Laboratory of Protein Folding


Associate Professor
Lee, Young-Ho

Functional Biomolecular Biology


Institute for Protein Research



Research Theme

Protein folding is a process in which an extended polypeptide chain acquires a unique folded conformation with a biological activity. Clarifying the mechanism of protein folding is essential for improving our understanding of the structure and function of proteins. It is also important because many critical biological processes and disease states involve protein misfolding and aggregation reactions. We are studying the mechanism of protein folding and misfolding, as well as its biological significance and related topics, with various physico-chemical and visualization methods, including circular dichroism, fluorescence, NMR, IR, X-ray scattering, calorimetry, ultracentrifuge, atomic force microscopy and fluorescence microscopy.

Folding and conformational stability of β-lactoglobulin

Bovine β-lactoglobulin (βLG) 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 βLG has been slow because its low reversibility from denaturation due to thiol-disulfide exchange and monomer-dimer equilibrium prevent a detailed NMR analysis. Previously, the expression of recombinant βLGs combined with heteronuclear NMR analysis has significantly improved our understanding of the physico-chemical properties of βLG. We study various topics including pH-dependent structural dynamics, ligand binding, and the complex folding mechanism with non-native intermediates [Sakurai et al., Biochim. Biophys. Acta, 1790, 527-537 (2009)]. In particular, it is a useful model for clarifying the mechanism of the β to α transition (Figure 1), which was observed in various critical biological processes. These unique properties might be brought about by conformational frustrations in the βLG structure, partly attributed to its relatively large molecular size. We expect studies with βLG to reveal various important findings, which are difficult to be obtained with small globular proteins, leading to a more comprehensive understanding of the conformational dynamics and folding of proteins.

Amyloid Fibril Formation of Proteins and Peptides

Amyloid fibrils are supramolecular assemblies exhibiting a long unbranched fibrillar morphology ~10 nanometers in diameter (Figure 2), the deposition of which is associated with over 30 degenerative diseases including Alzheimer’s disease, prion disease, and dialysis-related amyloidosis. Dialysis-related amyloidosis is a serious problem for patients receiving hemodialysis for more than ten years. β2-Microglobulin (β2-m), a light chain of the type I major histocompatibility antigen (MHC-1) with 99 amino acid residues, is the main component of the amyloid fibrils deposited in the synovia of the carpal tunnel. Because of its relatively small size, which makes the study of protein folding and misfolding possible, and because of its clinical importance, β2-m is one of the most extensively studied amyloidogenic proteins. We have been studying the conformation and stability of amyloid fibrils of recombinant human β2-m. We are also studying amyloid fibrils of amyloid β peptide, islet amyloid polypeptide (IAPP), and kelatoepithlin, associated with Alzheimer’s disease, type II diabetes, blinding corneal dystrophies, respectively. We previously reported that ultrasonication is useful for preparing minimum-sized and relatively monodispersed amyloid fibrils of β2-m, which is achieved by the free energy minimum under competition between ultrasonication-induced fibril production and breakdown. In this year, we reported that the ultrasonication produced β2-m fibrils with a minimum molecular size, their structural and chemical properties remaining unchanged. [Yoshimura et al., Protein Sci., 12, 2347-2355 (2010)] With the fragmented β2m fibrils, we succeeded in observing directly the solution NMR signals of β2-m fibrils, which were broadened beyond detection for the unfragmented fibrils. We have been studying the effects of a laser irradiation to the amyloid fibrils. We reported the laser irradiation-dependent destruction of preformed fibrils of kelatoepithelin [Ozawa et al., J. Biol. Chem., 286, 10856-10863 (2011)]. More interesting finding is that, although extensive irradiation destroyed the preformed Aβ fibrils, irradiation during the formation of fibrils resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils, leading to a bell-shaped profile of propagation against the laser energy [Yagi et al., J. Biol. Chem., 285, 19660-19667 (2010)]. The explosive propagation was caused by an increase in the number of active ends because of breakage. We propose that the effects of the irradiation are determined by a balance between the laser-induced acceleration of propagation and the destruction. These results suggest that the laser-induced destruction of amyloid fibrils coupled with an amyloid-specific dye is useful in the treatment or prevention of amyloid-related diseases, for which no effective method has yet been established.

Conformation and folding ferredoxin-NADP+ reductase

Ferredoxin-NADP+ reductase (FNR) is a key enzyme in photosynthetic electron transfer process. In collaboration with Professors Hase and Ikegami of IPR, we study the conformation and folding of FNR. Previously, to address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pDrs 8.0 and 6.0, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible [Lee et al., J. Biol. Chem., 282, 5959-5967 (2007)]. To obtain further insight into the nature of motions required for enzymatic function, we have been investigating the binding reaction between ferredoxin-NADP+ reductase (FNR) and ferredoxin (Fd) using isothermal titration calorimetry and NMR-based nuclear relaxation and hydrogen/deuterium exchange (HDex). We try to provide a dynamic structure-based explanation for the negative cooperativity between Fd and NADP+(H) via FNR.

0Figure 1. The folding process of bovie β-lactoglobulin.

1Figure 2. An image of amyloid fibrils taken by TIRFM.


Ozawa D, Kaji Y, Yagi H, Hasegawa K, Kawakami T, Naiki H and Goto Y. Destruction of amyloid fibrils of keratoepithelin peptides by laser irradiation coupled with amyloid-specific thiofravin T J. Biol. Chem. 286 , 10856 - 10863 (2011)

Ozawa D, Hasegawa K, Lee Y-H, Sakurai K, Yanagi K, Ookoshi T, Goto Y and Naiki H. Inhibition of β2-Microglobulin amyloid fibril formation by α2-Macroglobulin J. BIol. Chem. 286 , 9668 - 9676 (2011)

Konuma T, Kimura T, Matsumoto S, Goto Y, Fujisawa T, Fersht AR and Takahashi S. Time-Resolved Small-Angle X-ray Scattering Study of the Folding Dynamics of Barnase J. Mol. Biol. 405 , 1284 - 1294 (2011)

Konuma T, Chatani E, Yagi M, Sakurai K, Ikagemi T, Naiki H and Goto Y. Kinetic Intermediates of β2-Microglobulin Fibril Elongation Probed by Pulse-Labeling H/D Exchange Combined with NMR Analysis J. Mol. Biol. 405 , 851 - 862 (2011)

Yoshimura Y, Sakurai K, Lee Y-H, Ikegami T, Chatani E, Naiki H and Goto Y. Direct Observation of Minimum-Sized Amyloid Fibrils Using Solution NMR Spectroscopy Protein Sci. 12 , 2347 - 2355 (2010)

Chatani E, Ohnishi R, Konuma T, Sakurai K, Naiki H and Goto Y. Pre-steady-state kinetic analysis of the elongation of amyloid fibrils of β2-microglobulin with tryptophan mutagenesis J. Mol. Biol. 400 , 1057 - 1066 (2010)


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