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PMID: 17328679 Published · ppublish English Journal Article Review

High-resolution, single-molecule measurements of biomolecular motion.

Annual review of biophysics and biomolecular structure ·Vol. 36 ·2007-00-00 ·Pages 171-90

Greenleaf WJ, Woodside MT, Block SM

Abstract

Many biologically important macromolecules undergo motions that are essential to their function. Biophysical techniques can now resolve the motions of single molecules down to the nanometer scale or even below, providing new insights into the mechanisms that drive molecular movements. This review outlines the principal approaches that have been used for high-resolution measurements of single-molecule motion, including centroid tracking, fluorescence resonance energy transfer, magnetic tweezers, atomic force microscopy, and optical traps. For each technique, the principles of operation are outlined, the capabilities and typical applications are examined, and various practical issues for implementation are considered. Extensions to these methods are also discussed, with an eye toward future application to outstanding biological problems.

MeSH Terms
Biophysics/methods Chemistry, Physical/methods Equipment Design Fluorescence Resonance Energy Transfer/instrumentation,methods Fluorescent Dyes/pharmacology Macromolecular Substances Magnetics Microscopy, Atomic Force/instrumentation,methods Microscopy, Fluorescence/instrumentation Molecular Conformation Motion Optical Tweezers Research Design
Chemicals
Fluorescent Dyes Macromolecular Substances
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Greenleaf William J
Department of Applied Physics, Stanford University, Stanford, California 94305-5030, USA.
Woodside Michael T
Block Steven M
References (108)
108 references, click to expand
  1. Reverse engineering of the giant muscle protein titin.
    Nature. 2002 Aug 29;418(6901):998-1002 PMID: 12198551
  2. Tracking kinesin-driven movements with nanometre-scale precision.
    Nature. 1988 Feb 4;331(6155):450-3 PMID: 3123999
  3. Elastic properties of a single-stranded charged homopolymeric ribonucleotide.
    Phys Rev Lett. 2004 Sep 10;93(11):118102 PMID: 15447383
  4. Mechanism of force generation of a viral DNA packaging motor.
    Cell. 2005 Sep 9;122(5):683-92 PMID: 16143101
  5. Direct observation of steps in rotation of the bacterial flagellar motor.
    Nature. 2005 Oct 6;437(7060):916-9 PMID: 16208378
  6. Friction and torque govern the relaxation of DNA supercoils by eukaryotic topoisomerase IB.
    Nature. 2005 Mar 31;434(7033):671-4 PMID: 15800630
  7. Can non-mechanical proteins withstand force? Stretching barnase by atomic force microscopy and molecular dynamics simulation.
    Biophys J. 2001 Oct;81(4):2344-56 PMID: 11566804
  8. Direct observation of kinesin stepping by optical trapping interferometry.
    Nature. 1993 Oct 21;365(6448):721-7 PMID: 8413650
  9. DNA primase acts as a molecular brake in DNA replication.
    Nature. 2006 Feb 2;439(7076):621-4 PMID: 16452983
  10. Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins.
    Proc Natl Acad Sci U S A. 2006 Apr 18;103(16):6190-5 PMID: 16606839
  11. Cytoplasmic dynein functions as a gear in response to load.
    Nature. 2004 Feb 12;427(6975):649-52 PMID: 14961123
  12. Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules.
    Science. 1996 Feb 9;271(5250):795-9 PMID: 8628994
  13. Fast, DNA-sequence independent translocation by FtsK in a single-molecule experiment.
    EMBO J. 2004 Jun 16;23(12):2430-9 PMID: 15167891
  14. Transcription against an applied force.
    Science. 1995 Dec 8;270(5242):1653-7 PMID: 7502073
  15. Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited.
    Nat Chem Biol. 2006 Feb;2(2):87-94 PMID: 16415859
  16. Force-dependent chemical kinetics of disulfide bond reduction observed with single-molecule techniques.
    Proc Natl Acad Sci U S A. 2006 May 9;103(19):7222-7 PMID: 16645035
  17. Oriented loading of FtsK on KOPS.
    Nat Struct Mol Biol. 2006 Nov;13(11):1026-8 PMID: 17041597
  18. Bacterial chromatin organization by H-NS protein unravelled using dual DNA manipulation.
    Nature. 2006 Nov 16;444(7117):387-90 PMID: 17108966
  19. Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads.
    Science. 1992 Nov 13;258(5085):1122-6 PMID: 1439819
  20. Polysaccharide elasticity governed by chair-boat transitions of the glucopyranose ring.
    Nature. 1998 Dec 17;396(6712):661-4 PMID: 9872313
  21. Simultaneous, coincident optical trapping and single-molecule fluorescence.
    Nat Methods. 2004 Nov;1(2):133-9 PMID: 15782176
  22. Myosin VI is a processive motor with a large step size.
    Proc Natl Acad Sci U S A. 2001 Nov 20;98(24):13655-9 PMID: 11707568
  23. Equilibrium information from nonequilibrium measurements in an experimental test of Jarzynski's equality.
    Science. 2002 Jun 7;296(5574):1832-5 PMID: 12052949
  24. Quantitative measurements of force and displacement using an optical trap.
    Biophys J. 1996 Apr;70(4):1813-22 PMID: 8785341
  25. Kinesin walks hand-over-hand.
    Science. 2004 Jan 30;303(5658):676-8 PMID: 14684828
  26. Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase.
    Nature. 2001 Apr 19;410(6831):898-904 PMID: 11309608
  27. A molecular ruler based on plasmon coupling of single gold and silver nanoparticles.
    Nat Biotechnol. 2005 Jun;23(6):741-5 PMID: 15908940
  28. Dynein-mediated cargo transport in vivo. A switch controls travel distance.
    J Cell Biol. 2000 Mar 6;148(5):945-56 PMID: 10704445
  29. Reversible unfolding of single RNA molecules by mechanical force.
    Science. 2001 Apr 27;292(5517):733-7 PMID: 11326101
  30. Observing spontaneous branch migration of Holliday junctions one step at a time.
    Proc Natl Acad Sci U S A. 2005 Apr 19;102(16):5715-20 PMID: 15824311
  31. Measuring 0.1-nm motion in 1 ms in an optical microscope with differential back-focal-plane detection.
    Opt Lett. 2004 Nov 15;29(22):2611-3 PMID: 15552661
  32. Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering.
    Prog Biophys Mol Biol. 2000;74(1-2):63-91 PMID: 11106807
  33. The mechanism of myosin VI translocation and its load-induced anchoring.
    Cell. 2004 Mar 5;116(5):737-49 PMID: 15006355
  34. Mechanochemical analysis of DNA gyrase using rotor bead tracking.
    Nature. 2006 Jan 5;439(7072):100-4 PMID: 16397501
  35. Single molecule force spectroscopy in biology using the atomic force microscope.
    Prog Biophys Mol Biol. 2000;74(1-2):37-61 PMID: 11106806
  36. Protein conformational dynamics probed by single-molecule electron transfer.
    Science. 2003 Oct 10;302(5643):262-6 PMID: 14551431
  37. Optical trapping.
    Rev Sci Instrum. 2004 Sep;75(9):2787-809 PMID: 16878180
  38. Single-molecule fluorescence resonance energy transfer.
    Methods. 2001 Sep;25(1):78-86 PMID: 11558999
  39. Movement and force produced by a single myosin head.
    Nature. 1995 Nov 9;378(6553):209-12 PMID: 7477328
  40. Fluorescence imaging with one nanometer accuracy: application to molecular motors.
    Acc Chem Res. 2005 Jul;38(7):574-82 PMID: 16028892
  41. Single-molecule quantum-dot fluorescence resonance energy transfer.
    Chemphyschem. 2005 May;6(5):956-60 PMID: 15884082
  42. Tethered particle motion method for studying transcript elongation by a single RNA polymerase molecule.
    Biophys J. 1994 Dec;67(6):2468-78 PMID: 7696485
  43. Magnetic tweezers: micromanipulation and force measurement at the molecular level.
    Biophys J. 2002 Jun;82(6):3314-29 PMID: 12023254
  44. Ligand-induced conformational changes observed in single RNA molecules.
    Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9077-82 PMID: 10430898
  45. Kinesin moves by an asymmetric hand-over-hand mechanism.
    Science. 2003 Dec 19;302(5653):2130-4 PMID: 14657506
  46. Reversible unfolding of individual titin immunoglobulin domains by AFM.
    Science. 1997 May 16;276(5315):1109-12 PMID: 9148804
  47. Kinesin hydrolyses one ATP per 8-nm step.
    Nature. 1997 Jul 24;388(6640):386-90 PMID: 9237757
  48. Single-molecule studies highlight conformational heterogeneity in the early folding steps of a large ribozyme.
    Proc Natl Acad Sci U S A. 2004 Jan 13;101(2):534-9 PMID: 14704266
  49. Direct observation of base-pair stepping by RNA polymerase.
    Nature. 2005 Nov 24;438(7067):460-5 PMID: 16284617
  50. Backtracking by single RNA polymerase molecules observed at near-base-pair resolution.
    Nature. 2003 Dec 11;426(6967):684-7 PMID: 14634670
  51. A metal-chelating microscopy tip as a new toolbox for single-molecule experiments by atomic force microscopy.
    Biophys J. 2000 Jun;78(6):3275-85 PMID: 10828003
  52. A four-way junction accelerates hairpin ribozyme folding via a discrete intermediate.
    Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9308-13 PMID: 12883002
  53. Dynamic strength of molecular adhesion bonds.
    Biophys J. 1997 Apr;72(4):1541-55 PMID: 9083660
  54. Direct measurement of the full, sequence-dependent folding landscape of a nucleic acid.
    Science. 2006 Nov 10;314(5801):1001-4 PMID: 17095702
  55. Hidden complexity in the mechanical properties of titin.
    Nature. 2003 Mar 27;422(6930):446-9 PMID: 12660787
  56. Identification of the FtsK sequence-recognition domain.
    Nat Struct Mol Biol. 2006 Nov;13(11):1023-5 PMID: 17041598
  57. Single myosin molecule mechanics: piconewton forces and nanometre steps.
    Nature. 1994 Mar 10;368(6467):113-9 PMID: 8139653
  58. Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy.
    Nature. 2002 Oct 17;419(6908):743-7 PMID: 12384704
  59. Exploring the energy landscape of GFP by single-molecule mechanical experiments.
    Proc Natl Acad Sci U S A. 2004 Nov 16;101(46):16192-7 PMID: 15531635
  60. Single molecule force spectroscopy of spectrin repeats: low unfolding forces in helix bundles.
    J Mol Biol. 1999 Feb 19;286(2):553-61 PMID: 9973570
  61. Short-range spectroscopic ruler based on a single-molecule optical switch.
    Phys Rev Lett. 2005 Mar 18;94(10):108101 PMID: 15783528
  62. Nanospring behaviour of ankyrin repeats.
    Nature. 2006 Mar 9;440(7081):246-9 PMID: 16415852
  63. Single-molecule RNA science.
    Annu Rev Biophys Biomol Struct. 2005;34:399-414 PMID: 15869396
  64. Overlapping hand-over-hand mechanism of single molecular motility of cytoplasmic dynein.
    Proc Natl Acad Sci U S A. 2006 Apr 11;103(15):5741-5 PMID: 16585530
  65. Sequence-dependent pausing of single lambda exonuclease molecules.
    Science. 2003 Sep 26;301(5641):1914-8 PMID: 12947034
  66. Correlating structural dynamics and function in single ribozyme molecules.
    Science. 2002 May 24;296(5572):1473-6 PMID: 12029135
  67. Forward and reverse motion of single RecBCD molecules on DNA.
    Biophys J. 2004 Mar;86(3):1640-8 PMID: 14990491
  68. Pulling on the nascent RNA during transcription does not alter kinetics of elongation or ubiquitous pausing.
    Mol Cell. 2006 Jul 21;23(2):231-9 PMID: 16857589
  69. Differential detection of dual traps improves the spatial resolution of optical tweezers.
    Proc Natl Acad Sci U S A. 2006 Jun 13;103(24):9006-11 PMID: 16751267
  70. Anisotropic deformation response of single protein molecules.
    Proc Natl Acad Sci U S A. 2006 Aug 22;103(34):12724-8 PMID: 16908850
  71. Optical torque wrench: angular trapping, rotation, and torque detection of quartz microparticles.
    Phys Rev Lett. 2004 May 14;92(19):190801 PMID: 15169392
  72. Single-molecule studies of the effect of template tension on T7 DNA polymerase activity.
    Nature. 2000 Mar 2;404(6773):103-6 PMID: 10716452
  73. The elasticity of a single supercoiled DNA molecule.
    Science. 1996 Mar 29;271(5257):1835-7 PMID: 8596951
  74. Single molecule analysis of RNA polymerase elongation reveals uniform kinetic behavior.
    Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13538-43 PMID: 12370445
  75. Force-clamp spectroscopy monitors the folding trajectory of a single protein.
    Science. 2004 Mar 12;303(5664):1674-8 PMID: 15017000
  76. Using single-particle tracking to study nuclear trafficking of viral genes.
    Biophys J. 2004 Oct;87(4):2749-58 PMID: 15454466
  77. Optical measurement of picometer displacements of transparent microscopic objects.
    Appl Opt. 1990 Jun 1;29(16):2382-91 PMID: 20563179
  78. Direct observation of the three-state folding of a single protein molecule.
    Science. 2005 Sep 23;309(5743):2057-60 PMID: 16179479
  79. Mechanical and chemical unfolding of a single protein: a comparison.
    Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3694-9 PMID: 10097099
  80. Interlaced optical force-fluorescence measurements for single molecule biophysics.
    Biophys J. 2006 Aug 1;91(3):1069-77 PMID: 16648165
  81. Force and velocity measured for single kinesin molecules.
    Cell. 1994 Jun 3;77(5):773-84 PMID: 8205624
  82. Folding-unfolding transitions in single titin molecules characterized with laser tweezers.
    Science. 1997 May 16;276(5315):1112-6 PMID: 9148805
  83. The bacteriophage straight phi29 portal motor can package DNA against a large internal force.
    Nature. 2001 Oct 18;413(6857):748-52 PMID: 11607035
  84. Passive all-optical force clamp for high-resolution laser trapping.
    Phys Rev Lett. 2005 Nov 11;95(20):208102 PMID: 16384102
  85. RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP.
    Nature. 2006 Jan 5;439(7072):105-8 PMID: 16397502
  86. Sequence-resolved detection of pausing by single RNA polymerase molecules.
    Cell. 2006 Jun 16;125(6):1083-94 PMID: 16777599
  87. Ubiquitous transcriptional pausing is independent of RNA polymerase backtracking.
    Cell. 2003 Nov 14;115(4):437-47 PMID: 14622598
  88. The mechanical stability of ubiquitin is linkage dependent.
    Nat Struct Biol. 2003 Sep;10(9):738-43 PMID: 12923571
  89. Coordination of kinesin's two heads studied with mutant heterodimers.
    Proc Natl Acad Sci U S A. 2002 Dec 10;99(25):16058-63 PMID: 12451178
  90. Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism.
    Science. 2006 Nov 17;314(5802):1144-7 PMID: 17110578
  91. F1-ATPase is a highly efficient molecular motor that rotates with discrete 120 degree steps.
    Cell. 1998 Jun 26;93(7):1117-24 PMID: 9657145
  92. Direct observation of the rotation of F1-ATPase.
    Nature. 1997 Mar 20;386(6622):299-302 PMID: 9069291
  93. Probing protein-DNA interactions by unzipping a single DNA double helix.
    Biophys J. 2002 Aug;83(2):1098-105 PMID: 12124289
  94. Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy.
    Nat Struct Biol. 2000 Sep;7(9):724-9 PMID: 10966638
  95. Stretching single molecules into novel conformations using the atomic force microscope.
    Nat Struct Biol. 2000 Sep;7(9):719-24 PMID: 10966637
  96. Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching.
    Science. 2006 Nov 17;314(5802):1139-43 PMID: 17110577
  97. Biological applications of optical forces.
    Annu Rev Biophys Biomol Struct. 1994;23:247-85 PMID: 7919782
  98. Identifying kinetic barriers to mechanical unfolding of the T. thermophila ribozyme.
    Science. 2003 Mar 21;299(5614):1892-5 PMID: 12649482
  99. Characterization of photodamage to Escherichia coli in optical traps.
    Biophys J. 1999 Nov;77(5):2856-63 PMID: 10545383
  100. DNA overwinds when stretched.
    Nature. 2006 Aug 17;442(7104):836-9 PMID: 16862122
  101. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization.
    Science. 2003 Jun 27;300(5628):2061-5 PMID: 12791999
  102. Single-molecule analysis of DNA uncoiling by a type II topoisomerase.
    Nature. 2000 Apr 20;404(6780):901-4 PMID: 10786800
  103. The use of the avidin-biotin complex as a tool in molecular biology.
    Methods Biochem Anal. 1980;26:1-45 PMID: 7392958
  104. Nanometre-level analysis demonstrates that lipid flow does not drive membrane glycoprotein movements.
    Nature. 1989 Jul 27;340(6231):284-8 PMID: 2747796
  105. The binding change mechanism for ATP synthase--some probabilities and possibilities.
    Biochim Biophys Acta. 1993 Jan 8;1140(3):215-50 PMID: 8417777
  106. Extreme conformational diversity in human telomeric DNA.
    Proc Natl Acad Sci U S A. 2005 Dec 27;102(52):18938-43 PMID: 16365301
  107. Single molecule observation of liposome-bilayer fusion thermally induced by soluble N-ethyl maleimide sensitive-factor attachment protein receptors (SNAREs).
    Biophys J. 2004 Nov;87(5):3569-84 PMID: 15347585
  108. A single-molecule study of RNA catalysis and folding.
    Science. 2000 Jun 16;288(5473):2048-51 PMID: 10856219
Article Info
Journal
Annual review of biophysics and biomolecular structure
Abbr.
Annu Rev Biophys Biomol Struct
ISSN
1056-8700
DOI
10.1146/annurev.biophys.36.101106.101451
Published
2007-00-00
Pages
171-90
Language
English
Region
United States
NLM ID
9211097
PMCID
PMC1945240
Subset
IM
Grants
NIGMS NIH HHS · R01 GM057035 · United States
NIGMS NIH HHS · R01 GM057035-13 · United States
External Links
PubMed source DOI: 10.1146/annurev.biophys.36.101106.101451 PMC Full Text
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