title: Observing lysozyme's closing and opening motions by high-resolution single-molecule enzymology.
abstract: Single-molecule techniques can monitor the kinetics of transitions between enzyme open and closed conformations, but such methods usually lack the resolution to observe the underlying transition pathway or intermediate conformational dynamics. We have used a 1 MHz bandwidth carbon nanotube transistor to electronically monitor single molecules of the enzyme T4 lysozyme as it processes substrate. An experimental resolution of 2 μs allowed the direct recording of lysozyme's opening and closing transitions. Unexpectedly, both motions required 37 μs, on average. The distribution of transition durations was also independent of the enzyme's state: either catalytic or nonproductive. The observation of smooth, continuous transitions suggests a concerted mechanism for glycoside hydrolysis with lysozyme's two domains closing upon the polysaccharide substrate in its active site. We distinguish these smooth motions from a nonconcerted mechanism, observed in approximately 10% of lysozyme openings and closings, in which the enzyme pauses for an additional 40-140 μs in an intermediate, partially closed conformation. During intermediate forming events, the number of rate-limiting steps observed increases to four, consistent with four steps required in the stepwise, arrow-pushing mechanism. The formation of such intermediate conformations was again independent of the enzyme's state. Taken together, the results suggest lysozyme operates as a Brownian motor. In this model, the enzyme traces a single pathway for closing and the reverse pathway for enzyme opening, regardless of its instantaneous catalytic productivity. The observed symmetry in enzyme opening and closing thus suggests that substrate translocation occurs while the enzyme is closed.
authors.affiliation: Departments of †Physics and Astronomy, ‡Molecular Biology and Biochemistry, and §Chemistry, University of California, Irvine, California 92697, United States.
authors.forename: Maxim V
authors.initials: MV
authors.lastname: Akhterov
authors.affiliation: Departments of †Physics and Astronomy, ‡Molecular Biology and Biochemistry, and §Chemistry, University of California, Irvine, California 92697, United States.
authors.forename: Yongki
authors.initials: Y
authors.lastname: Choi
authors.affiliation: Departments of †Physics and Astronomy, ‡Molecular Biology and Biochemistry, and §Chemistry, University of California, Irvine, California 92697, United States.
authors.forename: Tivoli J
authors.initials: TJ
authors.lastname: Olsen
authors.affiliation: Departments of †Physics and Astronomy, ‡Molecular Biology and Biochemistry, and §Chemistry, University of California, Irvine, California 92697, United States.
authors.forename: Patrick C
authors.initials: PC
authors.lastname: Sims
authors.affiliation: Departments of †Physics and Astronomy, ‡Molecular Biology and Biochemistry, and §Chemistry, University of California, Irvine, California 92697, United States.
authors.forename: Mariam
authors.initials: M
authors.lastname: Iftikhar
authors.affiliation: Departments of †Physics and Astronomy, ‡Molecular Biology and Biochemistry, and §Chemistry, University of California, Irvine, California 92697, United States.
authors.forename: O Tolga
authors.initials: OT
authors.lastname: Gul
authors.affiliation: Departments of †Physics and Astronomy, ‡Molecular Biology and Biochemistry, and §Chemistry, University of California, Irvine, California 92697, United States.
authors.forename: Brad L
authors.initials: BL
authors.lastname: Corso
authors.affiliation: Departments of †Physics and Astronomy, ‡Molecular Biology and Biochemistry, and §Chemistry, University of California, Irvine, California 92697, United States.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Weiss
authors.affiliation: Departments of †Physics and Astronomy, ‡Molecular Biology and Biochemistry, and §Chemistry, University of California, Irvine, California 92697, United States.
authors.forename: Philip G
authors.initials: PG
authors.lastname: Collins
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry; D019943:Amino Acid Substitution; D017122:Bacteriophage T4 / Q000737:chemistry / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006868:Hydrolysis; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009038:Motion; D009112:Muramic Acids / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D009112:Muramic Acids; D011994:Recombinant Proteins; D014764:Viral Proteins; C031651:N-acetylmuramic acid; D009113:Muramidase; D000117:Acetylglucosamine