Detecting Sequential Bond Formation Using Three-Dimensional Thermal Fluctuation Analysis

Authors

  • Tobias F. Bartsch,

    1. Physics Department and Center for Nonlinear Dynamics, The University of Texas at Austin, 1 University Station, C1600, Austin, TX 78712-0264 (USA), Fax: (+ 1) 512-471-1558
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  • Samo Fišinger Dr.,

    1. Institute of Biophysics, Faculty of Medicine, University of Ljubljana, Lipičeva 2, 1000 Ljubljana (Slovenia)
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  • Martin D. Kochanczyk,

    1. Physics Department and Center for Nonlinear Dynamics, The University of Texas at Austin, 1 University Station, C1600, Austin, TX 78712-0264 (USA), Fax: (+ 1) 512-471-1558
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  • Rongxin Huang Dr.,

    1. Physics Department and Center for Nonlinear Dynamics, The University of Texas at Austin, 1 University Station, C1600, Austin, TX 78712-0264 (USA), Fax: (+ 1) 512-471-1558
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  • Alexandr Jonáš Dr.,

    1. Institute of Scientific Instruments of the ASCR, v.v.i. Academy of Sciences of the Czech Republic, Královopolská 147, 612 64 Brno (Czech Republic)
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  • Ernst-Ludwig Florin Prof. Dr.

    1. Physics Department and Center for Nonlinear Dynamics, The University of Texas at Austin, 1 University Station, C1600, Austin, TX 78712-0264 (USA), Fax: (+ 1) 512-471-1558
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Abstract

Detecting specific bond formation: The formation of specific bonds between a particle and a substrate results in an immobilization of the particle that occurs in discrete steps, even for nanometer-sized complexes. Each configuration of bonds corresponds to a characteristic distribution of the particle's thermal position fluctuations (see picture).

original image

We present a novel experimental method that solves two key problems in nondestructive mechanical studies of small biomolecules at the single-molecule level, namely the confirmation of single-molecule conditions and the discrimination against nonspecific binding. A biotin–avidin ligand–receptor couple is spanned between a glass slide and a 1 μm latex particle using short linker molecules. Optical tweezers are used to initiate bond formation and to follow the particle’s thermal position fluctuations with nanometer spatial and microsecond temporal resolution. Here we show that each step in the specific binding process leads to an abrupt change in the magnitude of the particle’s thermal position fluctuations, allowing us to count the number of bonds formed one by one. Moreover, three-dimensional position histograms calculated from the particle’s fluctuations can be separated into well-defined categories reflecting different binding conditions (single specific, multiple specific, nonspecific). Our method brings quantitative mechanical single-molecule studies to the majority of proteins, paving the way for the investigation of a wide range of phenomena at the single-molecule level.

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