Here is a brief overview of the techniques we use to characterize enzymes and small molecules. However, the Yokoyama lab is a hypothesis- and question-driven research group, meaning we don’t confine ourselves to a specific set of techniques or experiments. We continuously broaden our experimental scope, adopting new research methods and collaborating with experts in the field. Each student and postdoc has the opportunity to learn a range of state-of-the-art techniques, providing a comprehensive research experience.
Enzyme kinetics: Kinetics of enzyme-catalyzed reactions are essential to understanding the function and mechanism of enzymes. We have extensive experience in steady-state and pre-steady-state kinetics, such as stopped-flow and rapid quenching techniques. We use these methods in combination with other biochemical and biophysical techniques.
Protein preparation: Protein preparation is central to all of our projects. We have extensive skills in the preparation of various proteins, including metalloenzymes that require anaerobic techniques, and membrane proteins that require detergent solubilization. We also use various heterologous protein expression systems to purify these challenging proteins.
Small molecule and carbohydrate characterization: Characterization of bacterial metabolites (natural products) and enzyme reaction products is central to our study. Therefore, we use a variety of analytical methodologies, including NMR, HPLC (Hitachi and Agilent), MS, SEC (size exclusion chromatography), HPAEC-PAD (high-performance anion-exchange chromatography coupled with pulsed amperometric detection), and LCMS.
Microbiology: We routinely grow bacteria and fungi (both nonpathogenic and pathogenic) for protein expression, natural product preparation, and antimicrobial testing. We have access to ~6 shaker incubators and a biosafety cabinet (BSC).
Chemoenzymatic synthesis of small molecules: In many of our projects, we use small molecules as probes to characterize the enzymes of interest. These small-molecule probes are synthesized in the lab using a combination of chemical and enzymatic approaches, which provide us with significant flexibility.
Biophysical spectroscopy:
Electron paramagnetic resonance spectroscopy (EPR): Many of our target enzymes use redox-active metals and organic free radicals. Characterization of these paramagnetic species is critical to understand their functions in enzyme catalysis. EPR spectroscopy is a powerful approach to characterize the paramagnetic species in biological samples quantitatively.
Fluorescent microscope: Some of our questions, such as the mode of action of antifungal drugs, require studies in intact cells. In these cases, we use fluorescent microscopy combined with fluorescently labeled probes.
Structural biology: Protein structures are necessary to understand the mechanism and function of enzymes. We have ongoing collaborations with X-ray structural biologists within and outside the department (see PNAS 2015).
