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Detecting Sequential Bond Formation Using Three‐Dimensional Thermal Fluctuation Analysis
Author(s) -
Bartsch Tobias F.,
Fišinger Samo,
Kochanczyk Martin D.,
Huang Rongxin,
Jonáš Alexandr,
Florin ErnstLudwig
Publication year - 2009
Publication title -
chemphyschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.016
H-Index - 140
eISSN - 1439-7641
pISSN - 1439-4235
DOI - 10.1002/cphc.200900211
Subject(s) - microsecond , particle (ecology) , chemical physics , biomolecule , thermal fluctuations , chemistry , molecule , optical tweezers , avidin , position (finance) , substrate (aquarium) , linker , molecular physics , crystallography , nanotechnology , materials science , biotin , optics , thermodynamics , physics , biochemistry , oceanography , organic chemistry , finance , computer science , economics , geology , operating system
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).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.