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Single molecule mechanical manipulation for studying biological properties of proteins, DNA, and sugars

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For over 20 years there has been immense biological insight gained using single molecule mechanical measurements to understand properties of biomolecules. This review outlines the field of single molecule mechanics studies and focuses on the manipulation of proteins, DNA, and sugars by single molecule force spectroscopy (SMFS) by atomic force microscopy (AFM). The methods and examples of SMFS by AFM are illustrated using recent advances in protein science including titin elasticity, mechanical unfolding and refolding of α‐helical repeat proteins, mechanoenzymatics of thioredoxin and titin kinase, and intermolecular interactions of P‐selectin complexes. The possibilities of SMFS to investigate the mechanics of other biopolymers like double‐ and single‐stranded DNA and forced‐induced conformational changes in sugars are also discussed. Finally, SMFS and its application to biological processes, like DNA replication, packing and transcription, and DNA methylation are illustrated. These measurements provide a unique and integral part of the development of our knowledge of biochemistry and molecular mechanics. This article is categorized under: Nanotechnology Approaches to Biology > Cells at the Nanoscale Nanotechnology Approaches to Biology > Nanoscale Systems in Biology

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Schematic of the unfolding process of a polyI27 protein during single molecule force spectroscopy (SMFS) by atomic force microscopy (AFM). (a) The molecular snapshots of the process of unfolding that gives rise to (b) the force‐extension signature of the molecule. Description is in the main text.
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Examples of the different force‐extension spectrums of sugar molecules. The unique force‐extension fingerprint of the sugar molecules allows for their easy identification. The transitions that deviate from the FJC in the force‐extension recordings arise due to the forced conformational transition from the chair conformation to the boat conformation (inset in the top panel). This transition does not occur in cellulose, in which the ground energy chair conformation provides a maximum separation of the glycosidic oxygen atoms (inset in the bottom panel). (Reprinted with permission from Ref. 44 Copyright 2001 Nature Publishing Group)
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Normalized force‐extension spectrum of single‐stranded DNA composed of only thymines (poly(dT)) or only adenines (poly(dA)). The inset indicates atomic force microscopy (AFM) experiment uses nonspecific attachment to adhere the molecule to the surface and the cantilever. The normalization allows different length molecules to be compared under the assumption that at high force and high extension the conformations of the backbones of different single‐stranded DNA molecules are the same. This force spectrum clearly shows that poly(dA) does not behave as a freely jointed chain FJC as poly(dT) does, and undergoes two force transitions at ∼20 and ∼120 pN. (Reprinted with permission from Ref. 113 Copyright 2007 The American Physical Society)
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(a) Schematic of an optical trap experiment in which a DNA molecule is stretched between beads fixed on a pipette or in the laser trap. (b) Force‐extension spectrums for ssDNA and dsDNA. The arrows indicate the change in extension when the trap holds the complex at constant force during polymerization (Poly) or during exonuclease activity (Exo). (Reprinted with permission from Ref. 45 Copyright 2003 Nature Publishing Group)
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(a) Schematic of single molecule force spectroscopy (SMFS) assay for detecting DNA methylation. Single‐stranded DNA (blue line) containing methylated cytosines (red boxes) is attached to a glass substrate. The cantilever tip is crosslinked within antibody targeting 5‐methylcytosine and the Fab arms of the antibody bind to methylcytosines that are separated by a given number of nucleotides. The inset shows the force‐distance diagram and the numbers indicate the stages of stretching the antibody–DNA complex. (b) The probability density of the distances between force peaks can be used to determine the specific pattern of methylation. (See main text for details). (Reprinted with permission from Ref. 93 Copyright 2010 Nature Publishing Group)
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(a) Schematic of pulling experiment in which (I27ss)8 molecules were held at constant force (‘force‐clamp’). Upon unfolding, the disulphide bond is exposed to the solvent and remains undisturbed until it is reduced by Trx. (b) A single (I27ss)8 molecule is held in the force‐clamp in the absence of Trx. At least six unfolding events are observed. (c) Unfolding of (I27ss)8 in the presence of 8 µM Trx reveals steps of ∼13.2 nm which corresponds to the extension of the trapped residues that are released after the reduction of the individual disulphide bonds by single enzymes. (Reprinted with permission from Ref. 84 Copyright 2007 Nature Publishing Group)
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(a) PDB derived model of the full‐length model protein (I273‐NI6C‐I273) attached to substrate undergoing force. (b) Example of typical force‐extension spectrum of the model protein. Each small NI6C repeat unfolds and can give a contour length increment of 10.5 nm which is consistent with the number of amino acids in each repeat. (c) Example of folding and refolding force‐extension curve of NI6C. (d) Amino acid sequence of mutated consensus NI6C repeats and (e) the force‐extension profile of NI6C mutant showing weakened unfolding as compared to the wild‐type protein. (Reprinted with permission from Ref. 31 Copyright 2010 The American Society for Biochemistry and Molecular Biology and Ref. 32 Copyright 2012 The American Society for Biochemistry and Molecular Biology)
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Single molecule force spectroscopy (SMFS) can be used to extract biochemical parameters for protein kinetics. (a) The unfolding rate can be determined along a mechanical pathway using force as the denaturant and extrapolating to zero force. The unfolding force correlates with the pulling speed because the protein takes a finite time to cross the energy barrier from the folded state to the unfolded state and the faster velocity of the pulling speed allows the internal forces in the protein to accumulate before there is time to unfold. Simple models can be used to model this behavior and extract the unfolding rate. (b) Schematic of a double‐pulse experiment. Each pulse unfolds and relaxes the protein using a ramp that moves with constant velocity. The pulses are spaced by a time delay. Decreasing the time delay attenuates the proportion of domains that are refolded (c) in a way that can be fit using an exponential function and a parameter that describes the folding rate. (Reprinted with permission from Ref. 60 Copyright 1998 Nature Publishing Group)
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