Hidetaka Nakata

A protease inhibitor discovery method using fluorescence correlation spectroscopy with position-specific labeled protein substrates

We developed novel substrates for protease activity evaluation by fluorescence correlation spectroscopy (FCS). Substrates were labeled in a position-specific manner with a fluorophore near the N terminus and included a C-terminal, 30 kDa, highly soluble protein (elongation factor Ts [EF-Ts]). The C-terminal protein enhanced the substrate peptide solubility and increased the molecular weight, enabling sensitive detection by FCS. Using the labeled substrates, caspase-3 and matrix metalloproteinase-9 (MMP-9) activities were confirmed by FCS. To demonstrate the suitability of this FCS-based assay for high-throughput screening, we screened various chemical compounds for MMP-9 inhibitors. The screening results confirmed the inhibitory activity of one compound and also revealed another potential MMP-9 inhibitor. Thus, this combination of position-specific labeled protein substrates and FCS may serve as a useful tool for evaluating activities of various proteases and for protease inhibitor screening.

High-sensitivity single molecule fluorescence detection using scanning single-molecule counting

A new, simple technique for single molecule fluorescence detection has been developed and detection limit of less than 100 aM fluorophores has been demonstrated. The technique, similarly to Fluorescence Correlation Spectroscopy (FCS) and other related techniques, uses confocal optics, but differs in that it detects individual molecules crossing the inside of a scanning confocal volume without using statistical techniques as applied in FCS or similar methods. The scanning speed of the confocal volume is higher than the Brownian motion speed of the molecules. Thus, the time evolution of the light intensity data reflects the confocal volume intensity profile, which clearly shows the crossing of single molecules. The estimated total scanning volume enables the concentration or the density of molecules to be obtained. In addition, information related to the rotational and translational diffusion of the molecule was obtained for the purpose of identifying different molecules. It was shown that utilizing the plural characteristic properties of molecules passing through a confocal volume makes possible the discrimination of different molecules. The proposed technique is based on the simple principle of counting molecules one by one using a scanning confocal volume, and is hereafter referred to as Scanning Single-Molecule Counting (SSMC).