I use single-molecule optical microscopy to address a fundamental question in molecular biology: how does proteins sequence encode its conformational dynamics and function? The model system that we study is the enzyme adenylate kinase (AK) from Escherichia coli.AKs 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 AKs enzymatic function; yet,its mechanistic roles and energetics remain elusive.Using the high-resolution time-dependent single-molecule FRET (F(o)rster Resonance Energy Transfer) developed in our group,we have measured AKs lid movements on the millisecond scale and map out its entire conformational distribution along the FRET coordinate without a presumed model.