Ultrasonication-dependent production and breakdown lead to minimum-sized amyloid fibrils

Abstract

Because of the insolubility and polymeric properties of amyloid fibrils, techniques used conventionally to analyze protein structure and dynamics have often been hampered. Ultrasonication can induce the monomeric solution of amyloidogenic proteins to form amyloid fibrils. However, ultrasonication can break down preformed fibrils into shorter fibrils. Here, combining these 2 opposing effects on ␤2-microglobulin (␤2-m), a protein responsible for dialysisrelated amyloidosis, we present that ultrasonication pulses are useful for preparing monodispersed amyloid fibrils of minimal size with an average molecular weight of Ϸ1,660,000 (140-mer). The production of minimal and monodispersed fibrils is achieved by the free energy minimum under competition between fibril production and breakdown. The small homogeneous fibrils will be of use for characterizing the structure and dynamics of amyloid fibrils, advancing molecular understanding of amyloidosis.␤2-microglobulin ͉ dialysis-related amyloidosis ͉ protein misfolding ͉ analytical ultracentrifugation A myloid fibrils are supramolecular assemblies exhibiting a long unbranched fibrillar morphology Ϸ10 nanometers in diameter, the deposition of which is associated with Ͼ30 degenerative diseases including Alzheimer's disease, prion disease, and dialysis-related amyloidosis (1-3). The past decade has seen progress in our biophysical understanding of amyloid fibrils using various approaches including solution and solid state NMR (4-6) and X-ray crystallography (7,8). However, the polymeric properties of amyloid fibrils, where huge size and heterogeneous nature result in insoluble and noncrystalline assemblies, are an obstacle to techniques such as X-ray crystallography, solution NMR spectroscopy and other conventional spectroscopic measurements. To overcome the analytical problems confronting studies of amyloid fibrils, it is worth establishing a strategy for producing monodispersed samples of amyloid fibrils. If amyloid fibrils of a well-defined molecular weight and improved solubility are formed reproducibly, a more general application of various types of fibril samples to a series of preexisting spectroscopic measurements will be accomplished.As a strategy to produce amyloid fibrils of uniform and minimal size, ultrasonication has several potential applications. Although ultrasonication was originally used to prepare seeds from preformed fibrils (9), which were further applied to the amplification of infectious prion proteins (10, 11), ultrasonication-dependent fragmentation is becoming an important approach to analyzing the properties of fibrils (12, 13). However, its strong effect of agitation has recently been found to accelerate the fibril nucleation of several proteins and peptides (14-18). Interestingly, amyloid fibrils produced by ultrasonicationinduced fibrillation were very short with apparently similar lengths as determined by AFM (15,19), suggesting that short fibrils of homogeneous molecular size are formed efficiently under ultrasonication in combination with the...

Ultrasonication: An Efficient Agitation for Accelerating the Supersaturation-Limited Amyloid Fibrillation of Proteins

“…38) As expected, the overshoot became less pronounced as the pulse interval became shorter, and with a 6-s quiescent period, the size of amyloid fibrils monitored by sedimentation velocity with analytical ultracentrifugation reached equilibrium almost directly. The fibril size obtained with ultrasonic pluses of 1 min and quiescent periods of 6 s was the smallest revealing 38) a sedimentation coefficient value of 17 S, where the unit S is a Svedberg unit, defined as 10 À13 s.…”

“…To gain more information about the shape and molecular weight of monodispersed amyloid fibrils, AFM, electron microscopy (EM), and a sedimentation equilibrium analysis were performed with the fibrils formed at 2-min intervals 38) AFM images indicated uniform fibril lengths, which were further confirmed with EM images. No serious change in conformation or chemical properties was detected by far-UV circular dichroism, high performance liquid chromatography, and mass spectroscopic analysis.…”

“…The results obtained with a microplate were consistent with those obtained with an Eppendorf tube. 35,38) However, as anticipated from the relatively wide microplate used (i.e., 6 Â 10 cm 2 with 9 Â 12 wells), the lag-time for fibrillation (t lag ) varied significantly depending on the location in the microplate, and the final fluorescence intensity also varied, indicating that the formation of fibrils is predominantly determined by the ultrasonication power.…”

“…3(a)]. To confirm this, Chatani et al 38) examined ultrasonication-induced fibrillation by applying repetitive pulses of a fixed period of 1 min with various quiescent incubation periods from 6 s to 9 min.…”

Interplay Between Mechanochemistry and Sonochemistry

“…Without sonication, the energy landscape is broad and additional break down or re-agglomeration mechanisms would result in a wide range of fibril lengths. The elongational field triggered by sonication modulates the free energy profile and leads to monodispersed fibrils with uniform size, as shown in Figure 22 (average molecular weight of 1,660,000 daltons or 140 mer) [114]. Molecular dynamics simulations have recently been performed to ascertain the disruption of amyloid fibrils, which sheds light onto the cavitational event [115].…”