Biophysical Chem.

Proteins are the primary vehicles with diverse biological functions and catalytic activities. The function of a protein is determined by its three-dimensional structure. In our group, we focus on two topics:

1) Structural characterization of proteins

X-ray crystallography has provided high resolution structures of numerous rigid proteins. However, X-ray crystallography is inapplicable to flexible proteins without well-defined structures. Although nuclear magnetic resonance (NMR) spectroscopy can be more adequate for characterization of such proteins, its application is limited to small proteins. Therfore, new experimental techniques other than these conventional methods are necessary to understand the structure of flexible and disordered proteins. We use mass spectrometry (MS)-based techniques to reveal the structures of various proteins in solution.

2) Understanding the folding properties of proteins

Folding of proteins is strictly controlled in body. Molecular chaperons guide the folding process of newly synthesized proteins, and unfolded proteins are degraded in the proteasomes. However, proteins can be damaged and misfolded, and show abnormal behaviors such as amyloidosis. We seek to understand how protein misfolding occur and reveal the environmental factors that accelerate or decelerate such processes.

Many proteins are associated with lipid bilayer cell membranes. Some proteins are only partially inserted inside the membrane, while some other proteins are fully immersed. Protein-membrane interactions can render proteins structures to be different from that in aqueous solution. For example, membrane-associated proteins typically show helix-rich secondary structures while interaction with lipid membranes. Despite the importance of protein-membrane interactions, structural characterization of proteins in lipid membranes is challenging because the environment is highly heterogeneous for conventional techniques. We combine various solution-phase methods and MS-based techniques to characterize the structures of membrane associated proteins. We utilize circular dichroism (CD) spectroscopy and small-angle X-ray scattering (SAXS) in solution, and apply hydrogen-deuterium exchange (HDX) and ion mobility mass spectrometry (IM-MS) for more detailed characterization.

Mass spectrometry (MS) has become an important tool in elucidating structure of protein and protein complexes. The development of electrospray ionization (ESI) has enabled the transfer of biomolecules from solution to the gas phase with the covalent bonds and noncovalent interactions intact. Most notably, many studies have shown the correlation between the solution and gas-phase protein structures, which allowed the MS-based structural biology to prosper. However, many aspects of the protein transfer via ESI is still not well revealed, and this obscures direct application of MS-based structural biology without complementary data. We aim to reveal the various factors governing the ionization process of proteins, to provide more rigid methodologies that allow to correlate the gas-phase protein structures with the protein structures in solution. We utilize various solution-phase experimental methods including circular dichroism (CD) spectroscopy and small-angle X-ray scattering (SAXS) to probe the protein structure in solution, and use ion mobility mass spectrometry (IM-MS) to understand the gas-phase protein structures. We also combine molecular dynamics simulations (MD) to more deeply understand how a protein behaves in solution, in the gas phase, and in between.

Amyloidogenic proteins tend to be aggregated into unbranched β-sheet rich amyloid fibril in vivo. This phenomenon is closely related to amyloidogenic diseases such as Alzheimer’s disease, Parkinson’s disease, type 2 diabetes, and spongiform encephalopathy. Amyloid fibrillation is generally initiated by exposing hydrophobic residues of amyloidogenic proteins, and self-assembly process is promoted by hydrophobic interaction of the residues. Both factors are considered as a driving force for amyloid fibrillation, but their correlation is not yet fully understood. To understand the correlation between environmental factors and protein fibrillation, we have investigated protein-organic solvent interaction during fibrillation process of insulin. Formamide derivatives (formamide, N-methyl formamide, N,N-dimethyl formamide) were used as a model system to systematically control denaturing power and hydrophobicity of solvents. Thioflavin T (ThT) assay and transmission electron microscopy (TEM) shows that the binary mixture of water and organic solvents promotes fibrillation rates and β-sheet abundance of insulin fibril. Solution small-angle X-ray scattering (SAXS) combined with molecular dynamics (MD) simulation suggested that the structural conversion of B11-B17 core residues from α-helix to random coil is crucial for the fibrillation kinetics of insulin. Differential scanning calorimetry (DSC) results revealed that protein-protein interaction for amyloid fibrillation is governed by solvophobicity of the exposed core residues. Our study suggests that both denaturation and solvophobic interaction of the core residues are prerequisite for the fibrillation process of insulin.