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This paper is dedicated to the memory of Oleg B. Ptitsyn (1929-1999) and presents an answer to his question: “What is the role of conserved non-functional residues in protein folding?”. This answer follows from the experimental works of three labs. The role of non-functional but conserved residues of apomyoglobin (apoMb) in the formation of the native protein fold in the molten globule state has been experimentally revealed. This research proves that the non-functional but conserved residues of apoMb are necessary for the formation and maintenance of the correct topological arrangement of the main elements in the apoMb secondary structure already in the early folding intermediate.

Biomacromolecular folding kinetics involves fast folding events and broad timescales. Current techniques face limitations in either the required time resolution or the observation window. In this study, we developed the TeZla micromixer, integrating Tesla and Zigzag microstructures with a multistage velocity descending strategy. TeZla achieves a significant short mixing dead time (40 µs) and a wide time window covering four orders of magnitude (up to 300 ms). Using this unique micromixer, we explored the folding landscape of c-Myc G4 and its noncanonical-G4 derivatives with different loop lengths or G-vacancy sites. Our findings revealed that c-Myc can bypass folding intermediates and directly adopt a G4 structure in the cation-deficient buffer. Moreover, we found that the loop length and specific G-vacancy site could affect the folding pathway and significantly slow down the folding rates. These results were also cross-validated with real-time NMR and circular dichroism. In conclusion, TeZla represents a versatile tool for studying biomolecular folding kinetics, and our findings may ultimately contribute to the design of drugs targeting G4 structures. [ABSTRACT FROM AUTHOR]

To investigate how disulfide bonds can impact protein energy landscapes, we surveyed the effects of adding or removing a disulfide in two β‐lactamase enzymes, TEM‐1 and CTX‐M‐9. The homologs share a structure and 38% sequence identity, but only TEM‐1 contains a native disulfide bond. They also differ in thermodynamic stability and in the number of states populated at equilibrium: CTX‐M‐9 is two‐state whereas TEM‐1 has an additional intermediate state. We hypothesized that the disulfide bond is the major underlying determinant for these observed differences in their energy landscapes. To test this, we removed the disulfide bridge from TEM‐1 and introduced a disulfide bridge at the same location in CTX‐M‐9. This modest change to sequence modulates the stabilities—and therefore populations—of TEM‐1's equilibrium states and, more surprisingly, creates a novel third state in CTX‐M‐9. Unlike TEM‐1's partially folded intermediate, this third state is a higher‐order oligomer with reduced cysteines that retains the native fold and is fully active. Sub‐denaturing concentrations of urea shifts the equilibrium to the monomeric form, allowing the disulfide bond to form. Interestingly, comparing the stability of the oxidized monomer with a variant lacking cysteines reveals the disulfide is neither stabilizing nor destabilizing in CTX‐M‐9, in contrast with the observed stabilization in TEM‐1. Thus, we can conclude that engineering disulfide bonds is not always an effective stabilization strategy even when analogous disulfides exist in more stable structural homologs. This study also illustrates how homo‐oligomerization can result from a small number of mutations, suggesting complex formation might be easily accessed during a protein family's evolution. [ABSTRACT FROM AUTHOR]

Protein folding is essential for a polypeptide chain to acquire its proper structure and function. Globins are a superfamily of ubiquitous heme-binding α-helical proteins whose function is principally to regulate oxygen homoeostasis. In this review, we explore the hierarchical helical formation in the globin proteins apomyoglobin and leghemoglobin, and we discuss the existence of non-native and misfolded structures occurring during the course of folding to its native state. This review summarizes the research aimed at characterizing and comparing the equilibrium and kinetic intermediates, as well as delineating the complete folding pathway at a molecular level, in order to answer the following questions: "What is the mechanism of misfolding via a folding intermediate? Does the non-native structure stabilize the contemporary intermediate structure? Does the non-native structure induce slower folding?" The role of the non-native structures in the folding intermediate related to misfolding is also discussed. [ABSTRACT FROM AUTHOR]

Investigations of protein folding have largely involved the use of disulfide-containing proteins, since the disulfide-coupled folding of proteins allows folding intermediates to be trapped and their conformations determined. However, studies of the folding mechanisms of mid-size proteins face several problems, one of which is that detecting folding intermediates is difficult. Therefore, to solve this issue, a novel peptide reagent, maleimidohexanoyl-Arg5-Tyr-NH2, was designed and applied to the detection of folding intermediates of model proteins. BPTI was chosen as a model small protein to estimate the ability of the novel reagent to detect folding intermediates. In addition, a precursor protein (prococoonase) of Bombyx mori cocoonase was used as a model mid-size protein. Cocoonase is classified as a serine protease and has a high homology with trypsin. We recently found that the propeptide sequence of prococoonase (proCCN) is important for the folding of cocoonase. However, it was difficult to study the folding pathway of proCCN since the folding intermediates could not be separated on a reversed-phase HPLC (RP-HPLC). Therefore, to separate the folding intermediates by RP-HPLC, the novel labeling reagent was used to accomplish this for proCCN. The results indicated that the peptide reagent allowed the intermediates to be captured, separated on SDS-PAGE, and analyzed by RP-HPLC without the occurrence of undesirable disulfide-exchange reactions during the labeling reactions. The peptide reagent reported herein is a practical tool for investigating the mechanisms of disulfide-coupled folding of mid-size proteins. [ABSTRACT FROM AUTHOR]

The K-homology (KH) domains are small, structurally conserved domains found in proteins of different origins characterized by a central conserved βααβ "core" and a GxxG motif in the loop between the two helices of the KH core. In the eukaryotic KHI type, additional αβ elements decorate the "core" at the C-terminus. Proteins containing KH domains perform different functions and several diseases have been associated with mutations in these domains, including those in the fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein crucial for the control of RNA metabolism whose lack or mutations lead to fragile X syndrome (FXS). Among missense mutations, the R138Q substitution is in the KH0 degenerated domain lacking the classical GxxG motif. By combining equilibrium and kinetic experiments, we present a characterization of the folding mechanism of the KH0 domain from the FMRP wild-type and of the R138Q variant showing that in both cases the folding mechanism implies the accumulation of an on-pathway transient intermediate. Moreover, by exploiting a battery of biophysical techniques, we show that the KH0 domain has the propensity to form amyloid-like aggregates in mild conditions in vitro and that the R138Q mutation leads to a general destabilization of the protein and to an increased fibrillogenesis propensity. [ABSTRACT FROM AUTHOR]

AAA+ proteolytic machines play essential roles in maintaining and rebalancing the cellular proteome in response to stress, developmental cues, and environmental changes. Of the five AAA+ proteases in Escherichia coli, FtsH is unique in its attachment to the inner membrane and its function in degrading both membrane and cytosolic proteins. E. coli dihydrofolate reductase (DHFR) is a stable and biophysically well‐characterized protein, which a previous study found resisted FtsH degradation despite the presence of an ssrA degron. By contrast, we find that FtsH degrades DHFR fused to a long peptide linker and ssrA tag. Surprisingly, we also find that FtsH degrades DHFR with shorter linkers and ssrA tag, and without any linker or tag. Thus, FtsH must be able to recognize a sequence element or elements within DHFR. We find that FtsH degradation of DHFR is noncanonical in the sense that it does not rely upon recognition of an unstructured polypeptide at or near the N‐terminus or C‐terminus of the substrate. Results using peptide‐array experiments, mutant DHFR proteins, and fusion proteins suggest that FtsH recognizes an internal sequence in a species of DHFR that is partially unfolded. Overall, our findings provide insight into substrate recognition by FtsH and indicate that its degradation capacity is broader than previously reported. [ABSTRACT FROM AUTHOR]

The formation of ordered Z (Glu342Lys) α1‐antitrypsin polymers in hepatocytes is central to liver disease in α1‐antitrypsin deficiency. In vitro experiments have identified an intermediate conformational state (M*) that precedes polymer formation, but this has yet to be identified in vivo. Moreover, the mechanism of polymer formation and their fate in cells have been incompletely characterised. We have used cell models of disease in conjunction with conformation‐selective monoclonal antibodies and a small molecule inhibitor of polymerisation to define the dynamics of polymer formation, accumulation and secretion. Pulse‐chase experiments demonstrate that Z α1‐antitrypsin accumulates as short‐chain polymers that partition with soluble cellular components and are partially secreted by cells. These precede the formation of larger, insoluble polymers with a longer half‐life (10.9 ± 1.7 h and 20.9 ± 7.4 h for soluble and insoluble polymers, respectively). The M* intermediate (or a by‐product thereof) was identified in the cells by a conformation‐specific monoclonal antibody. This was completely abrogated by treatment with the small molecule, which also blocked the formation of intracellular polymers. These data allow us to conclude that the M* conformation is central to polymerisation of Z α1‐antitrypsin in vivo; preventing its accumulation represents a tractable approach for pharmacological treatment of this condition; polymers are partially secreted; and polymers exist as two distinct populations in cells whose different dynamics have likely consequences for the aetiology of the disease. [ABSTRACT FROM AUTHOR]

AAI, the major alpha-amylase inhibitor (AAI) present in the seeds of the Mexican crop plant Amaranthus hypocondriacus is a 32-residue-long polypeptide with three disulfide bridges. Its structure is most closely related to the plant amylase inhibitor subfamily of knottins characterized by a topological knot formed by one disulfide bridge threading through a loop formed by the peptide chain as well as a short three-stranded beta sandwich core. AAI is specific against insect amylases and does not act on corresponding human or mammalian enzymes. It was found that the oxidative folding of AAI seems to follow a hirudine-like pathway with many non-native intermediates, but notably it proceeds through a major folding intermediate (MFI) that contains a vicinal disulfide bridge. Based on a review of the pertinent literature, the known vicinal disulfides in native proteins as well as well as the network of disulfide interchanges, we propose that MFI is a kinetic trap corresponding to a compact molten globule-like state which constrains the peptide chain to a smaller number of conformations that in turn can be rapidly funneled toward the native state.

Single-stranded noncoding regulatory RNAs, as exemplified by bacterial riboswitches, are highly dynamic. The conformational dynamics allow the riboswitch to reach maximum switching efficiency under appropriate conditions. Here we characterize the conformational dynamics of preQ 1 riboswitches from mesophilic and thermophilic bacterial species at various temperatures. With the integrative use of small-angle X-ray scattering, NMR, and molecular dynamics simulations, we model the ensemble-structures of the preQ 1 riboswitch aptamers without or with a ligand bound. We show that the preQ 1 riboswitch is sufficiently dynamic and fluctuating among multiple folding intermediates only near the physiological temperature of the microorganism. The hierarchical folding dynamics of the RNA involves the docking of 3′-tail to form a second RNA helix and the helical stacking to form an H-type pseudoknot structure. Further, we show that RNA secondary and tertiary dynamics can be modulated by temperature and by the length of an internal loop. The coupled equilibria between RNA folding intermediates are essential for preQ 1 binding, and a four-state exchange model can account for the change of ligand-triggered switching efficiency with temperature. Together, we have established a relationship between the hierarchical dynamics and riboswitch function, and illustrated how the RNA adapts to high temperature. Unlabelled Image • preQ 1 riboswitch adopts an H-type pseudoknot structure upon cognate ligand binding. • 3′-Tail alternates between undocked and docked states with helix P2 formed. • RNA P1 and P2 helices alternate between unstacked and stacked conformations. • The length of an internal loop L2 affects RNA secondary and tertiary dynamics. • Hierarchical dynamics allows efficient switching at the "physiological" temperature. [ABSTRACT FROM AUTHOR]

Background: Human aldose reductase (hAR) is the first and rate-limiting enzyme of the polyol pathway. For the development of secondary complications of diabetes in chronic hyperglycemic conditions, one of the critical factors is the increased flux of glucose through the polyol pathway. Due to this clinical implication, hAR attracted considerable attention from the drug discovery perspective. In spite of extensive characterization in the context of biochemical and structural aspects, we know very little about the unfolding behavior of hAR. This study reports equilibrium unfolding studies of hAR. Methods: We carried out thermal denaturation and chemical-induced equilibrium unfolding studies of hAR monitored by circular dichroism and fluorescence spectroscopy. Results: Thermal denaturation studies presented a classical picture of two-state unfolding from native to the denatured state. The data was used to derive thermodynamic parameters and study the thermostability of hAR. Chemical induced equilibrium unfolding studies led us to discover an intermediate state, which gets populated at 3.5-4.0 M and 0.7-2.0 M of urea and GuHCl, respectively. Thermodynamic parameters derived from chemical-induced unfolding are in agreement with those obtained from thermal denaturation of hAR. Conclusion: This study revealed that aldose reductase unfolds from native to the unfolded state via an intermediate. Assessment of the thermodynamic stability of native, intermediate, and unfolded states shows that significant energy barriers separate these states, which ensures the cooperativity of unfolding. As hAR functions in cells that are under osmotic and oxidative stress, these in vitro findings may have implications for its native conformation under the physiological state.

Background: Human aldose reductase (hAR) converts glucose to sorbitol under hyperglycemic conditions. Aldose reductase is first and rate limiting enzyme of polyol pathway. Under hyperglycemia, increased flux of glucose through this pathway has been implicated in development of secondary complication in diabetes. Due to this clinical implication, aldose reductase attracted considerable attention from drug discovery perspective. In spite of extensive characterization of the biochemical and structural context, little is known about the unfolding behavior of aldose reductase. This study reports equilibrium unfolding studies of human aldose reductase. Methods: We carried out thermal and chemical induced equilibrium unfolding studies of human aldose reductase monitored by circular dichroism and tryptophan and ANS fluorescence spectroscopy. Results: Thermal unfolding studies present a classical picture of two state unfolding from native to unfolded state. The data was used to derive thermodynamic parameters and study thermostability of aldose reductase. Urea and GuHCl induced equilibrium unfolding studies led us to discover an intermediate state, which gets populated at 3.5-4.0 M and 0.7-2 M of urea and GuHCl, respectively. Thermodynamic parameters from chemical induced unfolding are in agreement with those obtained from thermal unfolding. Conclusion: This study revealed that aldose reductase unfolds from native to unfolded state via an intermediate. Assessment of thermodynamic stability of native, intermediate and unfolded state shows that three states are separated by significant energy barriers that ensure cooperativity of unfolding. As hAR functions in cells which are under osmotic and oxidative stress, these in vitro findings may have implications for its native conformation under physiological state.

Bromodomains (BRDs) are small protein domains often present in large multidomain proteins involved in transcriptional regulation in eukaryotic cells. They currently represent valuable targets for the development of inhibitors of aberrant transcriptional processes in a variety of human diseases. Here we report urea-induced equilibrium unfolding experiments monitored by circular dichroism (CD) and fluorescence on two structurally similar BRDs: BRD2(2) and BRD4(1), showing that BRD4(1) is more stable than BRD2(2). Moreover, we report a description of their kinetic folding mechanism, as obtained by careful analysis of stopped-flow and temperature-jump data. The presence of a high energy intermediate for both proteins, suggested by the non-linear dependence of the folding rate on denaturant concentration in the millisec time regime, has been experimentally observed by temperature-jump experiments. Quantitative global analysis of all the rate constants obtained over a wide range of urea concentrations, allowed us to propose a common, three-state, folding mechanism for these two BRDs. Interestingly, the intermediate of BRD4(1) appears to be more stable and structurally native-like than that populated by BRD2(2). Our results underscore the role played by structural topology and sequence in determining and tuning the folding mechanism.

A new crystal form of lysozyme with a very low solvent content (26.35%) has been obtained in the orthorhombic space group P212121 (with unit‐cell parameters a = 30.04, b = 51.68, c = 61.53 Å). The lysozyme structure obtained from these crystals does not show the typical overall fold. Instead, major conformational changes take place in some elements of the secondary structure and in the hydrophobic core of the protein. At the end of the central α‐helix (α2), Glu35 is usually buried in the catalytic site and shows an abnormally high pKa value, which is key to the activity of the enzyme. The high pKa value of this glutamate residue is favoured by the hydrophobic environment, particularly by its neighbour Trp108, which is important for structural stability and saccharide binding. In this new structure, Trp108 shows a 90° rotation of its side chain, which results in the rearrangement of the hydrophobic core. Conformational changes also result in the exposure of Glu35 to the solvent, which impairs the catalytic site by increasing the distance between Glu35 and Asp52 and lowering the pKa value of the glutamate. Altogether, this new lysozyme structure reveals major conformational changes in the hydrophobic core and catalytic site that might play a role in the folding and bactericidal function of the protein. [ABSTRACT FROM AUTHOR]

Drug discovery using small molecule inhibitors is reaching a stalemate due to low selectivity, adverse off-target effects and inevitable failures in clinical trials. Conventional chemical screening methods may miss potent small molecules because of their use of simple but outdated kits composed of recombinant enzyme proteins. Non-canonical inhibitors targeting a hidden pocket in a protein have received considerable research attention. Kii and colleagues identified an inhibitor targeting a transient pocket in the kinase DYRK1A during its folding process and termed it FINDY. FINDY exhibits a unique inhibitory profile; that is, FINDY does not inhibit the fully folded form of DYRK1A, indicating that the FINDY-binding pocket is hidden in the folded form. This intriguing pocket opens during the folding process and then closes upon completion of folding. In this review, we discuss previously established kinase inhibitors and their inhibitory mechanisms in comparison with FINDY. We also compare the inhibitory mechanisms with the growing concept of “cryptic inhibitor-binding sites.” These sites are buried on the inhibitor-unbound surface but become apparent when the inhibitor is bound. In addition, an alternative method based on cell-free protein synthesis of protein kinases may allow the discovery of small molecules that occupy these mysterious binding sites. Transitional folding intermediates would become alternative targets in drug discovery, enabling the efficient development of potent kinase inhibitors.

To investigate how disulfide bonds can impact protein energy landscapes, we surveyed the effects of adding or removing a disulfide in two β-lactamase enzymes, TEM-1 and CTX-M-9. The homologs share a structure and 38% sequence identity, but only TEM-1 contains a native disulfide bond. They also differ in thermodynamic stability and in the number of states populated at equilibrium: CTX-M-9 is two-state whereas TEM-1 has an additional intermediate state. We hypothesized that the disulfide bond is the major underlying determinant for these observed differences in their energy landscapes. To test this, we removed the disulfide bridge from TEM-1 and introduced a disulfide bridge at the same location in CTX-M-9. This modest change to sequence modulates the stabilities-and therefore populations-of TEM-1's equilibrium states and, more surprisingly, creates a novel third state in CTX-M-9. Unlike TEM-1's partially folded intermediate, this third state is a higher-order oligomer with reduced cysteines that retains the native fold and is fully active. Sub-denaturing concentrations of urea shifts the equilibrium to the monomeric form, allowing the disulfide bond to form. Interestingly, comparing the stability of the oxidized monomer with a variant lacking cysteines reveals the disulfide is neither stabilizing nor destabilizing in CTX-M-9, in contrast with the observed stabilization in TEM-1. Thus, we can conclude that engineering disulfide bonds is not always an effective stabilization strategy even when analogous disulfides exist in more stable structural homologs. This study also illustrates how homo-oligomerization can result from a small number of mutations, suggesting complex formation might be easily accessed during a protein family's evolution., (© 2023 The Protein Society.)

In general, small proteins rapidly fold on the timescale of milliseconds or less. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin, we demonstrate that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record 2D and 3D NMR spectra of the unfolded protein at atmospheric pressure, providing residuespecific information on the folding process. 15N and 13C chemical shifts measured immediately after dropping the pressure from 2.5 kbar (favoring unfolding) to 1 bar (native) are close to the random-coil chemical shifts observed for a large, disordered peptide fragment of the protein. However, 15N relaxation data show evidence for rapid exchange, on a ~100-µs timescale, between the unfolded state and unstable, structured states that can be considered as failed folding events. The NMR data also provide direct evidence for parallel folding pathways, with approximately one-half of the protein molecules efficiently folding through an on-pathway kinetic intermediate, whereas the other half fold in a single step. At protein concentrations above ~300 µM, oligomeric off-pathway intermediates compete with folding of the native state. [ABSTRACT FROM AUTHOR]

Transthyretin (TTR) is an extracellular protein able to deposit into well-defined protein aggregates called amyloid, in pathological conditions known as senile systemic amyloidosis, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and leptomeningeal amyloidosis. At least three distinct partially folded states have been described for TTR, including the widely studied amyloidogenic state at mildly acidic pH. Here, we have used fluorescence resonance energy transfer (FRET) experiments in a monomeric variant of TTR (M-TTR) and in its W41F and W79F mutants, taking advantage of the presence of a unique, solvent-exposed, cysteine residue at position 10, that we have labelled with a coumarin derivative (DACM, acceptor), and of the two natural tryptophan residues at positions 41 and 79 (donors). Trp41 is located in an ideal position as it is one of the residues of β-strand C, whose degree of unfolding is debated. We found that the amyloidogenic state at low pH has the same FRET efficiency as the folded state at neutral pH in both M-TTR and W79F-M-TTR, indicating an unmodified Cys10-Trp41 distance. The partially folded state populated at low denaturant concentrations also has a similar FRET efficiency, but other spectroscopic probes indicate that it is distinct from the amyloidogenic state at acidic pH. By contrast, the off-pathway state accumulating transiently during refolding has a higher FRET efficiency, indicating non-native interactions that reduce the Cys10-Trp41 spatial distance, revealing a third distinct conformational state. Overall, our results clarify a negligible degree of unfolding of β-strand C in the formation of the amyloidogenic state and establish the concept that TTR is a highly plastic protein able to populate at least three distinct conformational states. [ABSTRACT FROM AUTHOR]

Background: Proteins with low sequence identity but almost identical tertiary structure and function have been valuable to uncover the relationship between sequence, tertiary structure, folding mechanism and functions. Two homologous chemokines, CCL11 and CCL24, with low sequence identity but similar tertiary structure and function, provide an excellent model system for respective studies. Results: The kinetics and thermodynamics of the two homologous chemokines were systematically characterized. Despite their similar tertiary structures, CCL11 and CCL24 show different thermodynamic stability in guanidine hydrochloride titration, with D50% = 2.20 M and 4.96 M, respectively. The kinetics curves clearly show two phases in the folding/unfolding processes of both CCL11 and CCL24, which suggests the existence of an intermediate state in their folding/unfolding processes. The folding pathway of both CCL11 and CCL24 could be well described using a folding model with an on-pathway folding intermediate. However, the folding kinetics and stability of the intermediate state of CCL11 and CCL24 are obviously different. Conclusion: Our results suggest homologous proteins with low sequence identity can display almost identical tertiary structure, but very different folding mechanisms, which applies to homologues in the chemokine protein family, extending the general applicability of the above observation. [ABSTRACT FROM AUTHOR]

Bovine α-lactalbumin (αLA) has four disulfide (SS) bonds in the native form (N). On the oxidative folding pathways of this protein, two specific SS folding intermediates, i.e., (61-77, 73-91) and des[6-120], which have two and three native SS bonds, respectively, accumulate predominantly in the presence of Ca2+. In this study, we reinvestigated the pathways using a water-soluble cyclic selenoxide reagent, trans-3,4-dihydroxyselenolane oxide (DHSox), as a strong and quantitative oxidant to oxidize the fully reduced form (R). In the presence of ethylenediaminetetraacetic acid (EDTA) (under a metal-free condition), SS formation randomly proceeded, and N did not regenerate. On the other hand, two specific SS intermediates transiently generated in the presence of Ca2+. These intermediates could be assigned to (61-77, 73-91) and des[6-120] having two common SS bonds, i.e., Cys61-Cys77 and Cys73-Cys91, near the calcium binding pocket of the β-sheet domain. Much faster folding to N was observed in the presence of Mn2+, whereas Na+, K+, Mg2+, and Zn2+ did not affect the pathways. The two key intermediates were susceptible to temperature and a denaturant. The oxidative folding pathways revealed were significantly different from those of hen egg white lysozyme, which has the same SS-bonding pattern as αLA, suggesting that the folding pathways of SS-containing proteins can alter depending on the amino acid sequence and other factors, even when the SS-bond topologies are similar to each other. [ABSTRACT FROM AUTHOR]

Here we discuss studies of the structure, folding, oligomerization and amyloid fibril formation of several proline mutants of human stefin B, which is a protein inhibitor of lysosomal cysteine cathepsins and a member of the cystatin family. The structurally important prolines in stefin B are responsible for the slow folding phases and facilitate domain swapping (Pro 74) and loop swapping (Pro 79). Moreover, our findings are compared to β2-microglobulin, a protein involved in dialysis-related amyloidosis. The assessment of the contribution of proline residues to the process of amyloid fibril formation may shed new light on the critical molecular events involved in conformational disorders. [ABSTRACT FROM AUTHOR]