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Conformational Properties Relevant to the Amyloidogenicity of #-Microglobulin Analyzed Using Pressure- and Salt-Dependent Chemical Shift Data 2
Kazumasa Sakurai, Akihiro Maeno, Young-Ho Lee, and Kazuyuki Akasaka J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b11408 • Publication Date (Web): 03 Jan 2019 Downloaded from http://pubs.acs.org on January 3, 2019
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The Journal of Physical Chemistry Pressure and salt effect on chemical shift data of β2-microglobulin
Conformational Properties Relevant to the Amyloidogenicity of β2Microglobulin Analyzed Using Pressure- and Salt-Dependent Chemical Shift Data
Kazumasa Sakurai†,‡,*, Akihiro Maeno§, Young-Ho Lee‡,¶ and Kazuyuki Akasaka# †High
Pressure Protein Research Center, Institute of Advanced Technology, Kindai University,
930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan ‡Institute
§
for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Laboratory of Chemistry, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka 573-
1010, Japan ¶Protein
Structure Research Group, Division of Bioconvergence Analysis, Korea Basic Science
Institute, Chungcheongbuk-do 28119, South Korea #Kyoto
Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Japan
*Corresponding Author: High Pressure Protein Research Center, Institute of Advanced Technology, Kindai University, 930 Nishimitani, Kinokawa, Wakayama 649-6493, Japan. Phone: +81-736-77-0345 (ex 5004). E-mail:
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Pressure and salt effect on chemical shift data of β2-microglobulin
Abstract β2-Microglobulin (β2m) is associated with dialysis-related amyloidosis. In vitro experiments have shown that β2m forms amyloid fibrils at acidic pHs in the presence of moderate concentrations of salt. Previous studies suggested that acid-denatured β2m has hydrophobic residual structure, and exposure of the hydrophobic residues enhances the association with seeds or other β2m monomers. However, the nature of the residual structure relevant to its amyloidogenicity remains to be investigated. To understand the structural properties of acid-denatured β2m and the role of salt, we investigated pressure- and salt-concentration-dependent conformational changes by NMR and other methods. Here, pressure was utilized to characterize the conformers existing in a conformational equilibrium at ambient pressure. Obtained pressure- and salt-concentrationdependent chemical shift data were simultaneously subjected to principal component analysis to characterize individual conformational change events. Unexpectedly, the addition of salt induced an expansion of the β2m molecule, which likely resulted from the exclusion of the N-terminal region from the hydrophobic cluster region. The dissected chemical shift patterns for the saltinduced conformational change and other experimental data indicated that this conformational change caused a rigidification in the intrinsic hydrophobic cluster, leading to the observed amyloidogenicity.
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The Journal of Physical Chemistry Pressure and salt effect on chemical shift data of β2-microglobulin
Introduction A number of proteins have been reported to form amyloid fibrils, which are supramolecular assemblies exhibiting a long, unbranched morphology ~10 nm in diameter. Amyloid fibril formation is associated with a number of diseases, including Alzheimer's disease, type II diabetes, and dialysis-related amyloidosis.1 Among identified amyloidogenic proteins, β2-microglobulin (β2m) has been one of the most extensively investigated protein. β2m is the light chain of MHC class I. In patients on long-term hemodialysis, released β2m cannot be degraded because of dysfunction of the kidneys and forms amyloid fibrils in the synovial membrane of the carpal tunnel, causing dialysis-related amyloidosis.2 In the process of fibril formation, a partially structured conformation is generally considered to be necessary for amyloidogenicity.3, 4 At acidic pH in the presence of preformed fibril seed and a moderate concentration of salt (e.g., ~100 mM NaCl), where β2m forms rigid amyloid fibrils,5 β2m also contains some hydrophobic residual structures around the intramolecular disulfide bond (between Cys25 and Cys80) and the position 60's aromatic amino acid-rich region (Trp60– Phe70).6, 7 It was also known that the residual structure was stabilized by addition of salt, evidenced by the enhanced transverse relaxation rate (R2) upon the increase in the salt concentration.6, 7 In addition, we reported previously that these regions transiently assume a fully unfolded state, and
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Pressure and salt effect on chemical shift data of β2-microglobulin
suggested that this transient state is relevant to the nucleation and extension steps of its fibril growth.7 However, the partially structured state should be in equilibrium with such a transient state in acidic conditions, so that it is quite hard to detect this state solely and measure its structural information with conventional spectroscopic methods. To overcome this problem, we used pressure. It has been demonstrated that pressure can alter the relative populations of conformers that exist in equilibrium through differences in free energy (ΔG) based on the difference in their partial molar volume (ΔVm).8 There are several reports of pressure application to preformed amyloid fibrils of various proteins.9-13 Generally, the results of these studies show the dissociation of fibrils in high-pressure conditions, indicating that the fibril structure has larger Vm than the monomeric state. Of note, Chatani et al.11, 12 reported that β2m molecules in fibrils formed at ambient pressure can adopt different types of conformation in moderate-pressure conditions. However, the structural properties of the monomeric state in amyloidogenic conditions remain to be investigated. Thus, we performed high-pressure-NMR measurements to investigate the nature of the possible residual structure of β2m that is expected to be present in equilibrium with the fully denatured monomeric state. Another important factor for amyloidogenicity is the ionic strength of the solvent. Various amyloidogenic conditions reported so far basically include moderate concentrations of salt, whereas relatively high concentrations of salt tend to induce non-specific aggregation.5, 14, 15 From
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The Journal of Physical Chemistry Pressure and salt effect on chemical shift data of β2-microglobulin
these observations, a moderate screening of electrostatic repulsions by ions likely enhances the ordered association of precursor molecules. However, the effect of salt on conformational changes relevant to amyloidogenicity have not been widely considered. Historically, salt is important factor for the conformation of protein molecules.16, 17 Thus, salt effects on not only the intermolecular repulsions but also monomeric structure should be investigated carefully for a thorough understanding of amyloidogenicity. NMR is an appropriate tool to investigate condition-dependent conformational changes in protein molecules. When conformational fluctuations are rapid with respect to the NMR time scale (τ
