题　目：How does Enzyme work? A Single-Molecule Perspective

报告人：谭砚文教授 （复旦大学物理系）

时　间：10月19日(周三), 下午3:00

地　点：物理馆512会议室

Abstract:

The advent of single-molecule fluorescence techniques has provided a new perspective for the study of enzymes. For protein molecules working in a noisy environment such as in cells, the energetics associated with the random fluctuations and perturbations of are commensurate with the functional energy scale of protein molecules; consequently, the function of proteins are modulated by their environment and are stochastic in nature. For such complex systems, energy landscape is an effective way of correlating function with structure and free energies. I use single-molecule optical microscopy and enzymatic assays to measure the energy landscape of a model enzyme system, adenylate kinase (AK) from Escherichia coli. AK’s lid domain undergoes a large conformational change at the catalytic, millisecond timescale, which leads to a reasonable assumption that this lid dynamics is involved in AK’s enzymatic function; yet, its mechanistic roles and energetics remain elusive. Using the high-resolution time-dependent single-molecule FRET (Förster Resonance Energy Transfer), we have measured AK's lid movements on the millisecond scale and map out its entire conformational distribution along the FRET coordinate without a presumed model. Using this information, we have quantitatively recovered AK's energetic landscape and related its stochastic lid dynamics to its catalytic function. Finally, the relationship between AK’s genetic coding and its catalytic function is experimentally established by introducing targeted mutation on specific AK sites. This study provides new directions for protein engineering.