., Moir, J.W., Howlett, G., Ferguson, S.J., Radford, S.E., and Hadju, J. 1995. Pseudospecific docking surfaces on electron transport proteins as illustrated by pseudoazurin, cytochrome c550 and cytochrome cd1 nitrite reductase. Nat. Struct. Biol. 2: 975–982.[CrossRef][Medline]
Yang, J.T., Wu, C.S., and Martinez, H.M. 1986. Calculation of protein conformation from circular dichroism. Methods Enzymol. 130: 208–269.[Medline]
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