Conservation of folding pathways in evolutionarily distant globin sequences

To test the hypothesis that the folding pathways of evolutionarily related proteins with similar three-dimensional structures but widely different sequences should be similar, the folding pathway of apoleghemoglobin has been characterized using stopped-flow circular dichroism, heteronuclear NMR pulse labeling techniques and mass spectrometry. The pathway of folding was found to differ significantly from that of a protein of the same family, apomyoglobin, although both proteins appear to fold through helical burst phase intermediates. For leghemoglobin, the burst phase intermediate exhibits stable helical structure in the G and H helices, together with a small region in the center of the E helix. The A and B helices are not stabilized until later stages of the folding process. The structure of the burst phase folding intermediate thus differs from that of apomyoglobin, in which stable helical structure is formed in the A, B, G and H helix regions.     

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.     

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.     

Tryptophanyl contributions to apomyoglobin fluorescence resolved by site-directed mutagenesis

The individual emission properties of the two tryptophanyl residues of sperm-whale apomyoglobin have been resolved by examining the fluorescence variations induced by denaturants, i.e., acid and guanidine, on apomyoglobin mutants W7F and W14F. The fluorescence changes have been correlated to the conformational transitions undergone by apomyoglobin on increasing denaturant concentration. The results indicate that the fluorescence decrease, observed for sperm-whale apomyoglobin on going from pH 8.0 to pH 6.0, cannot be ascribed to the formation of a charge transfer complex between a nearby histidine residue and W14 as reported in earlier papers but rather to minor structural changes affecting the microenvironments of both residues. The formation of the acidic partly folded state around pH 4.0 determines an increase of the fluorescence yield and a small red shift (5 nm) of W7 due to removal of sterically interacting K79, which is able to attenuate the emission of this residue in the native state. The fluorescence intensity of the other residue, i.e., W14, is not affected by the acidic transition. Guanidine denaturation experiments revealed an increase of fluorescence yield of W14 upon the intermediate formation, whereas the fluorescence of the other residue remained constant. The results suggest that the unfolding pathway may be different depending on the chemical nature of the denaturant used.     

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.     

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.     

A catenated intermediate in plasmid replication

An intermediate in the replication of three different staphylococcal plasmids is a catenated mixed dimer consisting of an open circular and a closed circular monomer. The mixed dimer, having a buoyant density intermediate between that of circular duplex and relaxed DNA's in dye-buoyant density gradients is rapidly and transiently labeled and appears to be a precursor of mature closed circular monomers. Whether it is an obligatory intermediate or a step on an alternate replication pathway remains uncertain.     

Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments

In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0–2.2 M) than the formation of the native state (0–1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7–2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin.     

Equilibrium unfolding of mutant apomyoglobins with substitutions of conserved nonfunctional residues by alanine

The problems of protein aggregation and protein misfolding in the cell are connected with the appearance of many genetic diseases. Both processes can be a consequence of substitutions of certain amino acid residues in proteins. The substitutions can influence the protein stability and protein folding rates in both the intermediate and the native states. We have studied equilibrium urea unfolding of mutant forms of apomyoglobin with substitutions of conserved nonfunctional residues by Ala to estimate their influence on protein stability. These residues include Val10, Trp14, Ilel11, Leu115, Met131 and Leu135. Conformational transitions were monitored by intrinsic Trp fluorescence and by circular dichroism spectra in the far UV region. Free energy changes upon the transition from the native to intermediate state and from the intermediate to unfolded state were determined. It was shown that all substitutions used lead to an appreciable decrease of the apomyoglobin native state stability, whereas the stability of the intermediate state is affected substantially smaller.     

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.     

Intermediate states of apomyoglobin: Are they parts of the same area of conformations diagram?

Several research teams have reported detection and characterization of various apomyoglobin intermediate states different in their accumulation mode, thus putting a natural question as to proportions of these intermediates. The current report presents spectral properties of sperm whale apomyoglobin studied over a wide range of conditions with the use of circular dichroism and fluorescence techniques. Based on the experimental data, a diagram of apomyoglobin conformational states has been constructed. It shows that though induced by various denaturants, all the observed intermediates belong to one and the same area in the diagram.     

Cysteine is not an obligatory intermediate in the biosynthesis of cysteate by Cytophaga johnsonae

A cysteine auxotroph of Cytophaga johnsonae was able to incorporate sulfur from sulfate into cysteate, and thus into sulfonolipid, in the absence of cysteine synthesis. This indicates that cysteine is not an obligatory intermediate of the cysteate biosynthetic pathway even though cysteine sulfur can be utilized for cysteate synthesis.     

How strong are side chain interactions in the folding intermediate?

Influence of 12 nonpolar amino acids residues from the hydrophobic core of apomyoglobin on stability of its native state and folding intermediate was studied. Six of the selected residues are from the A, G and H helices; these are conserved in structure of the globin family, although nonfunctional, that is, not involved in heme binding. The rest are nonconserved hydrophobic residues that belong to the B, C, D, and E helices. Each residue was substituted by alanine, and equilibrium pH‐induced transitions in apomyoglobin and its mutants were studied by circular dichroism and fluorescent spectroscopy. The obtained results allowed estimating changes in their free energy during formation of the intermediate state. It was first shown that the strength of side chain interactions in the apomyoglobin intermediate state amounts to 15–50% of that in its native state for conserved residues, and practically to 0% for nonconserved residues. These results allow a better understanding of interactions occurring in the intermediate state and shed light on involvement of certain residues in protein folding at different stages.     

Glycation Accelerates Fibrillization of the Amyloidogenic W7FW14F Apomyoglobin

Neurodegenerative diseases are associated with misfolding and deposition of specific proteins, either intra or extracellularly in the nervous system. Advanced glycation end products (AGEs) originate from different molecular species that become glycated after exposure to sugars. Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. To this concern, in the present study we have investigated the effect of glycation on the aggregation pathway of the amyloidogenic W7FW14F apomyoglobin. Although this protein has not been related to any amyloid disease, it represents a good model to resemble proteins that intrinsically evolve toward the formation of amyloid aggregates in physiological conditions. We show that D-ribose, but not D-glucose, rapidly induces the W7FW14F apomyoglobin to generate AGEs in a time-dependent manner and protein ribosylation is likely to involve lysine residues on the polypeptide chain. Ribosylation of the W7FW14F apomyoglobin strongly affects its aggregation kinetics producing amyloid fibrils within few days. Cytotoxicity of the glycated aggregates has also been tested using a cell viability assay. We propose that ribosylation in the W7FW14F apomyoglobin induces the formation of a cross-link that strongly reduces the flexibility of the H helix and/or induce a conformational change that favor fibril formation. These results open new perspectives for AGEs biological role as they can be considered not only a triggering factor in amyloidosis but also a player in later stages of the aggregation process.     

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.     

Native and non-native secondary structure and dynamics in the pH 4 intermediate of apomyoglobin

The partly folded state of apomyoglobin at pH 4 represents an excellent model for an obligatory kinetic folding intermediate. The structure and dynamics of this intermediate state have been extensively examined using NMR spectroscopy. Secondary chemical shifts, (1)H-(1)H NOEs, and amide proton temperature coefficients have been used to probe residual structure in the intermediate state, and NMR relaxation parameters T(1) and T(2) and ?(1)H?-(15)N NOE have been analyzed using spectral densities to correlate motion of the polypeptide chain with these structural observations. A significant amount of helical structure remains in the pH 4 state, indicated by the secondary chemical shifts of the (13)C(alpha), (13)CO, (1)H(alpha), and (13)C(beta) nuclei, and the boundaries of this helical structure are confirmed by the locations of (1)H-(1)H NOEs. Hydrogen bonding in the structured regions is predominantly native-like according to the amide proton chemical shifts and their temperature dependence. The locations of the A, G, and H helix segments and the C-terminal part of the B helix are similar to those in native apomyoglobin, consistent with the early, complete protection of the amides of residues in these helices in quench-flow experiments. These results confirm the similarity of the equilibrium form of apoMb at pH 4 and the kinetic intermediate observed at short times in the quench-flow experiment. Flexibility in this structured core is severely curtailed compared with the remainder of the protein, as indicated by the analysis of the NMR relaxation parameters. Regions with relatively high values of J(0) and low values of J(750) correspond well with the A, B, G, and H helices, an indication that nanosecond time scale backbone fluctuations in these regions of the sequence are restricted. Other parts of the protein show much greater flexibility and much reduced secondary chemical shifts. Nevertheless, several regions show evidence of the beginnings of helical structure, including stretches encompassing the C helix-CD loop, the boundary of the D and E helices, and the C-terminal half of the E helix. These regions are clearly not well-structured in the pH 4 state, unlike the A, B, G, and H helices, which form a native-like structured core. However, the proximity of this structured core most likely influences the region between the B and F helices, inducing at least transient helical structure.     

The Molten Globule of β2-Microglobulin Accumulated at pH 4 and Its Role in Protein Folding

The acid transition of β₂-microglobulin (β2m) was studied by tryptophan fluorescence, peptide circular dichroism, and NMR spectroscopy. The protein exhibits a three-state transition with an equilibrium intermediate accumulated at pH 4 (25 °C). The pH 4 intermediate has typical characteristics of the molten globule (MG) state; it showed a native-like secondary structure without specific side-chain tertiary structure, and the hydrodynamic radius determined by pulse field gradient NMR was only 20% larger than that of the native state. The accumulation of the pH 4 intermediate is very analogous to the behavior of apomyoglobin, for which the pH 4 MG has been well characterized, although β2m, a β-protein, is structurally very different from α-helical apomyoglobin. NMR pH titration of histidine residues of β2m has also indicated that His84 has an abnormally low pK_a value in the native state. From the pH dependence of the unfolding transition, the protonations of this histidine and 10 weakly abnormal carboxylates triggered the transition from the native to the MG state. This behavior is again analogous to that of apomyoglobin, suggesting a common mechanism of production of the pH 4 MG. In contrast to the folding of apomyoglobin, in which the MG was equivalent to the burst-phase kinetic folding intermediate, the burst-phase refolding intermediate of β2m, detected by stopped-flow circular dichroism, was significantly more structured than the pH 4 intermediate. It is proposed that the folding of β2m from its acid-denatured state takes place in the following order: denatured state → MG → burst-phase intermediate → native state.     

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.     

The folding pathway of a fast-folding immunoglobulin domain revealed by single-molecule mechanical experiments

The F-actin crosslinker filamin from Dictyostelium discoideum (ddFLN) has a rod domain consisting of six structurally similar immunoglobulin domains. When subjected to a stretching force, domain 4 unfolds at a lower force than all the other domains in the chain. Moreover, this domain shows a stable intermediate along its mechanical unfolding pathway. We have developed a mechanical single-molecule analogue to a double-jump stopped-flow experiment to investigate the folding kinetics and pathway of this domain. We show that an obligatory and productive intermediate also occurs on the folding pathway of the domain. Identical mechanical properties suggest that the unfolding and refolding intermediates are closely related. The folding process can be divided into two consecutive steps: in the first step 60 C-terminal amino acids form an intermediate at the rate of 55 s(-1); and in the second step the remaining 40 amino acids are packed on this core at the rate of 179 s(-1). This division increases the overall folding rate of this domain by a factor of ten compared with all other homologous domains of ddFLN that lack the folding intermediate.     

Compartmentation of terpenoid biosynthesis in green plants. A proposed route of acetyl-coenzyme A synthesis in maize chloroplasts

On the basis of radioisotope-incorporation experiments it is suggested that acetyl-CoA, an obligatory intermediate in chloroplast terpenoid biosynthesis, may be formed in maize from photosynthetically fixed carbon dioxide by the route carbon dioxide-->glycollate-->glyoxylate-->glycine-->serine-->pyruvate-->acetyl-CoA. The proposed route is supported by conventional radioisotope-dilution studies and by experiments with inhibitors affecting reactions involved in the pathway. The proposed route appears to play little part in formation of extrachloroplastidic sterol.