Enhanced accessibility and hydrophobicity of amyloidogenic intermediates of the ß2-microglobulin D76N mutant revealed by high-pressure experiments.

ß2-Microglobulin (ß2m) is the causative protein of dialysis-related amyloidosis. Its unfolding mainly proceeds along the pathway of NC →UC ⇄ UT, whereas refolding follows the UT → IT (→NT) →NC pathway, in which N, I, and U are the native, intermediate, and unfolded states, respectively, with the Pro32 peptidyl-prolyl bond in cis or trans conformation as indicated by the subscript. It is noted that the IT state is a putative amyloidogenic precursor state. Several aggregation-prone variants of ß2m have been reported to date. One of these variants is D76N ß2m, which is a naturally occurring amyloidogenic mutant. To elucidate the molecular mechanisms contributing to the enhanced amyloidogenicity of the mutant, we investigated the equilibrium and kinetic transitions of pressure-induced folding/unfolding equilibria in the wild type and D76N mutant by monitoring intrinsic tryptophan and 1-anilino-8-naphthalene sulfonate fluorescence. An analysis of kinetic data revealed that the different folding/unfolding behaviors of the wild type and D76N mutant were due to differences in the activation energy between the unfolded and the intermediate states as well as stability of the native state, leading to more rapid accumulation of IT state for D76N in the refolding process. In addition, the IT state was found to assume more hydrophobic nature. These changes induced the enhanced amyloidogenicity of the D76N mutant and the distinct pathogenic symptoms of patients. Our results suggest that the stabilization of the native state will be an effective approach for suppressing amyloid fibril formation of this mutant.

Heating during agitation of ß2-microglobulin reveals that supersaturation breakdown is required for amyloid fibril formation at neutral pH.

Amyloidosis-associated amyloid fibrils are formed by denatured proteins when supersaturation of denatured proteins is broken. ß2-Microglobulin (ß2m) forms amyloid fibrils and causes dialysis-related amyloidosis in patients receiving long-term hemodialysis. Although amyloid fibrils of ß2m in patients are observed at neutral pH, formation of ß2m amyloids in vitro has been difficult to discern at neutral pH because of the amyloid-resistant native structure. Here, to further understand the mechanism underlying in vivo amyloid formation, we investigated the relationship between protein folding/unfolding and misfolding leading to amyloid formation. Using thioflavin T assays, CD spectroscopy, and transmission EM analyses, we found that ß2m efficiently forms amyloid fibrils even at neutral pH by heating with agitation at high-salt conditions. We constructed temperature- and NaCl concentration-dependent conformational phase diagrams in the presence or absence of agitation, revealing how amyloid formation under neutral pH conditions is related to thermal unfolding and breakdown of supersaturation. Of note, after supersaturation breakdown and following the law of mass action, the ß2m monomer equilibrium shifted to the unfolded state, destabilizing the native state and thereby enabling amyloid formation even under physiological conditions with a low amount of unfolded precursor. The amyloid fibrils depolymerized at both lower and higher temperatures, resembling cold- or heat-induced denaturation of globular proteins. Our results suggest an important role for heating in the onset of dialysis-related amyloidosis and related amyloidoses.

Amyloid Formation under Complicated Conditions in Which ß2-Microglobulin Coexists with Its Proteolytic Fragments.

Amyloid formation in vivo occurs under complicated conditions in which various amyloidogenic and non-amyloidogenic components coexist, often under crowding. Controversy surrounds the role of additional components under complicated conditions. They have been suggested to accelerate amyloid formation because molecular crowding or interactions with additives increase effective concentrations and, thus, break the supersaturation of amyloidogenic proteins. On the other hand, cellular crowding conditions with various heterogeneous components may retard or prevent amyloid formation because they impede homologous amyloidogenic associations. To elucidate the roles of these additional components, we examined the amyloid formation of ß2-microglobulin (ß2m), a protein responsible for dialysis-related amyloidosis, with a simplified model system in which intact ß2m and its proteolytic peptides coexist. Among the nine proteolytic peptides of ß2m produced in vitro with lysyl endopeptidase, the 22-residue K3 peptide is highly amyloidogenic. The amyloid formation of the K3 peptide, which occurred with a lag time of 1 h at pH 2 and 37 °C, was significantly retarded by the coexistence of ß2m or a mixture of the proteolytic digests. To identify the sites of inhibitory interactions, we performed paramagnetic relaxation enhancement measurements using spin-labeled K3 and uniformly 15N-labeled ß2m with nuclear magnetic resonance detection. The results revealed that K3 interacted weakly with a broad cluster of the hydrophobic residues of ß2m, which accommodated the residues located in some distant sequence, leading to competitive inhibition. The results showed that relatively weak and broad interactions formed a nonproductive complex, implying a role for heterogeneous interactions under complicated conditions.

Heparin-dependent aggregation of hen egg white lysozyme reveals two distinct mechanisms of amyloid fibrillation.

Heparin, a biopolymer possessing high negative charge density, is known to accelerate amyloid fibrillation by various proteins. Using hen egg white lysozyme, we studied the effects of heparin on protein aggregation at low pH, raised temperature, and applied ultrasonic irradiation, conditions under which amyloid fibrillation was promoted. Heparin exhibited complex bimodal concentration-dependent effects, either accelerating or inhibiting fibrillation at pH 2.0 and 60 °C. At concentrations lower than 20 µg/ml, heparin accelerated fibrillation through transient formation of hetero-oligomeric aggregates. Between 0.1 and 10 mg/ml, heparin rapidly induced amorphous heteroaggregation with little to no accompanying fibril formation. Above 10 mg/ml, heparin again induced fibrillation after a long lag time preceded by oligomeric aggregate formation. Compared with studies performed using monovalent and divalent anions, the results suggest two distinct mechanisms of heparin-induced fibrillation. At low heparin concentrations, initial hen egg white lysozyme cluster formation and subsequent fibrillation is promoted by counter ion binding and screening of repulsive charges. At high heparin concentrations, fibrillation is caused by a combination of salting out and macromolecular crowding effects probably independent of protein net charge. Both fibrillation mechanisms compete against amorphous aggregation, producing a complex heparin concentration-dependent phase diagram. Moreover, the results suggest an active role for amorphous oligomeric aggregates in triggering fibrillation, whereby breakdown of supersaturation takes place through heterogeneous nucleation of amyloid on amorphous aggregates.

Non-Native α-Helices in the Initial Folding Intermediate Facilitate the Ordered Assembly of the ß-Barrel in ß-Lactoglobulin.

The roles of non-native α-helices frequently observed in the initial folding stage of ß-sheet proteins have been examined for many years. We herein investigated the residue-level structures of several mutants of bovine ß-lactoglobulin (ßLG) in quenched-flow pH-pulse labeling experiments. ßLG assumes a collapsed intermediate with a non-native α-helical structure (I0) in the early stage of folding, although its native form is predominantly composed of ß-structures. The protection profile in I0 of pseudo-wild type (WT*) ßLG was found to deviate from the pattern of the "average area buried upon folding" (AABUF). In particular, the level of protection at the region of strand A, at which non-native α-helices form in the I0 state, was significantly low compared to AABUF. G17E, the mutant with an increased helical propensity, showed a similar protection pattern. In contrast, the protection pattern for I0 of E44L, the mutant with an increased ß-sheet propensity, was distinct from that of WT* and resembled the AABUF pattern. Transverse relaxation measurements demonstrated that the positions of the residual structures in the unfolded states of these mutants were consistent with those of the protected residues in the respective I0 states. On the basis of the slower conversion of I0 to the native state for E44L to that for WT*, non-native α-helices facilitate the ordered assembly of the ß-barrel by preventing interactions that trap folding.

Supersaturation-limited and Unlimited Phase Transitions Compete to Produce the Pathway Complexity in Amyloid Fibrillation.

Although amyloid fibrils and amorphous aggregates are two types of aggregates formed by denatured proteins, their relationship currently remains unclear. We used ß2-microglobulin (ß2m), a protein responsible for dialysis-related amyloidosis, to clarify the mechanism by which proteins form either amyloid fibrils or amorphous aggregates. When ultrasonication was used to accelerate the spontaneous fibrillation of ß2m at pH 2.0, the effects observed depended on ultrasonic power; although stronger ultrasonic power effectively accelerated fibrillation, excessively strong ultrasonic power decreased the amount of fibrils formed, as monitored by thioflavin T fluorescence. An analysis of the products formed indicated that excessively strong ultrasonic power generated fibrillar aggregates that retained ß-structures but without high efficiency as seeds. On the other hand, when the spontaneous fibrillation of ß2m was induced at higher concentrations of NaCl at pH 2.0 with stirring, amorphous aggregates became more dominant than amyloid fibrils. These apparent complexities in fibrillation were explained comprehensively by a competitive mechanism in which supersaturation-limited reactions competed with supersaturation-unlimited reactions. We link the kinetics of protein aggregation and a conformational phase diagram, in which supersaturation played important roles.

Effects of a reduced disulfide bond on aggregation properties of the human IgG1 CH3 domain.

Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. An IgG1 molecule, which is now mainly used for antibody preparation, consists of a total of 12 immunoglobulin domains. Each domain has one disulfide bond. The CH3 domain is the C-terminal domain of the heavy chain of IgG1. The disulfide bonds of some of the CH3 domains are known to be reduced in recombinant human monoclonal antibodies. The lack of intramolecular disulfide bonds may decrease the stability and increase the aggregation propensity of an antibody molecule. To investigate the effects of a reduced disulfide bond in the CH3 domain on conformational stability and aggregation propensity, we performed several physicochemical measurements including circular dichroism, differential scanning calorimetry (DSC), and 2D NMR. DSC measurements showed that both the stability and reversibility of the reduced form were lower than those of the oxidized form. In addition, detailed analyses of the thermal denaturation data revealed that, although a dominant fraction of the reduced form retained a stable dimeric structure, some fractions assumed a less-specifically associated oligomeric state between monomers. The results of the present study revealed the characteristic aggregation properties of antibody molecules.

Distinguishing crystal-like amyloid fibrils and glass-like amorphous aggregates from their kinetics of formation.

Amyloid fibrils and amorphous aggregates are two types of aberrant aggregates associated with protein misfolding diseases. Although they differ in morphology, the two forms are often treated indiscriminately. ß(2)-microglobulin (ß2m), a protein responsible for dialysis-related amyloidosis, forms amyloid fibrils or amorphous aggregates depending on the NaCl concentration at pH 2.5. We compared the kinetics of their formation, which was monitored by measuring thioflavin T fluorescence, light scattering, and 8-anilino-1-naphthalenesulfonate fluorescence. Thioflavin T fluorescence specifically monitors amyloid fibrillation, whereas light scattering and 8-anilino-1-naphthalenesulfonate fluorescence monitor both amyloid fibrillation and amorphous aggregation. The amyloid fibrils formed via a nucleation-dependent mechanism in a supersaturated solution, analogous to crystallization. The lag phase of fibrillation was reduced upon agitation with stirring or ultrasonic irradiation, and disappeared by seeding with preformed fibrils. In contrast, the glass-like amorphous aggregates formed rapidly without a lag phase. Neither agitation nor seeding accelerated the amorphous aggregation. Thus, by monitoring the kinetics, we can distinguish between crystal-like amyloid fibrils and glass-like amorphous aggregates. Solubility and supersaturation will be key factors for further understanding the aberrant aggregation of proteins.

A common mechanism underlying amyloid fibrillation and protein crystallization revealed by the effects of ultrasonication.

Protein crystals form in supersaturated solutions via a nucleation and growth mechanism. The amyloid fibrils of denatured proteins also form via a nucleation and growth mechanism. This similarity suggests that, although protein crystals and amyloid fibrils are distinct in their morphologies, both processes can be controlled in a similar manner. It has been established that ultrasonication markedly accelerates the formation of amyloid fibrils and simultaneously breaks them down into fragmented fibrils. In this study, we investigated the effects of ultrasonication on the crystallization of hen egg white lysozyme and glucose isomerase from Streptomyces rubiginosus. Protein crystallization was monitored by light scattering, tryptophan fluorescence, and light transmittance. Repeated ultrasonic irradiations caused the crystallization of lysozyme and glucose isomerase after cycles of irradiations. The size of the ultrasonication-induced crystals was small and homogeneous, and their numbers were larger than those obtained under quiescent conditions. Switching off ultrasonic irradiation when light scattering or tryptophan fluorescence began to change resulted in the formation of larger crystals due to the suppression of the further nucleation and fractures in preformed crystals. The results indicate that protein crystallization and amyloid fibrillation are explained on the basis of a common phase diagram in which ultrasonication accelerates the formation of crystals or crystal-like amyloid fibrils as well as fragmentation of preformed crystals or fibrils.

Principal component analysis of chemical shift perturbation data of a multiple-ligand-binding system for elucidation of respective binding mechanism.

Chemical shift perturbations (CSPs) in NMR spectra provide useful information about the interaction of a protein with its ligands. However, in a multiple-ligand-binding system, determining quantitative parameters such as a dissociation constant (K(d) ) is difficult. Here, we used a method we named CS-PCA, a principal component analysis (PCA) of chemical shift (CS) data, to analyze the interaction between bovine ß-lactoglobulin (ßLG) and 1-anilinonaphthalene-8-sulfonate (ANS), which is a multiple-ligand-binding system. The CSP on the binding of ANS involved contributions from two distinct binding sites. PCA of the titration data successfully separated the CSP pattern into contributions from each site. Docking simulations based on the separated CSP patterns provided the structures of ßLG-ANS complexes for each binding site. In addition, we determined the K(d) values as 3.42 × 10⁻4 M² and 2.51 × 10⁻³ M for Sites 1 and 2, respectively. In contrast, it was difficult to obtain reliable K(d) values for respective sites from the isothermal titration calorimetry experiments. Two ANS molecules were found to bind at Site 1 simultaneously, suggesting that the binding occurs cooperatively with a partial unfolding of the ßLG structure. On the other hand, the binding of ANS to Site 2 was a simple attachment without a significant conformational change. From the present results, CS-PCA was confirmed to provide not only the positions and the K(d) values of binding sites but also information about the binding mechanism. Thus, it is anticipated to be a general method to investigate protein-ligand interactions.

GaInAsP/InP MZI waveguide optical isolator integrated with spot size converter.

We fabricated a waveguide optical isolator with a GaInAsP guiding layer integrated with spot size converters (SSCs) for efficient coupling to optical fibers. The isolator is constructed with a Mach-Zehnder interferometer (MZI), which is composed of multi-mode interference (MMI) couplers, as well as nonreciprocal and reciprocal phase shifters. The nonreciprocal phase shifter is constructed with a magneto-optical cladding layer directly bonded to a semiconductor guiding layer. The performance of the GaInAsP waveguide optical isolator was demonstrated with a maximum optical isolation of 28.3 dB at a wavelength of 1558 nm for the TM mode.

The residual structure of acid-denatured ß2 -microglobulin is relevant to an ordered fibril morphology.

ß2 -Microglobulin (ß2m) forms amyloid fibrils in vitro under acidic conditions. Under these conditions, the residual structure of acid-denatured ß2m is relevant to seeding and fibril extension processes. Disulfide (SS) bond-oxidized ß2m has been shown to form rigid, ordered fibrils, whereas SS bond-reduced ß2m forms curvy, less-ordered fibrils. These findings suggest that the presence of an SS bond affects the residual structure of the monomer, which subsequently influences the fibril morphology. To clarify this process, we herein performed NMR experiments. The results obtained revealed that oxidized ß2m contained a residual structure throughout the molecule, including the N- and C-termini, whereas the residual structure of the reduced form was localized and other regions had a random coil structure. The range of the residual structure in the oxidized form was wider than that of the fibril core. These results indicate that acid-denatured ß2m has variable conformations. Most conformations in the ensemble cannot participate in fibril formation because their core residues are hidden by residual structures. However, when hydrophobic residues are exposed, polypeptides competently form an ordered fibril. This conformational selection phase may be needed for the ordered assembly of amyloid fibrils.

A back hydrogen exchange procedure via the acid-unfolded state for a large protein.

A deuterated protein sample is required for nuclear magnetic resonance (NMR) measurements of a large protein because severe signal broadenings occur because of the high molecular weight. The deuterated sample expressed in (2)H(2)O should subsequently be subjected to a back hydrogen exchange at amide groups. To perform the back exchange, the protein molecule is unfolded or destabilized so that internal residues become accessible to the solvent. However, the refolding yield from the destabilized or unfolded state of a large protein is usually low, leading to a dilemma in NMR measurements of large proteins. In our previous paper [Suzuki, M., et al. (2011) Biochemistry 50, 10390-10398], we suggested that an acid-denatured microbial transglutaminase (MTG) consisting of 331 amino acid residues can be recovered effectively under low-salt conditions, escaping from the aggregation-prone intermediate. Here, we demonstrate that proMTG, the pro form of MTG consisting of 376 amino acid residues, can be refolded perfectly from the acid-unfolded state under low-salt conditions, as confirmed by circular dichroism and NMR spectroscopies. By performing the same procedure with a deuterated proMTG expressed in (2)H(2)O, we observed complete back exchanges for internal residues by NMR spectroscopy. Our procedure has potential applications to the back hydrogen exchange of large proteins for NMR measurements.

Destruction of amyloid fibrils of keratoepithelin peptides by laser irradiation coupled with amyloid-specific thioflavin T.

Mutations in keratoepithelin are associated with blinding ocular diseases, including lattice corneal dystrophy type 1 and granular corneal dystrophy type 2. These diseases are characterized by deposits of amyloid fibrils and/or granular non-amyloid aggregates in the cornea. Removing the deposits in the cornea is important for treatment. Previously, we reported the destruction of amyloid fibrils of ß(2)-microglobulin K3 fragments and amyloid ß by laser irradiation coupled with the binding of an amyloid-specific thioflavin T. Here, we studied the effects of this combination on the amyloid fibrils of two 22-residue fragments of keratoepithelin. The direct observation of individual amyloid fibrils was performed in real time using total internal reflection fluorescence microscopy. Both types of amyloid fibrils were broken up by the laser irradiation, dependent on the laser power. The results suggest the laser-induced destruction of amyloid fibrils to be a useful strategy for the treatment of these corneal dystrophies.

Hexafluoroisopropanol induces amyloid fibrils of islet amyloid polypeptide by enhancing both hydrophobic and electrostatic interactions.

Although amyloid fibrils deposit with various proteins, the comprehensive mechanism by which they form remains unclear. We studied the formation of fibrils of human islet amyloid polypeptide associated with type II diabetes in the presence of various concentrations of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) under acidic and neutral pH conditions using CD, amyloid-specific thioflavin T fluorescence, fluorescence imaging with thioflavin T, and atomic force microscopy. At low pH, the formation of fibrils was promoted by HFIP with an optimum at 5% (v/v). At neutral pH in the absence of HFIP, significant amounts of amorphous aggregates formed in addition to the fibrils. The addition of HFIP suppressed the formation of amorphous aggregates, leading to a predominance of fibrils with an optimum effect at 25% (v/v). Under both conditions, higher concentrations of HFIP dissolved the fibrils and stabilized the α-helical structure. The results indicate that fibrils and amorphous aggregates are different types of precipitates formed by exclusion from water-HFIP mixtures. The exclusion occurs through the combined effects of hydrophobic interactions and electrostatic interactions, both of which are strengthened by low concentrations of HFIP, and a subtle balance between the two types of interactions determines whether the fibrils or amorphous aggregates dominate. We suggest a general view of how the structure of precipitates varies dramatically from single crystals to amyloid fibrils and amorphous aggregates.

Inhibition of beta2-microglobulin amyloid fibril formation by alpha2-macroglobulin.

The relationship between various amyloidoses and chaperones is gathering attention. In patients with dialysis-related amyloidosis, α(2)-macroglobulin (α2M), an extracellular chaperone, forms a complex with ß(2)-microglobulin (ß2-m), a major component of amyloid fibrils, but the molecular mechanisms and biological implications of the complex formation remain unclear. Here, we found that α2M substoichiometrically inhibited the ß2-m fibril formation at a neutral pH in the presence of SDS, a model for anionic lipids. Binding analysis showed that the binding affinity between α2M and ß2-m in the presence of SDS was higher than that in the absence of SDS. Importantly, SDS dissociated tetrameric α2M into dimers with increased surface hydrophobicity. Western blot analysis revealed that both tetrameric and dimeric α2M interacted with SDS-denatured ß2-m. At a physiologically relevant acidic pH and in the presence of heparin, α2M was also dissociated into dimers, and both tetrameric and dimeric α2M interacted with ß2-m, resulting in the inhibition of fibril growth reaction. These results suggest that under conditions where native ß2-m is denatured, tetrameric α2M is also converted to dimeric form with exposed hydrophobic surfaces to favor the hydrophobic interaction with denatured ß2-m, thus dimeric α2M as well as tetrameric α2M may play an important role in controlling ß2-m amyloid fibril formation.

A circumventing role for the non-native intermediate in the folding of ß-lactoglobulin.

Folding experiments have suggested that some proteins have kinetic intermediates with a non-native structure. A simple G ̅o model does not explain such non-native intermediates. Therefore, the folding energy landscape of proteins with non-native intermediates should have characteristic properties. To identify such properties, we investigated the folding of bovine ß-lactoglobulin (ßLG). This protein has an intermediate with a non-native α-helical structure, although its native form is predominantly composed of ß-structure. In this study, we prepared mutants whose α-helical and ß-sheet propensities are modified and observed their folding using a stopped-flow circular dichroism apparatus. One interesting finding was that E44L, whose ß-sheet propensity was increased, showed a folding intermediate with an amount of ß-structure similar to that of the wild type, though its folding took longer. Thus, the intermediate seems to be a trapped intermediate. The high α-helical propensity of the wild-type sequence likely causes the folding pathway to circumvent such time-consuming intermediates. We propose that the role of the non-native intermediate is to control the pathway at the beginning of the folding reaction.

Laser-induced propagation and destruction of amyloid beta fibrils.

The amyloid deposition of amyloid beta (Abeta) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of beta(2)-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on Abeta fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed Abeta fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.

Binding energetics of ferredoxin-NADP+ reductase with ferredoxin and its relation to function.

To obtain insight into the motional features of proteins for enzymatic function, we studied binding reactions between ferredoxin-NADP(+) reductase (FNR) and ferredoxin (Fd) using isothermal titration calorimetry and NMR-based magnetic relaxation and hydrogen/deuterium exchange (HD(ex)). Fd-FNR binding was accompanied by endothermic reactions and driven by the entropy gain. Component-wise analysis of the net entropy change revealed that increases in the conformational entropy of the Fd-FNR complex contributed largely to stabilizing the complex. Intriguingly, analyses of magnetic relaxation and HD(ex) rates with X-ray B factor implied that Fd binding led to both structural stiffening and softening of FNR. Enhanced FNR backbone fluctuations suggest favorable contributions to the net conformational entropy. Fd-bound FNR further showed that relatively large-scale motions of the C terminus, a gatekeeper for interactions of NADP(+) (H), were quenched in the closed form, thereby facilitating exit of NADP(+) (H). This can provide a first dynamic structure-based explanation for the negative cooperativity between Fd and NADP(+) (H) via FNR.

Structural dynamics and folding of beta-lactoglobulin probed by heteronuclear NMR.

Bovine beta-lactoglobulin (beta LG) has been one of the most extensively studied proteins in the history of protein science mainly because its abundance in cow's milk makes it readily available to researchers. However, compared to other textbook proteins, progress in the study of beta LG has been slow because of obstacles such as a low reversibility from denaturation linked with thiol-disulfide exchange or monomer-dimer equilibrium preventing a detailed NMR analysis. Recently, the expression of various types of recombinant beta LGs combined with heteronuclear NMR analysis has significantly improved understanding of the physico-chemical properties of beta LG. In this review, we address several topics including pH-dependent structural dynamics, ligand binding, and the complex folding mechanism with non-native intermediates. These unique properties might be brought about by conformational frustration of the beta LG structure, partly attributed to the relatively large molecular size of beta LG. We expect studies with beta LG to continue to reveal various important findings, difficult to obtain with small globular proteins, leading to a more comprehensive understanding of the conformation, dynamics and folding of proteins.