Conformational analysis of intermediates involved in the in vitro folding pathways of recombinant human macrophage colony stimulating factor beta by sulfhydryl group trapping and hydrogen/deuterium pulsed labeling

In vitro oxidative folding of reduced recombinant human macrophage colony stimulating factor beta (rhm-CSFbeta) involves two major events: disulfide isomerization in the monomeric intermediates and disulfide-mediated dimerization. Kinetic analysis of rhm-CSFbeta folding indicated that monomer isomerization is slower than dimerization and is, in fact, the rate-determining step. A time-dependent determination of the number of free cysteines remaining was made after refolding commence. The folding intermediates revealed that rhm-CSFbeta folds systematically, forming disulfide bonds via multiple pathways. Mass spectrometric evidence indicates that native as well as non-native intrasubunit disulfide bonds form in monomeric intermediates. Initial dimerization is assumed to involve formation of disulfide bonds, Cys 157/159-Cys' 157/159. Among six intrasubunit disulfide bonds, Cys 48-Cys 139 and Cys' 48-Cys' 139 are assumed to be the last to form, while Cys 31-Cys' 31 is the last intersubunit disulfide bond that forms. Conformational properties of the folding intermediates were probed by H/D exchange pulsed labeling, which showed the coexistence of noncompact dimeric and monomeric species at early stages of folding. As renaturation progresses, the noncompact dimer undergoes significant structural rearrangement, forming a native-like dimer while the monomer maintains a noncompact conformation.     

Characterization of the folding pathway of recombinant human macrophage-colony stimulating-factor β (rhM-CSF β) by bis-cysteinyl modification and mass spectrometry

Melarsen oxide [p-(4,6-diamino-1,3,5-triazin-2-yl)aminophenylarsonous acid (MEL)], which selectively bridges spatially neighboring bis-cysteinyl residues in (reduced) proteins, was used to trap folding intermediates chemically during 1) time-dependent renaturation of recombinant human macrophage colony-stimulating factor (rhM-CSF); by redox refolding in vitro; 2) reductive unfolding in the presence of the trapping reagent; and 3) denaturing unfolding reactions in urea and guanidinium hydrochloride. Characterization of intermediates from folding and unfolding reactions was performed by electrospray ionization mass spectometry (ESI-MS). In all folding and unfolding reactions a characteristic dimeric intermediate with two attached melarsen oxide (MEL) groups was observed, suggesting that these rhM-CSF β species were important refolding intermediates. These intermediates presented a characteristic “charge structure” in ESI spectra with a most abundant 26+ charged molecular ion whereas the mature homodimeric rhM-CSF β showed a most abundant 23+ molecular ion, indicating that the final product was more compact. The major locations of the two MEL groups were identified by mass spectrometric peptide mapping at cysteine residues C157 and C159 from each monomer. Cysteine residues C7 and C90 were minor modification sites. The mass spectrometric results from the in vitro folding reactions of rhM-CSF β are in agreement with intrinsic tryptophan fluorescence measurements and are consistent with the folding pathway that starts with a fully reduced monomer (R), includes partially folded monomeric intermediates (M) and dimeric intermediates (D), and yields a final product with the native tertiary structure (N): 2R ⇒ 2M ⇒ D ⇒ N. Our results show that selective chemical trapping of bis-thiol groups of proteins with MEL permits study of folding pathways by mass spectrometric structure characterization of intermediates with otherwise transient conformations. Proteins Suppl. 2:50–62, 1998. © 1998 Wiley-Liss, Inc.     

Cold rescue of the thermolabile tailspike intermediate at the junction between productive folding and off-pathway aggregation.

Off-pathway intermolecular interactions between partially folded polypeptide chains often compete with correct intramolecular interactions, resulting in self-association of folding intermediates into the inclusion body state. Intermediates for both productive folding and off-pathway aggregation of the parallel beta-coil tailspike trimer of phage P22 have been identified in vivo and in vitro using native gel electrophoresis in the cold. Aggregation of folding intermediates was suppressed when refolding was initiated and allowed to proceed for a short period at 0 degrees C prior to warming to 20 degrees C. Yields of refolded tailspike trimers exceeding 80% were obtained using this temperature-shift procedure, first described by Xie and Wetlaufer (1996, Protein Sci 5:517-523). We interpret this as due to stabilization of the thermolabile monomeric intermediate at the junction between productive folding and off-pathway aggregation. Partially folded monomers, a newly identified dimer, and the protrimer folding intermediates were populated in the cold. These species were electrophoretically distinguished from the multimeric intermediates populated on the aggregation pathway. The productive protrimer intermediate is disulfide bonded (Robinson AS, King J, 1997, Nat Struct Biol 4:450-455), while the multimeric aggregation intermediates are not disulfide bonded. The partially folded dimer appears to be a precursor to the disulfide-bonded protrimer. The results support a model in which the junctional partially folded monomeric intermediate acquires resistance to aggregation in the cold by folding further to a conformation that is activated for correct recognition and subunit assembly.     

Mapping the folding free energy surface for metal-free human Cu,Zn superoxide dismutase

Mutations at many different sites in the gene encoding human Cu,Zn superoxide dismutase (SOD) are known to be causative agents in amyotrophic lateral sclerosis (ALS). One explanation for the molecular basis of this pathology is the aggregation of marginally soluble, partially structured states whose populations are enhanced in the protein variants. As a benchmark for testing this hypothesis, the equilibrium and kinetic properties of the reversible folding reaction of a metal-free variant of SOD were investigated. Reversibility was achieved by replacing the two non-essential cysteine residues with non-oxidizable analogs, C6A/C111S, to produce apo-AS-SOD. The metal-free pseudo-wild-type protein is folded and dimeric in the absence of chemical denaturants, and its equilibrium folding behavior is well described by an apparent two-state mechanism involving the unfolded monomer and the native dimer. The apparent free energy of folding in the absence of denaturant and at standard state is -20.37(+/- 1.04) kcal (mol dimer)(-1). A global analysis of circular dichroism kinetic traces for both unfolding and refolding reactions, combined with results from small angle X-ray scattering and time-resolved fluorescence anisotropy measurements, supports a sequential mechanism involving the unfolded monomer, a folded monomeric intermediate, and the native dimer. The rate-limiting monomer folding reaction is followed by a near diffusion-limited self-association reaction to form the native dimer. The relative population of the folded monomeric intermediate is predicted not to exceed 0.5% at micromolar concentrations of protein under equilibrium and both strongly unfolding and refolding conditions for metal-free pseudo-wild-type SOD.     

Rough energy landscapes in protein folding: dimeric E. coli Trp repressor folds through three parallel channels

The folding mechanism of the dimeric Escherichia coli Trp repressor (TR) is a kinetically complex process that involves three distinguishable stages of development. Following the formation of a partially folded, monomeric ensemble of species, within 5 ms, folding to the native dimer is controlled by three kinetic phases. The rate-limiting step in each phase is either a non-proline isomerization reaction or a dimerization reaction, depending on the final denaturant concentration. Two approaches have been employed to test the previously proposed folding mechanism of TR through three parallel channels: (1) unfolding double-jump experiments demonstrate that all three folding channels lead directly to native dimer; and (2) the differential stabilization of the transition state for the final step in folding and the native dimer, by the addition of salt, shows that all three channels involve isomerization of a dimeric species. A refined model for the folding of Trp repressor is presented, in which all three channels involve a rapid dimerization reaction between partially folded monomers followed by the isomerization of the dimeric intermediates to yield native dimer. The ensemble of partially folded monomers can be captured at equilibrium by low pH; one-dimensional proton NMR spectra at pH 2.5 demonstrate that monomers exist in two distinct, slowly interconverting conformations. These data provide a potential structural explanation for the three-channel folding mechanism of TR: random association of two different monomeric forms, which are distinguished by alternative packing modes of the core dimerization domain and the DNA-binding, helix-turn-helix, domain. One, perhaps both, of these packing modes contains non-native contacts.     

Evolution of the folding landscape of effector caspases

Caspases are a family of cysteinyl proteases that control programmed cell death and maintain homeostasis in multicellular organisms. The caspase family is an excellent model to study protein evolution because all caspases are produced as zymogens (procaspases [PCPs]) that must be activated to gain full activity; the protein structures are conserved through hundreds of millions of years of evolution; and some allosteric features arose with the early ancestor, whereas others are more recent evolutionary events. The apoptotic caspases evolved from a common ancestor (CA) into two distinct subfamilies: monomers (initiator caspases) or dimers (effector caspases). Differences in activation mechanisms of the two subfamilies, and their oligomeric forms, play a central role in the regulation of apoptosis. Here, we examine changes in the folding landscape by characterizing human effector caspases and their CA. The results show that the effector caspases unfold by a minimum three-state equilibrium model at pH 7.5, where the native dimer is in equilibrium with a partially folded monomeric (PCP-7, CA) or dimeric (PCP-6) intermediate. In comparison, the unfolding pathway of PCP-3 contains both oligomeric forms of the intermediate. Overall, the data show that the folding landscape was first established with the CA and was retained for >650 million years. Partially folded monomeric or dimeric intermediates in the ancestral ensemble provide mechanisms for evolutionary changes that affect stability of extant caspases. The conserved folding landscape allows for the fine-tuning of enzyme stability in a species-dependent manner while retaining the overall caspase–hemoglobinase fold.     

Modeling the role of disulfide bonds in protein folding: Entropic barriers and pathways

The role of disulfide bonds in directing protein folding is studied using lattice models. We find that the stability and the specificity of the disulfide bond interactions play quite different roles in the folding process: Under some conditions, the stability decreases the overall rate of folding; the specificity, however, by yielding a simpler connectivity of intermediates, always increases the rate of folding. This conclusion is intimately related to the selection mechanism entailed by entropic driving forces, such as the loop formation probability, and entropic barriers separating the native and the many native-like metastable states. The folding time is found to be a minimum for a certain range of the effective disulfide bond interaction. Examination of a model, which allows for the formation of disulfide bonded intermediates, suggests that folding proceeds via a threestage multiple pathways kinetics. We show that there are pathways to the native state involving only native-like intermediates, as well as those that are mediated by nonnative intermediates. These findings are interpreted in terms of the appropriate energy landscape describing the barriers connecting low energy conformations. The consistency of our conclusions with several experimental studies is also discussed. © 1995 Wiley-Liss, Inc.     

Conformation of P22 tailspike folding and aggregation intermediates probed by monoclonal antibodies.

The partitioning of partially folded polypeptide chains between correctly folded native states and off-pathway inclusion bodies is a critical reaction in biotechnology. Multimeric partially folded intermediates, representing early stages of the aggregation pathway for the P22 tailspike protein, have been trapped in the cold and isolated by nondenaturing polyacrylamide gel electrophoresis (PAGE) (speed MA, Wang DIC, King J. 1995. Protein Sci 4:900-908). Monoclonal antibodies against tailspike chains discriminate between folding intermediates and native states (Friguet B, Djavadi-Ohaniance L, King J, Goldberg ME. 1994. J Biol Chem 269:15945-15949). Here we describe a nondenaturing Western blot procedure to probe the conformation of productive folding intermediates and off-pathway aggregation intermediates. The aggregation intermediates displayed epitopes in common with productive folding intermediates but were not recognized by antibodies against native epitopes. The nonnative epitope on the folding and aggregation intermediates was located on the partially folded N-terminus, indicating that the N-terminus remained accessible and nonnative in the aggregated state. Antibodies against native epitopes blocked folding, but the monoclonal directed against the N-terminal epitope did not, indicating that the conformation of the N-terminus is not a key determinant of the productive folding and chain association pathway.     

Conformational stability and folding mechanisms of dimeric proteins

The folding of multisubunit proteins is of tremendous biological significance since the large majority of proteins exist as protein-protein complexes. Extensive experimental and computational studies have provided fundamental insights into the principles of folding of small monomeric proteins. Recently, important advances have been made in extending folding studies to multisubunit proteins, in particular homodimeric proteins. This review summarizes the equilibrium and kinetic theory and models underlying the quantitative analysis of dimeric protein folding using chemical denaturation, as well as the experimental results that have been obtained. Although various principles identified for monomer folding also apply to the folding of dimeric proteins, the effects of subunit association can manifest in complex ways, and are frequently overlooked. Changes in molecularity typically give rise to very different overall folding behaviour than is observed for monomeric proteins. The results obtained for dimers have provided key insights pertinent to understanding biological assembly and regulation of multisubunit proteins. These advances have set the stage for future advances in folding involving protein-protein interactions for natural multisubunit proteins and unnatural assemblies involved in disease.     

The Folding Free-Energy Surface of HIV-1 Protease: Insights into the Thermodynamic Basis for Resistance to Inhibitors

Spontaneous mutations at numerous sites distant from the active site of human immunodeficiency virus type 1 protease enable resistance to inhibitors while retaining enzymatic activity. As a benchmark for probing the effects of these mutations on the conformational adaptability of this dimeric β-barrel protein, the folding free-energy surface of a pseudo-wild-type variant, HIV-PR^?, was determined by a combination of equilibrium and kinetic experiments on the urea-induced unfolding/refolding reactions. The equilibrium unfolding reaction was well described by a two-state model involving only the native dimeric form and the unfolded monomer. The global analysis of the kinetic folding mechanism reveals the presence of a fully folded monomeric intermediate that associates to form the native dimeric structure. Independent analysis of a stable monomeric version of the protease demonstrated that a small-amplitude fluorescence phase in refolding and unfolding, not included in the global analysis of the dimeric protein, reflects the presence of a transient intermediate in the monomer folding reaction. The partially folded and fully folded monomers are only marginally stable with respect to the unfolded state, and the dimerization reaction provides a modest driving force at micromolar concentrations of protein. The thermodynamic properties of this system are such that mutations can readily shift the equilibrium from the dimeric native state towards weakly folded states that have a lower affinity for inhibitors but that could be induced to bind to their target proteolytic sites. Presumably, subsequent secondary mutations increase the stability of the native dimeric state in these variants and, thereby, optimize the catalytic properties of the resistant human immunodeficiency virus type 1 protease.     

Zinc binding modulates the entire folding free energy surface of human Cu,Zn superoxide dismutase

Over 100 amino acid replacements in human Cu,Zn superoxide dismutase (SOD) are known to cause amyotrophic lateral sclerosis, a gain-of-function neurodegenerative disease that destroys motor neurons. Supposing that aggregates of partially folded states are primarily responsible for toxicity, we determined the role of the structurally important zinc ion in defining the folding free energy surface of dimeric SOD by comparing the thermodynamic and kinetic folding properties of the zinc-free and zinc-bound forms of the protein. The presence of zinc was found to decrease the free energies of a peptide model of the unfolded monomer, a stable variant of the folded monomeric intermediate, and the folded dimeric species. The unfolded state binds zinc weakly with a micromolar dissociation constant, and the folded monomeric intermediate and the native dimeric form both bind zinc tightly, with subnanomolar dissociation constants. Coupled with the strong driving force for the subunit association reaction, the shift in the populations toward more well-folded states in the presence of zinc decreases the steady-state populations of higher-energy states in SOD under expected in vivo zinc concentrations (approximately nanomolar). The significant decrease in the population of partially folded states is expected to diminish their potential for aggregation and account for the known protective effect of zinc. The ∼ 100-fold increase in the rate of folding of SOD in the presence of micromolar concentrations of zinc demonstrates a significant role for a preorganized zinc-binding loop in the transition-state ensemble for the rate-limiting monomer folding reaction in this β-barrel protein.     

In vitro folding of methionine‐arginine human lyspro‐proinsulin S‐sulfonate—Disulfide formation pathways and factors controlling yield

We investigated the in vitro folding of an oxidized proinsulin (methionine‐arginine human lyspro‐proinsulin S‐sulfonate), using cysteine as a reducing agent at 5°C and high pH (10.5–11). Folding intermediates were detected and characterized by means of matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS), reversed‐phase chromatography (RPC), size‐exclusion chromatography, and gel electrophoresis. The folding kinetics and yield depended on the protein and cysteine concentrations. RPC coupled with MALDI‐MS analyses indicated a sequential formation of intermediates with one, two, and three disulfide bonds. The MALDI‐MS analysis of Glu‐C digested, purified intermediates indicated that an intra‐A‐chain disulfide bond formed first among A6, A7, and A11. Various non‐native intra‐A (A20 with A6, A7, or A11), intra‐B (between B7 and B19), and inter‐A‐B disulfide bonds were observed in the intermediates with two disulfide bonds. The intermediates with three disulfide bonds had mainly the non‐native intra‐A and intra‐B bonds. At a cysteine‐to‐proinsulin‐SH ratio of 3.5, all intermediates with the non‐native disulfide bonds were converted to properly folded proinsulin via disulfide bond reshuffling, which was the slowest step. Aggregation via the formation of intermolecular disulfide bonds of early intermediates was the major cause of yield loss. At a higher cysteine‐to‐proinsulin‐SH ratio, some intermediates and folded MR‐KPB‐hPI were reduced to proteins with thiolate anions, which caused unfolding and even more yield loss than what resulted from aggregation of the early intermediates. Reducing protein concentration, while keeping an optimal cysteine‐to‐protein ratio, can improve folding yield significantly. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Disulfide bonds and protein folding

The applications of disulfide-bond chemistry to studies of protein folding, structure, and stability are reviewed and illustrated with bovine pancreatic ribonuclease A (RNase A). After surveying the general properties and advantages of disulfide-bond studies, we illustrate the mechanism of reductive unfolding with RNase A, and discuss its application to probing structural fluctuations in folded proteins. The oxidative folding of RNase A is then described, focusing on the role of structure formation in the regeneration of the native disulfide bonds. The development of structure and conformational order in the disulfide intermediates during oxidative folding is characterized. Partially folded disulfide species are not observed, indicating that disulfide-coupled folding is highly cooperative. Contrary to the predictions of "rugged funnel" models of protein folding, misfolded disulfide species are also not observed despite the potentially stabilizing effect of many nonnative disulfide bonds. The mechanism of regenerating the native disulfide bonds suggests an analogous scenario for conformational folding. Finally, engineered covalent cross-links may be used to assay for the association of protein segments in the folding transition state, as illustrated with RNase A.     

Mutational analysis of the BPTI folding pathway: II. Effects of aromatic-->leucine substitutions on folding kinetics and thermodynamics.

The rates of the individual steps in the disulfide-coupled folding and unfolding of eight BPTI variants, each containing a single aromatic to leucine amino acid replacement, were measured. From this analysis, the contributions of the four phenylalanine and four tyrosine residues to the stabilities of the native protein and the disulfide-bonded folding intermediates were determined. While the substitutions were found to destabilize the native protein by 2 to 7 kcal/mol, they had significantly smaller effects on the intermediates that represent the earlier stages of folding, even when the site of the substitution was located within the ordered regions of the intermediates. These results suggest that stabilizing interactions contribute less to conformational stability in the context of a partially folded intermediate than in a fully folded native protein, perhaps because of decreased cooperativity among the individual interactions. The kinetic analysis also provides new information about the transition states associated with the slowest steps in folding and unfolding, supporting previous suggestions that these transition states are extensively unfolded. Although the substitutions caused large changes in the distribution of folding intermediates and in the rates of some steps in the folding pathway, the kinetically-preferred pathway for all of the variants involved intramolecular disulfide rearrangements, as observed previously for the wild-type protein. These results suggest that the predominance of the rearrangement mechanism reflects conformational constraints present relatively early in the folding pathway.     

Intermediates control domain swapping during folding of p13suc1

The 13-kDa protein p13(suc1) has two folded states, a monomer and a structurally similar domain-swapped dimer formed by exchange of a beta-strand. The refolding reaction of p13(suc1) is multiphasic, and in this paper we analyze the kinetics as a function of denaturant and protein concentration and compare the behavior of wild type and a set of mutants previously designed with dimerization propensities that span 9 orders of magnitude. We show that the folding reactions of wild type and all mutants produce the monomer predominantly despite their very different equilibrium behavior. However, the addition of low concentrations of denaturant in the refolding buffer leads to thermodynamic control of the folding reaction with products that correspond to the wild type and mutant equilibrium dimerization propensities. We present evidence that the kinetic control in the absence of urea arises because of the population of the folding intermediates. Intermediates are usually considered to be detrimental to folding because they slow down the reaction; however, our work shows that intermediates buffer the monomeric folding pathway against the effect of mutations that favor the nonfunctional, dimeric state at equilibrium.     

Roles of dimerization in folding and stability of ketosteroid isomerase from Pseudomonas putida biotype B

Equilibrium and kinetic analyses have been performed to elucidate the roles of dimerization in folding and stability of KSI from Pseudomonas putida biotype B. Folding was reversible in secondary and tertiary structures as well as in activity. Equilibrium unfolding transition, as monitored by fluorescence and ellipticity measurements, could be modeled by a two-state mechanism without thermodynamically stable intermediates. Consistent with the two-state model, one dimensional (1D) NMR spectra and gel-filtration chromatography analysis did not show any evidence for a folded monomeric intermediate. Interestingly enough, Cys 81 located at the dimeric interface was modified by DTNB before unfolding. This inconsistent result might be explained by increased dynamic motion of the interface residues in the presence of urea to expose Cys 81 more frequently without the dimer dissociation. The refolding process, as monitored by fluorescence change, could best be described by five kinetic phases, in which the second phase was a bimolecular step. Because <30% of the total fluorescence change occurred during the first step, most of the native tertiary structure may be driven to form by the bimolecular step. During the refolding process, negative ellipticity at 225 nm increased very fast within 80 msec to account for >80% of the total amplitude. This result suggests that the protein folds into a monomer containing most of the alpha-helical structures before dimerization. Monitoring the enzyme activity during the refolding process could estimate the activity of the monomer that is not fully active. Together, these results stress the importance of dimerization in the formation and maintenance of the functional native tertiary structure.     

Kinetic characterization of early intermediates in the folding of E. coli tryptophan-synthase beta 2 subunit

This report describes the use of fluorescence energy transfer between an intrinsic energy donor (tryptophan 177) and two chemically added acceptors to study intermediates in the folding of the beta 2 subunit of E. coli tryptophan-synthase. Two early folding steps are thus identified and characterized. One is very rapid (its rate constant at 12 degrees C is 0.02 sec-1) and corresponds to the folding of the N-terminal domain into a structure whose overall features approximate well those of the native domain. The second step is somewhat slower (its rate constant at 12 degrees C is 0.008 sec-1) and involves a conformational rearrangement of the N-terminal domain brought about by the interactions between the N- and C-terminal domains within a monomeric beta chain. This brings to five the number of intermediates which have been identified and ordered on the folding pathway of the dimeric beta 2 subunit.     

Folding of the yeast prion protein Ure2: kinetic evidence for folding and unfolding intermediates.

The Saccharomyces cerevisiae non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The N-terminal prion domain (PrD) of Ure2 is required for prion activity in vivo and amyloid formation in vitro. However, the molecular mechanism of the prion-like activity remains obscure. Here we measure the kinetics of folding of Ure2 and two N-terminal variants that lack all or part of the PrD. The kinetic folding behaviour of the three proteins is identical, indicating that the PrD does not change the stability, rates of folding or folding pathway of Ure2. Both unfolding and refolding kinetics are multiphasic. An intermediate is populated during unfolding at high denaturant concentrations resulting in the appearance of an unfolding burst phase and "roll-over" in the denaturant dependence of the unfolding rate constants. During refolding the appearance of a burst phase indicates formation of an intermediate during the dead-time of stopped-flow mixing. A further fast phase shows second-order kinetics, indicating formation of a dimeric intermediate. Regain of native-like fluorescence displays a distinct lag due to population of this on-pathway dimeric intermediate. Double-jump experiments indicate that isomerisation of Pro166, which is cis in the native state, occurs late in refolding after regain of native-like fluorescence. During protein refolding there is kinetic partitioning between productive folding via the dimeric intermediate and a non-productive side reaction via an aggregation prone monomeric intermediate. In the light of this and other studies, schemes for folding, aggregation and prion formation are proposed.     

Oxidative folding of Amaranthus alpha-amylase inhibitor: disulfide bond formation and conformational folding

Oxidative folding is the fusion of native disulfide bond formation with conformational folding. This complex process is guided by two types of interactions: first, covalent interactions between cysteine residues, which transform into native disulfide bridges, and second, non-covalent interactions giving rise to secondary and tertiary protein structure. The aim of this work is to understand both types of interactions in the oxidative folding of Amaranthus alpha-amylase inhibitor (AAI) by providing information both at the level of individual disulfide species and at the level of amino acid residue conformation. The cystine-knot disulfides of AAI protein are stabilized in an interdependent manner, and the oxidative folding is characterized by a high heterogeneity of one-, two-, and three-disulfide intermediates. The formation of the most abundant species, the main folding intermediate, is favored over other species even in the absence of non-covalent sequential preferences. Time-resolved NMR and photochemically induced dynamic nuclear polarization spectroscopies were used to follow the oxidative folding at the level of amino acid residue conformation. Because this is the first time that a complete oxidative folding process has been monitored with these two techniques, their results were compared with those obtained at the level of an individual disulfide species. The techniques proved to be valuable for the study of conformational developments and aromatic accessibility changes along oxidative folding pathways. A detailed picture of the oxidative folding of AAI provides a model study that combines different biochemical and biophysical techniques for a fuller understanding of a complex process.