Study on the molecular recognition mechanism of proteins by single-molecule fluorescence resonance energy transfer (smFRET)

Abstract

Single-molecule analysis has expanded our knowledge of various biological processes in molecular level. Particularly, the understandings of protein dynamics related to key biological reaction, such as molecular recognition, catalytic turnover, and regulatory process, have provided great insight into the functions of proteins. Although there are some limitations in the site-specific labeling and the time resolution for the analysis of protein dynamics, single-molecule FRET approaches have great potential to unravel many questions that are undetectable in ensemble, heterogeneous system. The coupling between the conformational change of protein and the ligand binding has been explained by two representative hypotheses, the “induced fit” and the “conforma-tional selection”. Though much effort has focused on elucidating these coupling mechan-isms, there are few direct experimental evidences and this issue is still controversial. Here we show which mechanism is involved in the molecular recognition of maltose-binding protein (MBP) as a model protein. To date, the structures of MBP indirectly displayed the induced fit mechanism. However, since recent NMR relaxation experiments discovered the existence of minor partial closed conformer in the absence of ligand, an alternative confor-mational selection model has been suggested. To answer this fundamental question, we per-formed single-molecule FRET analysis, based on a prism-type total internal reflection fluo-rescence (TIRF) microscopy. To clearly observe the dynamics of MBP upon ligand binding, we utilized the MBP mutants with one or two mutations in hinge region. By directly observing the dynamics of single molecules in real time and analyzing the dwell times of the open conformation in the absence and presence of ligand, we demonstrated that the ligand binding of MBP was governed by induced fit mechanism. We also found that there were no differences in induced fit capability between maltose and maltotriose, despite...