Alternatively folded states of an immunoglobulin

Well-defined, non-native protein structures of low stability have been increasingly observed as intermediates in protein folding or as equilibrium structures populated under specific solvent conditions. These intermediate structures, frequently referred to as molten globule states, are characterized by the presence of secondary structure, a lack of significant tertiary contacts, increased hydrophobicity and partial specific volume as compared to native structures, and low cooperativity in thermal unfolding. The present study demonstrates that under acidic conditions (pH less than 3) the antibody MAK33 can assume a folded stable conformation. This A-state is characterized by a high degree of secondary structure, increased hydrophobicity, a native-like maximum wavelength of fluorescence emission, and a tendency toward slow aggregation. A prominent feature of this low-pH conformation is the stability against denaturant and thermal unfolding that is manifested in highly cooperative reversible phase transitions indicative of the existence of well-defined tertiary contacts. These thermodynamic results are corroborated by the kinetics of folding from the completely unfolded chain to the alternatively folded state at pH 2. The given data suggest that MAK33 at pH 2 adopts a cooperative structure that differs from the native immunoglobulin fold at pH 7. This alternatively folded state exhibits certain characteristics of the molten globule but differs distinctly from it by its extraordinary structural stability that is characteristic for native protein structures.     

Structural heterogeneity of the various forms of apomyoglobin: implications for protein folding.

Temperature-induced denaturation transitions of different structural forms of apomyoglobin were studied monitoring intrinsic tryptophan fluorescence. It was found that the tryptophans are effectively screened from solvent both in native and acid forms throughout most of the temperature range tested. Thus, the tryptophans' surrounding do not show a considerable change in structure where major protein conformational transitions have been found in apomyoglobin using other techniques. At high temperatures and under strong destabilizing conditions, the tryptophans' fluorescence parameters show sigmoidal thermal denaturation. These results, combined with previous studies, show that the structure of this protein is heterogeneous, including native-like (tightly packed) and molten globule-like substructures that exhibit conformation (denaturation) transitions under different conditions of pH and temperature (and denaturants). The results suggest that the folding of this protein proceeds via two "nucleation" events whereby native-like contacts are formed. One of these events, which involves AGH "core" formation, appears to occur very early in the folding process, even before significant hydrophobic collapse in the rest of the protein molecule. From the current studies and other results, a rather detailed picture of the folding of myoglobin is presented, on the level of specific structures and their thermodynamical properties as well as formation kinetics.     

Comparison of solution structures and stabilities of native, partially unfolded and partially refolded pepsin

A zymogen-derived protein, pepsin, appears to be incapable of folding to the native state without the presence of the prosegment. To better understand the nature of the irreversible denaturation of pepsin, the present study reports on the characterization of the stability and low-resolution tertiary and secondary structures of native, alkaline unfolded and acid refolded porcine pepsin. Through a combination of small-angle neutron scattering (SANS), CD, and DSC, acid refolded pepsin (Rp) was shown to have secondary and tertiary structures intermediate between the alkaline denatured and native forms but was found to be thermodynamically stable relative to the native state. It was also observed that the acid refolded state of pepsin was dependent on the protein concentration during refolding because CD and SANS data revealed that both the secondary and tertiary structures of concentrated-refolded pepsin (>10 mg/mL) (CRp) were native-like, in contrast to the intermediate nature of Rp, refolded under dilute concentration (<10 mg/mL). Despite a native-like conformation, CRp was more stable and had substantially reduced activity compared to that of the native state, suggesting that the protein was misfolded. It is proposed that the stable but misfolded, acid-refolded states are evidence that pepsin in its native conformation was metastable. Furthermore, the disruption of the active site cleft in the denatured states could be discerned by modeling of the SANS data.     

Conformational restrictions on the pathway of folding and unfolding of the pancreatic trypsin inhibitor

The kinetic roles of the partially folded, intermediate protein species with two disulphide bonds in folding and unfolding of the pancreatic trypsin inhibitor have been investigated further. Formation of a second disulphide bond between Cys5 and Cys55 during refolding of the reduced inhibitor, which would yield the species with the 30–51 and 5–55 disulphide bonds and, possibly, the native-like conformation of the protein, is not significant. Instead, three other second disulphide bonds (5–14, 5–38 and 14–38) are formed approximately 10⁵ times more readily, but each of these two-disulphide species then rearranges intramolecularly to the native-like, two-disulphide intermediate. Therefore, the reduced protein does not simply form sequentially the three disulphide bonds of the native state. Unfolding of the native state takes place by the reverse of this process.The kinetic importance for folding and unfolding of this transition between two-disulphide intermediates under the conditions used here was illustrated experimentally by a modified form of the inhibitor in which the thiols of Cys14 and Cys38 were blocked irreversibly. In the folded conformation, this modified protein is more stable to unfolding than normal, but after unfolding cannot readily regain the native-like conformation, because Cys14 or Cys38 are required to be involved in disulphide bonds during the interconversion of the two-disulphide intermediates.Some conception of the conformational transitions that take place at each stage of the folding transition may be inferred from the relative propensities of the six cysteine residues to make or rearrange disulphide bonds. It is concluded that the inhibitor probably does not refold by sequential adoption of the native conformation by the unfolded polypeptide chain. Instead, it appears that essentially all elements of the native conformation are attained simultaneously in the final stage of folding, within an unstable and flexible, yet relatively compact, form of the entire polypeptide chain produced by weak interactions between groups distant in the primary structure.     

Effects of urea and guanidine · HCl on the folding and unfolding of pancreatic trypsin inhibitor

Concentrated solutions of urea and of guanidine · HCl produced a random spectrum of single-disulphide forms of the polypeptide chain of the pancreatic trypsin inhibitor. Guanidine · HCl also unfolded completely, with accompanying interchange of disulphide bonds, the two-disulphide form of this protein in the native-like conformation; urea produced an equilibrium mixture in which one-quarter of the molecules had the native-like conformation and disulphide bonds. The unfolded forms of the protein in the denaturants were very flexible polypeptide chains. The observations suggest that urea and guanidine · HCl are denaturants because they produce essentially equally favourable solvation of all portions of a polypeptide.The energetics of the conformational transitions involved in folding and unfolding of the inhibitor were determined in urea and compared with those observed in its absence. The denaturant lowers the stability of the native, folded inhibitor relative to that of the reduced, unfolded state by 6.5 kilocalories per mole; the greatest part of this apparent free-energy difference was expressed at the two-disulphide stage of folding. The results are consistent with other indications that most of the favourable interactions stabilizing the native conformation of this protein are not encountered until the final stage of folding, when all may occur simultaneously.The unfolded one- and two-disulphide species produced in guanidine · HCl were trapped, and their rearrangement to the normal intermediates followed after removal of the denaturant. The random single-disulphide species, with one exception, reverted very rapidly to the non-random spectrum of intermediates normally observed during folding; this confirms that these species are normally rapidly interconverted and that normal refolding of the reduced protein is not dependent kinetically upon residual stable conformation in the reduced protein. The unfolded two-disulphide species refolded to the native-like conformation more slowly, but appeared to pass through the same intermediates normally observed during refolding from the fully reduced state.     

Transition state contact orders correlate with protein folding rates

We have used molecular dynamics simulations restrained by experimental phi values derived from protein engineering experiments to determine the structures of the transition state ensembles of ten proteins that fold with two-state kinetics. For each of these proteins we then calculated the average contact order in the transition state ensemble and compared it with the corresponding experimental folding rate. The resulting correlation coefficient is similar to that computed for the contact orders of the native structures, supporting the use of native state contact orders for predicting folding rates. The native contacts in the transition state also correlate with those of the native state but are found to be about 30% lower. These results show that, despite the high levels of heterogeneity in the transition state ensemble, the large majority of contributing structures have native-like topologies and that the native state contact order captures this phenomenon.     

A tale of two secondary structure elements: when a beta-hairpin becomes an alpha-helix.

In this work, we have analyzed the relative importance of secondary versus tertiary interactions in stabilizing and guiding protein folding. For this purpose, we have designed four different mutants to replace the alpha-helix of the GB1 domain by a sequence with strong beta-hairpin propensity in isolation. In particular, we have chosen the sequence of the second beta-hairpin of the GB1 domain, which populates the native conformation in aqueous solution to a significant extent. The resulting protein has roughly 30 % of its sequence duplicated and maintains the 3D-structure of the wild-type protein, but with lower stability (up to -5 kcal/mol). The loss of intrinsic helix stability accounts for about 80 % of the decrease in free energy, illustrating the importance of local interactions in protein stability. Interestingly enough, all the mutant proteins, included the one with the duplicated beta-hairpin sequence, fold with similar rates as the GB1 domain. Essentially, it is the nature of the rate-limiting step in the folding reaction that determines whether a particular interaction will speed up, or not, the folding rates. While local contacts are important in determining protein stability, residues involved in tertiary contacts in combination with the topology of the native fold, seem to be responsible for the specificity of protein structures. Proteins with non-native secondary structure tendencies can adopt stable folds and be as efficient in folding as those proteins with native-like propensities.     

Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

Osmolytes are low molecular weight organic molecules accumulated by organisms to assist proper protein folding, and to provide protection to the structural integrity of proteins under denaturing stress conditions. It is known that osmolyte-induced protein folding is brought by unfavorable interaction of osmolytes with the denatured/unfolded states. The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding. We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding. We observed that osmolyte-assisted refolding of protein obtained from heat-induced denatured state produces native molecules with higher enzyme activity than those initiated from GdmCl- or urea-induced denatured state indicating that the structural property of the initial denatured state during refolding by osmolytes determines the catalytic efficiency of the folded protein molecule. These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (K _m and k _cat), thermodynamic stability (T _m and ΔH _m) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes.     

Unfolding and refolding of cytochrome c driven by the interaction with lipid micelles

Binding of native cyt c to L-PG micelles leads to a partially unfolded conformation of cyt c. This micelle-bound state has no stable tertiary structure, but remains as alpha-helical as native cyt c in solution. In contrast, binding of the acid-unfolded cyt c to L-PG micelles induces folding of the polypeptide, resulting in a similar helical state to that originated from the binding of native cyt c to L-PG micelles. Far-ultraviolet (UV) circular dichroism (CD) spectra showed that this common micelle-associated helical state (HL) has a native-like alpha-helix content, but is highly expanded without a tightly packed hydrophobic core, as revealed by tryptophan fluorescence, near-UV, and Soret CD spectroscopy. The kinetics of the interaction of native and acid-unfolded cyt c was investigated by stopped-flow tryptophan fluorescence. Formation of H(L) from the native state requires the disruption of the tightly packed hydrophobic core in the native protein. This micelle-induced unfolding of cyt c occurs at a rate approximately 0.1 s(-1), which is remarkably faster in the lipid environment compared with the expected rate of unfolding in solution. Refolding of acid-unfolded cyt c with L-PG micelles involves an early highly helical collapsed state formed during the burst phase (<3 ms), and the observed main kinetic event reports on the opening of this early compact intermediate prior to insertion into the lipid micelle.     

Non-native interactions play an effective role in protein folding dynamics

Systematic Monte Carlo simulations of simple lattice models show that the final stage of protein folding is an ordered process where native contacts get locked (i.e., the residues come into contact and remain in contact for the duration of the folding process) in a well‐defined order. The detailed study of the folding dynamics of protein‐like sequences designed as to exhibit different contact energy distributions, as well as different degrees of sequence optimization (i.e., participation of non‐native interactions in the folding process), reveals significant differences in the corresponding locking scenarios—the collection of native contacts and their average locking times, which are largely ascribable to the dynamics of non‐native contacts. Furthermore, strong evidence for a positive role played by non‐native contacts at an early folding stage was also found. Interestingly, for topologically simple target structures, a positive interplay between native and non‐native contacts is observed also toward the end of the folding process, suggesting that non‐native contacts may indeed affect the overall folding process. For target models exhibiting clear two‐state kinetics, the relation between the nucleation mechanism of folding and the locking scenario is investigated. Our results suggest that the stabilization of the folding transition state can be achieved through the establishment of a very small network of native contacts that are the first to lock during the folding process.     

Diffusing and colliding: the atomic level folding/unfolding pathway of a small helical protein

Proteins with ultra-fast folding/unfolding kinetics are excellent candidates for study by molecular dynamics. Here, we describe such simulations of a three helix bundle protein, the engrailed homeodomain (En-HD), which folds via the diffusion-collision model. The unfolding pathway of En-HD was characterized by seven simulations of the protein and 12 simulations of its helical fragments yielding over 1.1 micros of simulation time in water. Various conformational states along the unfolding pathway were identified. There is the compact native-like transition state, a U-shaped helical intermediate and an unfolded state with dynamic helical segments. Each of these states is in good agreement with experimental data. Examining these states as well as the transitions between them, we find the role of long-range tertiary contacts, specifically salt-bridges, important in the folding/unfolding pathway. In the folding direction, charged residues form long-range tertiary contacts before the hydrophobic core is formed. The formation of HII is assisted by a specific salt-bridge and by non-specific (fluctuating) tertiary contacts, which we call contact-assisted helix formation. Salt-bridges persist as the protein approaches the transition state, stabilizing HII until the hydrophobic core is formed. To complement this information, simulations of fragments of En-HD illustrate the helical propensities of the individual segments. By thermal denaturation, HII proved to be the least stable helix, unfolding in less than 450 ps at high temperature. We observed the low helical propensity of C-terminal residues from HIII in fragment simulations which, when compared to En-HD unfolding simulations, link the unraveling of HIII to the initial event that drives the unfolding of En-HD.     

Oligomeric States along the Folding Pathways of β2-Microglobulin: Kinetics,Thermodynamics, and Structure

The transition of proteins from their soluble functional state to amyloid fibrils and aggregates is associated with the onset of several human diseases. Protein aggregation often requires some structural reshaping and the subsequent formation of intermolecular contacts. Therefore, the study of the conformation of excited protein states and their ability to form oligomers is of primary importance for understanding the molecular basis of amyloid fibril formation. Here, we investigated the oligomerization processes that occur along the folding of the amyloidogenic human protein β2-microglobulin. The combination of real-time two-dimensional NMR data with real-time small-angle X-ray scattering measurements allowed us to derive thermodynamic and kinetic information on protein oligomerization of different conformational states populated along the folding pathways. In particular, we could demonstrate that a long-lived folding intermediate (I-state) has a higher propensity to oligomerize compared to the native state. Our data agree well with a simple five-state kinetic model that involves only monomeric and dimeric species. The dimers have an elongated shape with the dimerization interface located at the apical side of β2-microglobulin close to Pro32, the residue that has a trans conformation in the I-state and a cis conformation in the native (N) state. Our experimental data suggest that partial unfolding in the apical half of the protein close to Pro32 leads to an excited state conformation with enhanced propensity for oligomerization. This excited state becomes more populated in the transient I-state due to the destabilization of the native conformation by the trans-Pro32 configuration.     

Equilibrium folding and unfolding pathways for a model protein

The folding–unfolding process of reduced bovine pancreatic trypsin inhibitor was investigated with an idealized model employing approximate free energies. The protein is regarded to consist of only C^α and C^β atoms. The backbone dihedral angles are the only conformational variables and are permitted to take discrete values at every 10°. Intraresidue energies consist of two terms: an empirical part taken from the observed frequency distributions of (?,ψ) and an additional favorable energy assigned to the native conformation of each residue. Interresidue interactions are simplified by assuming that there is an attractive energy operative only between residue pairs in close contact in the native structure. A total of 230,000 molecular conformations, with no atomic overlaps, ranging from the native state to the denatured state, are randomly generated by changing the sampling bias. Each conformation is classified according to its conformational energy, F; a conformational entropy, S(F) is estimated for each value of F from the number of samples. The dependence of S(F) on energy reveals that the folding–unfolding transition for this idealized model is an “all-or-none” type; this is attributable to the specific long-range interactions. Interresidue contact probabilities, averaged over samples representing various stages of folding, serve to characterize folding intermediates. Most probable equilibrium pathways for the folding–unfolding transition are constructed by connecting conformationally similar intermediates. The specific details obtained for bovine pancreatic trypsin inhibitor are as follows: (1) Folding begins with the appearance of nativelike medium-range contacts at a β-turn and at the α-helix. (2) These grow to include the native pair of interacting β-strands. This state includes intact regular secondary conformations, as well as the interstrand sheet contacts, and corresponds to an activated state with the highest free energy on the pathway. (3) Additional native long-range contacts are completely formed either toward the amino terminus or toward the carboxyl terminus. (4) In a final step, the missing contacts appear. Although these folding pathways for this model are not consistent with experimental reports, it does indicate multiple folding pathways. The method is general and can be applied to any set of calculated conformational energies and furthermore permits investigation of gross folding features.     

The Folding Pathway of the Antibody VL Domain

Antibodies are modular proteins consisting of domains that exhibit a β-sandwich structure, the so-called immunoglobulin fold. Despite structural similarity, differences in folding and stability exist between different domains. In particular, the variable domain of the light chain V_L is unusual as it is associated with misfolding diseases, including the pathologic assembly of the protein into fibrillar structures. Here, we have analysed the folding pathway of a V_L domain with a view to determine features that may influence the relationship between productive folding and fibril formation. The V_L domain from MAK33 (murine monoclonal antibody of the subtype κ/IgG1) has not previously been associated with fibrillisation but is shown here to be capable of forming fibrils. The folding pathway of this V_L domain is complex, involving two intermediates in different pathways. An obligatory early molten globule-like intermediate with secondary structure but only loose tertiary interactions is inferred. The native state can then be formed directly from this intermediate in a phase that can be accelerated by the addition of prolyl isomerases. However, an alternative pathway involving a second, more native-like intermediate is also significantly populated. Thus, the protein can reach the native state via two distinct folding pathways. Comparisons to the folding pathways of other antibody domains reveal similarities in the folding pathways; however, in detail, the folding of the V_L domain is striking, with two intermediates populated on different branches of the folding pathway, one of which could provide an entry point for molecules diverted into the amyloid pathway.     

The denatured state dictates the topology of two proteins with almost identical sequence but different native structure and function

The protein folding problem is often studied by comparing the mechanisms of proteins sharing the same structure but different sequence. The recent design of the two proteins G_A88 and G_B88, displaying different structures and functions while sharing 88% sequence identity (49 out of 56 amino acids), allows the unique opportunity for a complementary approach. At which stage of its folding pathway does a protein commit to a given topology? Which residues are crucial in directing folding mechanisms to a given structure? By using a combination of biophysical and computational techniques, we have characterized the folding of both G_A88 and G_B88. We show that, contrary to expectation, G_B88, characterized by a native α+β fold, displays in the denatured state a content of native-like helical structure greater than G_A88, which is all-α in its native state. Both experiments and simulations indicate that such residual structure may be tuned by changing pH. Thus, despite the high sequence identity, the folding pathways for these two proteins appear to diverge as early as in the denatured state. Our results suggest a mechanism whereby protein topology is committed very early along the folding pathway, being imprinted in the residual structure of the denatured state.     

Structure of a partially unfolded form of Escherichia coli dihydrofolate reductase provides insight into its folding pathway

Proteins frequently fold via folding intermediates that correspond to local minima on the conformational energy landscape. Probing the structure of the partially unfolded forms in equilibrium under native conditions can provide insight into the properties of folding intermediates. To elucidate the structures of folding intermediates of Escherichia coli dihydrofolate reductase (DHFR), we investigated transient partial unfolding of DHFR under native conditions. We probed the structure of a high‐energy conformation susceptible to proteolysis (cleavable form) using native‐state proteolysis. The free energy for unfolding to the cleavable form is clearly less than that for global unfolding. The dependence of the free energy on urea concentration (m‐value) also confirmed that the cleavable form is a partially unfolded form. By assessing the effect of mutations on the stability of the partially unfolded form, we found that native contacts in a hydrophobic cluster formed by the F‐G and Met‐20 loops on one face of the central β‐sheet are mostly lost in the partially unfolded form. Also, the folded region of the partially unfolded form is likely to have some degree of structural heterogeneity. The structure of the partially unfolded form is fully consistent with spectroscopic properties of the near‐native kinetic intermediate observed in previous folding studies of DHFR. The findings suggest that the last step of the folding of DHFR involves organization in the structure of two large loops, the F‐G and Met‐20 loops, which is coupled with compaction of the rest of the protein.     

Protein unfolding transitions in an intrinsically unstable annexin domain: molecular dynamics simulation and comparison with nuclear magnetic resonance data

Unfolding transitions of an intrinsically unstable annexin domain and the unfolded state structure have been examined using multiple approximately 10-ns molecular dynamics simulations. Three main basins are observed in the configurational space: native-like state, compact partially unfolded or intermediate compact state, and the unfolded state. In the native-like state fluctuations are observed that are nonproductive for unfolding. During these fluctuations, after an initial loss of approximately 20% of the core residue native contacts, the core of the protein transiently completely refolds to the native state. The transition from the native-like basin to the partially unfolded compact state involves approximately 75% loss of native contacts but little change in the radius of gyration or core hydration properties. The intermediate state adopts for part of the time in one of the trajectories a novel highly compact salt-bridge stabilized structure that can be identified as a conformational trap. The intermediate-to-unfolded state transition is characterized by a large increase in the radius of gyration. After an initial relaxation the unfolded state recovers a native-like topology of the domain. The simulated unfolded state ensemble reproduces in detail experimental nuclear magnetic resonance data and leads to a convincing complete picture of the unfolded domain.     

Structural Rearrangements Linked to Global Folding Pathways of the Azoarcus Group I Ribozyme

Stable RNAs must fold into specific three-dimensional structures to be biologically active, yet many RNAs form metastable structures that compete with the native state. Our previous time-resolved footprinting experiments showed that Azoarcus group I ribozyme forms its tertiary structure rapidly (τ < 30 ms) without becoming significantly trapped in kinetic intermediates. Here, we use stopped-flow fluorescence spectroscopy to probe the global folding kinetics of a ribozyme containing 2-aminopurine in the loop of P9. The modified ribozyme was catalytically active and exhibited two equilibrium folding transitions centered at 0.3 and 1.6 mM Mg²⁺, consistent with previous results. Stopped-flow fluorescence revealed four kinetic folding transitions with observed rate constants of 100, 34, 1, and 0.1 s^− 1 at 37 °C. From comparison with time-resolved Fe(II)-ethylenediaminetetraacetic acid footprinting of the modified ribozyme under the same conditions, these folding transitions were assigned to formation of the I_C intermediate, tertiary folding and docking of the nicked P9 tetraloop, reorganization of the P3 pseudoknot, and refolding of nonnative conformers, respectively. The footprinting results show that 50-60% of the modified ribozyme folds in less than 30 ms, while the rest of the RNA population undergoes slow structural rearrangements that control the global folding rate. The results show how small perturbations to the structure of the RNA, such as a nick in P9, populate kinetic folding intermediates that are not observed in the natural ribozyme.     

Monte Carlo simulation of denaturation of adsorbed proteins

Denaturation of model proteinlike molecules at the liquid–solid interface is simulated over a wide temperature range by employing the lattice Monte Carlo technique. Initially, the molecule containing 27 monomers of two types (A and B) is assumed to be adsorbed in the native folded state (a 3 × 3 × 3 cube) so that one of its sides is in contact with the surface. The details of the denaturation kinetics are found to be slightly dependent on the choice of the side, but the main qualitative conclusions hold for all the sides. In particular, the kinetics obey approximately the conventional first-order law at T > T_c (T_c is the collapse temperature for solution). With decreasing temperature, below T_c but above T_f (T_f is the folding temperature for solution), deviations appear from the first-order kinetics. For the most interesting temperatures, that is, below T_f, the denaturation kinetics are shown to be qualitatively different from the conventional ones. In particular, the denaturation process occurs via several intermediate steps due to trapping in metastable states. Mathematically, this means that (i) the transition to the denatured state of a given molecule is nonexponential, and (ii) the denaturation process cannot be described by a single rate constant k_r. One should rather introduce a distribution of values of this rate constant (different values of k_r correspond to the transitions to the altered state via different metastable states). Proteins 30:168–176, 1998. © 1998 Wiley-Liss, Inc.