Structural characterization of partially folded intermediates of apomyoglobin H64F

We present a detailed investigation of unfolded and partially folded states of a mutant apomyoglobin (apoMb) where the distal histidine has been replaced by phenylalanine (H64F). Previous studies have shown that substitution of His64, located in the E helix of the native protein, stabilizes the equilibrium molten globule and native states and leads to an increase in folding rate and a change in the folding pathway. Analysis of changes in chemical shift and in backbone flexibility, detected via [1H]-15N heteronuclear nuclear Overhauser effect measurements, indicates that the phenylalanine substitution has only minor effects on the conformational ensemble in the acid- and urea-unfolded states, but has a substantial effect on the structure, dynamics, and stability of the equilibrium molten globule intermediate formed near pH 4. In H64F apomyoglobin, additional regions of the polypeptide chain are recruited into the compact core of the molten globule. Since the phenylalanine substitution has negligible effect on the unfolded ensemble, its influence on folding rate and stability comes entirely from interactions within the compact folded or partly folded states. Replacement of His64 with Phe leads to favorable hydrophobic packing between the helix E region and the molten globule core and leads to stabilization of helix E secondary structure and overall thermodynamic stabilization of the molten globule. The secondary structure of the equilibrium molten globule parallels that of the burst phase kinetic intermediate; both intermediates contain significant helical structure in regions of the polypeptide that comprise the A, B, E, G, and H helices of the fully folded protein.     

Role of the B helix in early folding events in apomyoglobin: evidence from site-directed mutagenesis for native-like long range interactions

The folding pathways of four mutants in which bulky hydrophobic residues in the B helix of apomyoglobin (ApoMb) are replaced by alanine (I28A, L29A, I30A, and L32A) have been analyzed using equilibrium and kinetic methods employing NMR, CD, fluorescence and mass spectrometry. Hydrogen exchange pulse-labeling followed by mass spectrometry reveals detectable intermediates in the kinetic folding pathways of each of these mutants. Comparison of the quench-flow data analyzed by NMR for the wild-type protein and the mutants showed that the substitutions I28A, L29A and L32A lead to destabilization of the B helix in the burst phase kinetic intermediate, relative to wild-type apomyoglobin. In contrast, the I30A mutation apparently has a slight stabilizing effect on the B helix in the burst phase intermediate; under weak labeling conditions, residues in the C helix region were also relatively stabilized in the mutant compared to the wild-type protein. This suggests that native-like helix B/helix C packing interactions occur in the folding intermediate. The L32A mutant showed significantly lower proton occupancies in the burst phase for several residues in the G helix, specifically F106, I107, E109 and A110, which are in close proximity to L32 in the X-ray structure of myoglobin, providing direct evidence that native-like helix B/helix G contacts are formed in the apomyoglobin burst phase intermediate. The L29A mutation resulted in an increase in burst phase proton occupancies for several residues in the E helix. Since these regions of the B and E helices are not in contact in the native myoglobin structure, these effects suggest the possibility of non-native B/E packing interactions in the kinetic intermediate. The differing effects of these B helix mutations on the apomyoglobin folding process suggests that each side-chain plays a different and important role in forming stable structure in the burst phase intermediate, and points to a role for both native-like and non-native contacts in stabilization of the folding intermediate.     

The apomyoglobin folding pathway revisited: structural heterogeneity in the kinetic burst phase intermediate

Extensive analysis of accurate quench-flow hydrogen exchange results indicates that the burst phase kinetic intermediate in the folding of apomyoglobin (apoMb) from urea is structurally heterogeneous. The structural variability is associated with the partial folding of the E helix during the burst phase (<6.4ms) of the folding process. Analysis of the effects of exchange-out of amide proton labels during the labeling pulse ( approximately pH 10) of the quench-flow process indicates that three of the amide protons in the E helix are in fact largely protected in the burst phase of folding, while the remainder of the E helix has a substantial complement of amide protons that show biphasic kinetics, i.e. are protected partly during the burst phase and partly during the slow phase of folding. The locations of these amide protons can be used to map the sites of structural heterogeneity in the kinetic molten globule. These sites include, besides the E helix, the ends of the A and B helices and part of the C helix. Our results give significant support to the hypothesis that the kinetic molten globule intermediate of apoMb is native-like.     

Energetic Frustration of Apomyoglobin Folding: Role of the B Helix

Apomyoglobin folds by a sequential mechanism in which the A, G, and H helix regions undergo rapid collapse to form a compact intermediate onto which the central portion of the B helix subsequently docks. To investigate the factors that frustrate folding, we have made mutations in the N-terminus of the B helix to stabilize helical structure (in the mutant G23A/G25A) and to promote native-like hydrophobic packing interactions with helix G (in the mutant H24L/H119F). The kinetic and equilibrium intermediates of G23A/G25A and H24L/H119F were studied by hydrogen exchange pulse labeling and interrupted hydrogen/deuterium exchange combined with NMR. For both mutants, stabilization of helical structure in the N-terminal region of the B helix is confirmed by increased exchange protection in the equilibrium molten globule states near pH 4. Increased protection is also observed in the GH turn region in the G23A/G25A mutant, suggesting that stabilization of the B helix facilitates native-like interactions with the C-terminal region of helix G. These interactions are further enhanced in H24L/H119F. The kinetic burst phase intermediates of both mutants show increased protection, relative to wild-type protein, of amides in the N-terminus of the B helix and in part of the E helix. Stabilization of the E helix in the intermediate is attributed to direct interactions between E helix residues and the newly stabilized N-terminus of helix B. Stabilization of native packing between the B and G helices in H24L/H119F also favors formation of native-like interactions in the GH turn and between the G and H helices in the ensemble of burst phase intermediates. We conclude that instability at the N-terminus of the B helix of apomyoglobin contributes to the energetic frustration of folding by preventing docking and stabilization of the E helix.     

Identification of native and non-native structure in kinetic folding intermediates of apomyoglobin

Site-directed mutagenesis has been used to probe the interactions that stabilize the equilibrium and burst phase kinetic intermediates formed by apomyoglobin. Nine bulky hydrophobic residues in the A, E, G and H helices were replaced by alanine, and the effects on protein stability and kinetic folding pathways were determined. Hydrogen exchange pulse-labeling experiments, with NMR detection, were performed for all mutants. All of the alanine substitutions resulted in changes in proton occupancy or an increased rate of hydrogen-deuterium exchange for amides in the immediate vicinity of the mutation. In addition, most mutations affected residues in distant parts of the amino acid sequence, providing insights into the topology of the burst phase intermediate and the interactions that stabilize its structure. Differences between the pH 4 equilibrium molten globule and the kinetic intermediate are evident: the E helix region plays no discernible role in the equilibrium intermediate, but contributes significantly to stabilization of the ensemble of compact intermediates formed during kinetic refolding. Mutations that interfere with docking of the E helix onto the preformed A/B/G/H helix core substantially decrease the folding rate, indicating that docking and folding of the E helix region occurs prior to formation of the apomyoglobin folding transition state. The results of the mutagenesis experiments are consistent with rapid formation of an ensemble of compact burst phase intermediates with an overall native-like topological arrangement of the A, B, E, G, and H helices. However, the experiments also point to disorder in docking of the E helix and to non-native contacts in the kinetic intermediate. In particular, there is evidence for translocation of the H helix by approximately one helical turn towards its N terminus to maximize hydrophobic interactions with helix G. Thus, the burst phase intermediate observed during kinetic refolding of apomyoglobin consists of an ensemble of compact, kinetically trapped states in which the helix docking appears to be topologically correct, but in which there are local non-native interactions that must be resolved before the protein can fold to the native structure.     

TEM-1 beta-lactamase folds in a nonhierarchical manner with transient non-native interactions involving the C-terminal region

The conformational stability and kinetics of refolding and unfolding of the W290F mutant of TEM-1 beta-lactamase have been determined as a function of guanidinium chloride concentration. The activity and spectroscopic properties of the mutant enzyme did not differ significantly from those of the wild type, indicating that the mutation has only a very limited effect on the structure of the protein. The stability of the folded protein is reduced, however, by 5-10 kJ mol-1 relative to that of the molten globule intermediate (H), but the values of the folding rate constants are unchanged, suggesting that Trp-290 becomes organized in its nativelike environment only after the rate-limiting step; i.e., the C-terminal region of the enzyme folds very late. In contrast to the significant increase in fluorescence intensity seen in the dead time (3-4 ms) of refolding of the wild-type protein, no corresponding burst phase was observed with the mutant enzyme, enabling the burst phase to be attributed specifically to the C-terminal Trp-290. This residue is suggested to be buried in a nonpolar environment from which it has to escape during subsequent folding steps. With both proteins, fast early collapse leads to a folding intermediate in which the C-terminal region of the polypeptide chain is trapped in a non-native structure, consistent with a nonhierarchical folding process.     

Conformational and dynamic characterization of the molten globule state of an apomyoglobin mutant with an altered folding pathway.

Kinetic and equilibrium studies of apomyoglobin folding pathways and intermediates have provided important insights into the mechanism of protein folding. To investigate the role of intrinsic helical propensities in the apomyoglobin folding process, a mutant has been prepared in which Asn132 and Glu136 have been substituted with glycine to destabilize the H helix. The structure and dynamics of the equilibrium molten globule state formed at pH 4.1 have been examined using NMR spectroscopy. Deviations of backbone (13)C(alpha) and (13)CO chemical shifts from random coil values reveal high populations of helical structure in the A and G helix regions and in part of the B helix. However, the H helix is significantly destabilized compared to the wild-type molten globule. Heteronuclear [(1)H]-(15)N NOEs show that, although the polypeptide backbone in the H helix region is more flexible than in the wild-type protein, its motions are restricted by transient hydrophobic interactions with the molten globule core. Quench flow hydrogen exchange measurements reveal stable helical structure in the A and G helices and part of the B helix in the burst phase kinetic intermediate and confirm that the H helix is largely unstructured. Stabilization of structure in the H helix occurs during the slow folding phases, in synchrony with the C and E helices and the CD region. The kinetic and equilibrium molten globule intermediates formed by N132G/E136G are similar in structure. Although both the wild-type apomyoglobin and the mutant fold via compact helical intermediates, the structures of the intermediates and consequently the detailed folding pathways differ. Apomyoglobin is therefore capable of compensating for mutations by using alternative folding pathways within a common basic framework. Tertiary hydrophobic interactions appear to play an important role in the formation and stabilization of secondary structure in the H helix of the N132G/E136G mutant. These studies provide important insights into the interplay between secondary and tertiary structure formation in protein folding.     

Effect of an alternative disulfide bond on the structure, stability, and folding of human lysozyme

Human lysozyme has four disulfide bonds, one of which, Cys65-Cys81, is included in a long loop of the beta-domain. A cysteine-scanning mutagenesis in which the position of Cys65 was shifted within a continuous segment from positions 61 to 67, with fixed Cys81, has previously shown that only the mutant W64CC65A, which has a nonnative Cys64-Cys81 disulfide, can be correctly folded and secreted by yeast. Here, using the W64CC65A mutant, we investigated the effects of an alternative disulfide bond on the structure, stability, and folding of human lysozyme using circular dichroism (CD) and fluorescence spectroscopy combined with a stopped-flow technique. Although the mutant is expected to have a different main-chain structure from that of the wild-type protein around the loop region, far- and near-UV CD spectra show that the native state of the mutant has tightly packed side chains and secondary structure similar to that of the wild-type. Guanidine hydrochloride-induced equilibrium unfolding transition of the mutant is reversible, showing high stability and cooperativity of folding. In the kinetic folding reaction, both proteins accumulate a similar burst-phase intermediate having pronounced secondary structure within the dead time of the measurement and fold into the native structure by means of a similar folding mechanism. Both the kinetic refolding and unfolding reactions of the mutant protein are faster than those of the wild-type, but the increase in the unfolding rate is larger than that of the refolding rate. The Gibbs' free-energy diagrams obtained from the kinetic analysis suggest that the structure around the loop region in the beta-domain of human lysozyme is formed after the transition state of folding, and thus, the effect of the alternative disulfide bond on the structure, stability, and folding of human lysozyme appears mainly in the native state.     

Use of site-directed mutagenesis to destabilize native apomyoglobin relative to folding intermediates

Site-directed mutagenesis has been used to study the effect on the stability of human apomyoglobin (apoMb) of modifying the size, hydrophobicity, and charge of a central residue in the G.B helix-helix packing interface. Some stability measurements have also been made on the corresponding holomyoglobins (heme present). Cys-110, a central helix pairing residue in the G helix, has been changed to Ala, Ser, Asp, and Leu. Stability to low-pH-induced unfolding has been measured for both native apoMb and the compact folding intermediate discovered by Griko et al. [Griko, Y. V., Privalov, P. L., Venyaminov, S. Y., & Kutyshenko, V. P. (1988) J. Mol. Biol. 202, 127-138]. As judged by its circular dichroism spectrum, this intermediate has a substantial helix content (about 35%). Whether or not this inferred helical structure is closely related to the myoglobin structure is not yet known. The mutational evidence shows that integrity of G.B helix pairing is important for the stability of apoMb as well as of myoglobin and that this helix pairing site is very sensitive to both steric and electrostatic disruption. Our results also suggest that G.B helix pairing does not stabilize the compact intermediate; hence, disrupting this site destabilizes the native protein relative to the compact intermediate. Such selective destabilization of the native state relative to equilibrium folding intermediates is not restricted to acid denaturation: urea denaturation of the Leu mutant appears to display at least one stable intermediate, while wild-type and the remaining mutant apoMbs undergo two-state urea unfolding transitions.     

High-pressure denaturation of apomyoglobin

The pressure denaturation of wild type and mutant apomyoglobin (apoMb) was investigated using a high-pressure, high-resolution nuclear magnetic resonance and high-pressure fluorescence techniques. Wild type apoMb is resistant to pressures up to 80 MPa, and denatures to a high-pressure intermediate, I(p), between 80 and 200 MPa. A further increase of pressure to 500 MPa results in denaturation of the intermediate. The two tryptophans, both in the A helix, remain sequestered from solvent in the high-pressure intermediate, which retains some native NOESY cross peaks in the AGH core as well as between F33 and F43. High-pressure fluorescence shows that the tryptophans remain inaccessible to solvent in the I(p) state. Thus the high-pressure intermediate has some structural properties in common with the apoMb I(2) acid intermediate. The resistance of the AGH core to pressures up to 200 MPa provides further evidence that the intrinsic stability of these alpha-helices is responsible for their presence in a number of equilibrium intermediates as well as in the earliest kinetic folding intermediate. Mutations in the AGH core designed to disrupt packing by burying a charge or increasing the size of a hydrophobic residue significantly perturbed the unfolding of native apoMb to the high-pressure intermediate. The F123W and S108L mutants both unfolded at lower pressures, while retaining some resistance to pressures below 50 MPa. The charge burial mutants, A130K and S108K, are not stable at very low pressures and both denature to the intermediate by 100 MPa, half of the pressure required for wild type apoMb. Thus a similar intermediate state is created independent of the method of perturbation, and mutations have similar effects on native state destabilization for both methods of denaturation. These data suggest that equilibrium intermediates that can be formed through different means are likely to resemble a kinetic intermediate.     

Consequences of stabilizing the natively disordered f helix for the folding pathway of apomyoglobin

The F helix region of sperm whale apomyoglobin is disordered, undergoing conformational fluctuations between a folded helical conformation and one or more locally unfolded states. To examine the effects of F helix stabilization on the folding pathway of apomyoglobin, we have introduced mutations to augment intrinsic helical structure in the F helix of the kinetic folding intermediate and to increase its propensity to fold early in the pathway, using predictions based on plots of the average area buried upon folding (AABUF) derived from the primary sequence. Two mutant proteins were prepared: a double mutant, P88K/S92K (F2), and a quadruple mutant, P88K/A90L/S92K/A94L (F4). Whereas the AABUF for F2 predicts that the F helix will not fold early in the pathway, the F helix in F4 shows a significantly increased AABUF and is therefore predicted to fold early. Protection of amide protons by formation of hydrogen-bonded helical structure during the early folding events has been analyzed by pH-pulse labeling. Consistent with the AABUF prediction, many of the F helix residues for F4 are significantly protected in the kinetic intermediate but are not protected in the F2 mutant. F4 folds via a kinetically trapped burst-phase intermediate that contains stabilized secondary structure in the A, B, F, G, and H helix regions. Rapid folding of the F helix stabilizes the central core of the misfolded intermediate and inhibits translocation of the H helix back to its native position, thereby decreasing the overall folding rate.     

Structure and function in bacteriorhodopsin: the effect of the interhelical loops on the protein folding kinetics

The loops connecting the seven transmembrane helices of bacteriorhodopsin have each been replaced in turn by structureless linkers of Gly-Gly-Ser repeat sequences, and the effect on the protein folding kinetics has been determined. An SDS-denatured state of each loop mutant bacterio-opsin was folded in l-alpha-1,2-dihexanoylphosphatidylcholine/l-alpha-1,2-dimyristoylphosphatidylcholine micelles, containing retinal, to give functional bacteriorhodopsin. Stopped-flow mixing was used to initiate the folding reaction, giving a time resolution of milliseconds, and changes in protein fluorescence were used to monitor folding. All loop mutant proteins folded according to the same reaction scheme as wild-type protein. The folding kinetics of the AB, BC and DE loop mutants were the same as wild-type protein, despite the blue-shifted chromophore band of the BC loop mutant bR state. A partially folded apoprotein intermediate state of the AB loop mutant did however appear to decay in the absence of retinal. The most significant effects on the folding kinetics were seen for mutant protein with structureless linkers in place of the CD, EF and FG loops. The rate-limiting apoprotein folding step of the CD loop mutant was about ten times slower than wild-type, whilst that of the EF loop mutant was almost four times slower than wild-type. Wild-type behaviour was observed for the other folding and retinal binding events of the CD and EF loop mutant proteins. These effects of the CD and EF loop mutations on apoprotein folding correlate with the fact that these two loop mutants also have the least stable, partially folded apoprotein intermediate of all the loop mutants, and are the most affected by a decrease in lipid lateral pressure. In contrast, the FG loop mutant exhibited wild-type apoprotein folding, but altered covalent binding of retinal and final folding to bacteriorhodopsin. This correlates with the fact that the FG loop mutant bacteriorhodopsin is the most susceptible to denaturation by SDS of all the loop mutants, but its partially folded apoprotein intermediate is more stable than that of the CD and EF mutants. Thus the CD and EF loops may contribute to the transition state for the rate-limiting apoprotein folding step and the FG loop to that for final folding and covalent binding of retinal.     

Folding of Aplysia limacina apomyoglobin involves an intermediate in common with other evolutionarily distant globins

In the globin family, similarities in the folding mechanism have been found among different mammalian apomyoglobins (apoMb). The best-characterized intermediate of sperm whale apoMb, called I(AGH), is mainly stabilized by nativelike contacts among the A, G, and H helices involving a cluster of hydrophobic residues that includes two conserved tryptophans. To verify the hypothesis of a common intermediate in the folding of all members of the globin family, we have extensively studied a site-directed mutant of the myoglobin from Aplysia limacina, distantly related to the mammalian counterpart, in which one of the two tryptophans in the A-G-H cluster [i.e., Trp(H8)130] has been mutated to tyrosine. The results presented here show that this mutation destabilizes both the native state and the acid intermediate I(A) but exerts little or no effect on the thermally stable core of an intermediate species (called I(T)) peculiar to Aplysia apomyoglobin. Dynamic quenching of Trp emission by acrylamide provides information on the accessibility of the chromophores at the native and the intermediate states of wild-type and mutant Aplysia apomyoglobin, consistent with the thermodynamics. Our results agree well with those obtained for the corresponding topological position of apomyoglobin from sperm whale and clearly show that the H8 position is involved in the stabilization of the main intermediate in both apoproteins. This residue thus plays a role which is evolutionarily conserved in the globin family from invertebrates to mammals; our results support the contention that the A-G-H cluster is important in the folding pathway of different globins.     

Protein phosphorylation corrects the folding defect of the neuroblastoma (S120G) mutant of human nucleoside diphosphate kinase A/Nm23-H1

Human nucleoside diphosphate (NDP) kinase A is a 'house-keeping' enzyme essential for the synthesis of nonadenine nucleoside (and deoxynucleoside) 5'-triphosphate. It is involved in complex cellular regulatory functions including the control of metastatic tumour dissemination. The mutation S120G has been identified in high-grade neuroblastomas. We have shown previously that this mutant has a folding defect: the urea-denatured protein could not refold in vitro. A molten globule folding intermediate accumulated, whereas the wild-type protein folded and associated into active hexamers. In the present study, we report that autophosphorylation of the protein corrected the folding defect. The phosphorylated S120G mutant NDP kinase, either autophosphorylated with ATP as donor, or chemically prosphorylated by phosphoramidate, refolded and associated quickly with high yield. Nucleotide binding had only a small effect. ADP and the non-hydrolysable ATP analogue 5'-adenyly-limido-diphosphate did not promote refolding. ATP-promoted refolding was strongly inhibited by ADP, indicating protein dephosphorylation. Our findings explain why the mutant enzyme is produced in mammalian cells and in Escherichia coli in a soluble form and is active, despite the folding defect of the S120G mutant observed in vitro. We generated an inactive mutant kinase by replacing the essential active-site histidine residue at position 118 with an asparagine residue, which abrogates the autophosphorylation. The double mutant H118N/S120G was expressed in inclusion bodies in E. coli. Its renaturation stops at a folding intermediate and cannot be reactivated by ATP in vitro. The transfection of cells with this double mutant might be a good model to study the cellular effects of folding intermediates.     

Replacement of proline with valine does not remove an apparent proline isomerization-dependent folding event in CRABP I

Site-directed mutagenesis has frequently been used to replace proline with other amino acids in order to determine if proline isomerization is responsible for a slow phase during refolding. Replacement of Pro 85 with alanine in cellular retinoic acid binding protein I (CRABP-I) abolished the slowest refolding phase, suggesting that this phase is due to proline isomerization in the unfolded state. To further test this assumption, we mutated Pro 85 to valine, which is the conservative replacement in the two most closely related proteins in the family (cellular retinoic acid binding protein II and cellular retinol binding protein I). The mutant protein was about 1 kcal/mole more stable than wild type. Retinoic acid bound equally well to wild type and P85V-CRABP I, confirming the functional integrity of this mutation. The refolding and unfolding kinetics of the wild-type and mutant proteins were characterized by stopped flow fluorescence and circular dichroism. The mutant P85V protein refolded with three kinetic transitions, the same number as wild-type protein. This result conflicts with the P85A mutant, which lost the slowest refolding rate. The P85V mutation also lacked a kinetic unfolding intermediate found for wild-type protein. These data suggest that proline isomerization may not be responsible for the slowest folding phase of CRABP I. As such, the loss of a slow refolding phase upon mutation of a proline residue may not be diagnostic for proline isomerization effects on protein folding.     

The kinetic and equilibrium molten globule intermediates of apoleghemoglobin differ in structure

An important question in protein folding is whether molten globule states formed under equilibrium conditions are good structural models for kinetic folding intermediates. The structures of the kinetic and equilibrium intermediates in the folding of the plant globin apoleghemoglobin have been compared at high resolution by quench-flow pH-pulse labeling and interrupted hydrogen/deuterium exchange analyzed in dimethyl sulfoxide. Unlike its well studied homolog apomyoglobin, where the equilibrium and kinetic intermediates are quite similar, there are striking structural differences between the intermediates formed by apoleghemoglobin. In the kinetic intermediate, formed during the burst phase of the quench-flow experiment, protected amides and helical structure are found mainly in the regions corresponding to the G and H helices of the folded protein, and in parts of the E helix and CE loop regions, whereas in the equilibrium intermediate, amide protection and helical structure are seen in parts of the A and B helix regions, as well as in the G and H regions, and the E helix remains largely unfolded. These results suggest that the structure of the molten globule intermediate of apoleghemoglobin is more plastic than that of apomyoglobin, so that it is readily transformed depending on the solution conditions, particularly pH. Thus, in the case of apoleghemoglobin at least, the equilibrium molten globule formed under destabilizing conditions at acid pH is not a good model for the compact intermediate formed during kinetic refolding experiments. Our high-precision kinetic analysis also reveals an additional slow phase during the folding of apoleghemoglobin, which is not observed for apomyoglobin. Hydrogen exchange pulse-labeling experiments show that the slow-folding phase is associated with residues in the CE loop, which probably forms non-native structure in the intermediate that must be resolved before folding can proceed to completion.     

Relationship between kinetic and equilibrium folding intermediates of creatine kinase

Creatine kinase (CK) is a dimeric enzyme important in ATP regeneration in cells where energy demands are high. The folding of CK under equilibrium and transient conditions has been studied in detail and is found to be complex. At equilibrium in 0.8 M GuHCl, 90% of CK molecules are in the form of a partially structured, monomeric intermediate. We exploit this property to measure kinetics of refolding and unfolding to and from this equilibrium intermediate (EI), using far-UV circular dichroism and intrinsic fluorescence as structural probes. We are thus able to compare the properties of EI and the kinetic intermediate formed during the burst phase in refolding. Native CK and EI unfold with rate constants in seconds and milliseconds, respectively. As is observed for refolding of fully-denatured CK, refolding from EI to the native state shows a burst phase followed by two exponential phases. The burst phase refolding intermediate is inferred to have more structure and greater stability than the equilibrium intermediate. When refolding from the fully-denatured state in 0.8 M GuHCl, the equilibrium intermediate is formed within the dead-time of mixing in the stopped-flow apparatus. The equilibrium intermediate may thus represent a kinetic intermediate formed early during folding.     

Acid denaturation and refolding of green fluorescent protein

Green fluorescent protein from the jellyfish Aequorea victoria can serve as a good model protein to understand protein folding in a complex environment with molecular chaperones and other macromolecules such as those in biological cells, but little is known about the detailed mechanisms of the in vitro folding of green fluorescent protein itself. We therefore investigated the kinetic refolding of a mutant (F99S/M153T/V163A) of green fluorescent protein, which is known to mature more efficiently than the wild-type protein, from the acid-denatured state; refolding was observed by chromophore fluorescence, tryptophan fluorescence, and far-UV CD, using a stopped-flow technique. In this study, we demonstrated that the kinetics of the refolding of the mutant have at least five kinetic phases and involve nonspecific collapse within the dead time of a stopped-flow apparatus and the subsequent formation of an on-pathway intermediate with the characteristics of the molten globule state. We also demonstrated that the slowest phase and a major portion of the second slowest phase were rate-limited by slow prolyl isomerization in the intermediate state, and this rate limitation accounts for a major portion of the observed kinetics in the folding of green fluorescent protein.     

On the role of some conserved and nonconserved amino acid residues in the transitional state and intermediate of apomyoglobin folding

The contributions of some amino acid residues in the A, B, G, and H helices to the formation of the folding nucleus and folding intermediate of apomyoglobin were estimated. The effects of point substitutions of Ala for hydrophobic amino acid residues on the structural stability of the native (N) protein and its folding intermediate (I), as well as on the folding/unfolding rates for four mutant apomyoglobin forms, were studied. The equilibrium and kinetic studies of the folding/unfolding rates of these mutant proteins in a wide range of urea concentrations demonstrated that their native state was considerably destabilized as compared with the wild-type protein, whereas the stability of the intermediate state changed moderately. It was shown that the amino acid residues in the A, G, and H helices contributed insignificantly to the stabilization of the apomyoglobin folding nucleus in the rate-limiting I ? N transition, taking place after the formation of the intermediate, whereas the residue of the B helix was of great importance in the formation of the folding nucleus in this transition.     

Modulation of the structural integrity of helix F in apomyoglobin by single amino acid replacements

The conformational features of native and mutant forms of sperm-whale apomyoglobin (apoMb) at neutral pH were probed by limited proteolysis experiments utilizing up to eight proteases of different substrate specificities. It was shown that all proteases selectively cleave apoMb at the level of chain segment 82-94 (HEAELKPLAQSHA), encompassing helix F in the X-ray structure of the holo form of the native protein; for example, thermolysin cleaves the Pro 88-Leu 89 peptide bond. These results indicate that helix F is highly flexible or largely disrupted in apoMb. Because helix F contains the helix-breaking Pro 88 residue, we propose that helix F is kept in place in the native holo protein by a variety of helix-heme stabilizing interactions. To modulate the stability of helix F, the Pro88Ala and Pro88Gly mutants were prepared by site-directed mutagenesis, and their conformational properties investigated by both far-UV circular dichroism spectroscopy and limited proteolysis. The helix content of the Pro88Ala mutant was somewhat enhanced with respect to that of both native and Pro88Gly mutant, as expected from the fact that Ala is the strongest helix inducer among the 20 amino acid residues. The rate of limited proteolysis of the three apoMb variants by thermolysin and proteinase K was in the order native > Pro88Gly > Pro88Ala, in agreement with the scale of helix propensity of Ala, Gly, and Pro. The possible role of the flexible/unfolded chain segment 82-94 for the function and fate of apoMb at the cellular level is discussed.