Kazumasa Sakurai

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. β2-microglobulin (β2m), 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.

Salt‐dependent monomer–dimer equilibrium of bovine β‐lactoglobulin at pH 3

Although bovine β‐lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer–dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO4 was more effective than other salts by about 20‐fold, suggesting that anion binding is responsible for the salt‐induced dimer formation, as observed for acid‐unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO4, the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (ΔHD) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric β‐lactoglobulin at pH 3.0 with NaClO4. The net heat effects after subtraction of the heat of salt dilution, corresponding to ΔHD, were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the ΔHD value at 20°C and the heat capacity change (ΔCp) of dimer formation. In both NaCl and NaClO4, the obtained ΔCp value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed ΔCp values were consistent with the calculated value from the X‐ray dimeric structure using a method of accessible surface area. These results indicated that monomer–dimer equilibrium of β‐lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.

Structural dynamics and folding of β-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 cows 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.

Principal component analysis of the pH-dependent conformational transitions of bovine β-lactoglobulin monitored by heteronuclear NMR

To clarify the pH-dependent conformational transitions of proteins, we propose an approach in which structural changes monitored by heteronuclear sequential quantum correlation (HSQC) spectroscopy were analyzed by using a principal component analysis (PCA). We use bovine β-lactoglobulin, a protein widely used in protein folding studies, as a target. First, we measured HSQC spectra at various pH values and subjected them to a PCA. The analysis revealed three apparent transitions with pKa values of 2.9, 4.9, and 6.8, consistent with previous reports using different methods. Next, Gdn-HCl-induced unfolding was examined by measuring tryptophan fluorescence at various pH values. Between pH 2 and 8, β-lactoglobulin exhibited a number of structural transitions as well as changes in stability represented by the free energy change of unfolding, ΔGU. By combining the NMR and fluorescence results, the change in ΔGU was suggested to result from the decreased pKa of some acidic residues. Notably, the native state at neutral pH is destabilized by deprotonation of Glu-89, leading to an increase in the relative population of the intermediate. Thus, the PCA of pH-dependent HSQC spectra provides a more comprehensive understanding of the stability and function of proteins.

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.

Conformational stability of amyloid fibrils of β2-microglobulin probed by guanidine-hydrochloride-induced unfolding

Although the stability of globular proteins has been studied extensively, that of amyloid fibrils is scarcely characterized. β2‐microglobulin (β2‐m) is a major component of the amyloid fibrils observed in patients with dialysis‐related amyloidosis. We studied the effects of guanidine hydrochloride on the amyloid fibrils of β2‐m, revealing a cooperative unfolding transition similar to that of the native state. The stability of amyloid fibrils increased on the addition of ammonium sulfate, consistent with a role of hydrophobic interactions. The results indicate that the analysis of unfolding transition is useful to obtain insight into the structural stability of amyloid fibrils.

Kinetic Coupling of Folding and Prolyl Isomerization of β2-Microglobulin Studied by Mutational Analysis

Beta(2)-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis, adopts a typical immunoglobulin domain fold with the N-terminal peptide bond of Pro32 in a cis isomer. The refolding of beta2-m is limited by the slow trans-to-cis isomerization of Pro32, implying that intermediates with a non-native trans-Pro32 isomer are precursors for the formation of amyloid fibrils. To obtain further insight into the Pro-limited folding of beta2-m, we studied the Gdn-HCl-dependent unfolding/refolding kinetics using two mutants (W39 and P32V beta2-ms) as well as the wild-type beta2-m. W39 beta2-m is a triple mutant in which both of the authentic Trp residues (Trp60 and Trp95) are replaced by Phe and a buried Trp common to other immunoglobulin domains is introduced at the position of Leu39 (i.e., L39W/W60F/W95F). W39 beta2-m exhibits a dramatic quenching of fluorescence upon folding, enabling a detailed analysis of Pro-limited unfolding/refolding. On the other hand, P32V beta2-m is a mutant in which Pro32 is replaced by Val, useful for probing the kinetic role of the trans-to-cis isomerization of Pro32. A comparative analysis of the unfolding/refolding kinetics of these mutants including three types of double-jump experiments revealed the prolyl isomerization to be coupled with the conformational transitions, leading to apparently unusual kinetics, particularly for the unfolding. We suggest that careful consideration of the kinetic coupling of unfolding/refolding and prolyl isomerization, which has tended to be neglected in recent studies, is essential for clarifying the mechanism of protein folding and, moreover, its biological significance.

Laser-induced propagation and destruction of amyloid β fibrils

The amyloid deposition of amyloid β (Aβ) 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 β2-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 Aβ fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed Aβ 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.

Flow-induced Alignment of Amyloid Protofilaments Revealed by Linear Dichroism

Amyloid fibrils underlying various serious amyloidoses including Alzheimer and prion diseases form characteristic deposits in which linear fibrils with an unbranched and rigid morphology associate laterally or radially, e.g. radial senile amyloid plaques of amyloid β. To clarify the formation of these high order amyloid deposits, studying the rheology is important. A 22-residue K3 peptide fragment of β2-microglobulin, a protein responsible for dialysis-related amyloidosis, forms long and homogeneous protofilament-like fibrils in 20% (v/v) 2,2,2-trifluoroethanol and 10 mm HCl (pH ∼2). Here, using circular dichroism and linear dichroism, we observed the flow-induced alignment of fibrils. Analysis of far- and near-UV linear dichroism spectra suggested that both the net π-π* transition moment of the backbone carbonyl group and Lb transition moment of the Tyr26 side chain are oriented in parallel to the fibril axis, revealing the structural details of amyloid protofilaments. Moreover, the intensities of flow-induced circular dichroism or linear dichroism signals depended critically on the length and type of fibrils, suggesting that they are useful for detecting and characterizing amyloid fibrils.

Supersaturation-limited and unlimited phase transitions compete to produce the pathway complexity in amyloid fibrillation

Background: Relationship between amyloid fibrils and amorphous aggregates has not yet been elucidated. Results: A competitive mechanism of amyloid fibrillation and amorphous aggregation reproduced the observed aggregation kinetics of β2-microglobulin. Conclusion: Apparent complexities in amyloid fibrillation are explained assuming supersaturation-limited crystal-like amyloid fibrils and unlimited glass-like amorphous aggregates. Significance: Linkage of the kinetics of protein aggregation and a conformational phase diagram improves the understanding of protein aggregation. Although amyloid fibrils and amorphous aggregates are two types of aggregates formed by denatured proteins, their relationship currently remains unclear. We used β2-microglobulin (β2m), 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 β2m 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 β-structures but without high efficiency as seeds. On the other hand, when the spontaneous fibrillation of β2m 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.