BMB Class 17, November 30, Single Molecule Biophysics (II)

Transcription

1 BMB Class 17, November 30, Single Molecule Biophysics (II)

2 New Advances in Single Molecule Techniques Atomic Force Microscopy Single Molecule Manipulation - optical traps and tweezers Single Molecule Fluorescence

3 Challenges for Single Molecules Studies 1. Establish conditions so that a single molecule is detected 2. Sensitivity - signal from a single molecule must be detected above background

4 Detection of Single Molecule Fluorescence Brighter, more stable dyes An ideal SM fluorescence dye should: - have high absorption coefficient and quantum yield; - have high photostability; - does not perturb the host molecule

5 1 st Generation SM fluorophores l ex = 550 nm l em = 570 nm l ex = 649 nm l em = 670 nm TMR l ex = 540 nm l em = 568 nm Rhodamin 6G l ex = 530 nm l em = 555 nm Texas Red l ex = 587 nm l em = 602 nm

6 Favorite SM fluorophores: AlexaFluor Molecular Probes Handbook: Molecular-Probes-The-Handbook.html

7 Favorite Single Molecule Probes: Atto Dyes

8 Detection of Single Molecule Fluorescence For freely diffusing molecules Confocal or Two photon Microscopy Signal detected by a point detector with ps resolution For surface-tethered molecules TIRF Microscopy Signal detected by CCDs with ms resolution.

9 SM Fluorescence of freely diffusing particles Diffraction limited laser beam illuminates a volume of 0.2 fl. => Statistically, a single molecule is illuminated within the laser beam at concentrations <1 nm.

10 Alternating Laser Excitation Spectroscopy Majumdar et al, PNAS 104: (2007)

11 Alternating Laser Excitation Spectroscopy FRET efficiency Stoichiometry

12 ALEX study of FRET distance in model DNA

13 Monitor the global conformation of Get3 by single molecule FRET

14 Monitor the global conformation of Get3 by single molecule FRET

15 Monitor the global conformation of Get3 by single molecule FRET Chio et al, PNAS 2017

16 Sequential Get3 opening during the targeting cycle Chio et al, PNAS 2017

17 Pros and Cons of SM studies with freely diffusing molecules Pros - No surface immobilization is needed. - Minimal perturbation to the molecule of interest. - Accurate determination of reaction equilibrium. Cons - Concentration needs to be kept low - Ideal for studying intramolecular events or high affinity complexes, but not for weaker complexes - Can not perform real-time kinetic analysis for processes t > 1 ms

18 SM Fluorescence of surface-immobilized molecules Allows analysis of the time trajectory of molecular events

19 SRP RNA Mediates Movement of GTPase Complex at Distinct Stages of Targeting Complex assembly GTP hydrolysis (Complex disassembly)

20 Single molecule FRET Setup Shen et al, Nature 2012

21 High FRET bursts support the transient docking model

22 HMM Analysis Identifies Short-lived States H state M states L state

23 Quantify Equilbria of GTPase Movement on SRP RNA

24 Quantify Kinetics of Transitions between states Dt

25 Pathway / Mechanism of Transition No. From To 1 L M2 2 M2 M1 3 M1 M2 4 M2 L 5 L H 6 H M1 7 M1 H 8 H L 9 L M2 10 M2 M1 11 M1 H

26 Transition density plot (TDP) L M1 M2 H L M1 M2 H Go through hundreds of traces No. From To 1 L M2 2 M2 M1 3 M1 M2 4 M2 L 5 L H 6 H M1 7 M1 H 8 H L 9 L M2 10 M2 M1 11 M1 H

27 Transition density plot (TDP): Hypothetical Cases Sequential Random L M1 M2 H L M1 M2 H L M1 M2 H L M1 M2 H

28 Search for Distal Site Is a trial-and-error process L M1 M2 H

29 SM Analysis of RNA Catalysis and Folding: Hairpin Ribozyme Liu et al, PNAS 104: (2007)

30 Fluorescence Traces of Single Ribozymes undergoing folding and cleavage Liu et al, PNAS 104: (2007)

31 Docking kinetics of Hairpin Ribozyme Liu et al, PNAS 104: (2007)

32 Docking kinetics of Hairpin Ribozyme Liu et al, PNAS 104: (2007)

33 However, Not all reaction intermediates have distinct FRET values Additional ways to distinguish between distinct states: - More colors - Kinetic Fingerprint - Fluorescence lifetime - Fluorescence polarization up to eight dimensions of information

34 Fluorescence Quencher to Measure Binding and Dissociation of 3 P FRET = 0.2 FRET = 0.3 Liu et al, PNAS 104: (2007)

35 However, Not all reaction intermediates have distinct FRET values Additional ways to distinguish between distinct states: More colors Kinetic Fingerprint Fluorescence lifetime Fluorescence polarization

36 Kinetic Fingerprint of Reaction Intermediates

37 Establish Internal Equibrium between Different Intermediate States Out of 824 molecules, 21 in U L, 591 in D L, 47 in D c, 165 in U C K L dock = k L dock / k L undock = 28 6 K C dock = k C dock / k C undock = K int = k lig / k cleav = 13 2 Liu et al, PNAS 104: (2007)

38 Additional ways to distinguish between distinct states More colors Kinetic Fingerprint Fluorescence lifetime Fluorescence polarization

39 Fluorescence Lifetime Time that a molecule stays in the excited state before decay F(t) = F 0 e -t/t Typical lifetime: ns A property of the fluorophore and its environment - dipolar interactions - electrostatic environment - FRET

40 SM lifetime measurement of DNA conformation Cy3 TMR No FRET FRET Edel, Eid, Meller, J. Phys. Chem. B 111: 2986 (2007)

41 Additional ways to distinguish between More colors distinct states Kinetic Fingerprint Fluorescence lifetime Fluorescence polarization: Determining rotational movements

42 Measure and Quantitate FP

43 SM Study of Rotary Motion in F 1 ATPase Noji et al, Nature 386: 299 (1997)

44 Does Actin Filament Impose a frictional drag to rotation of the g-subunit? Rotation rate estimated from ATPase activity: ~17 rev s -1 Measured: ~ 4 rev. s -1 Torque for rotating actin filament: > 45 pn nm Need to measure rotation without actin filament

45 SM FP Measurement of F1 ATPase Rotation 120 rotation is a genuine property of F 1 ATPase Rotational rate approaches 20 rev s -1 Adachi et al, PNAS 97: 7243 (2000)

46 Detect single molecule events at high sample concentrations 1. Nanovesicle trapping - Effective concentration ~ 3 µm for a single molecule - Avoids protein-glass interactions Benitez et al, JACS 130: 2446 (2008)

47 Detect single molecule events at high sample concentrations 2. Zero-Mode Waveguides - nanophotonic structure cuts off propagating light above a threshold wavelength - exponential decay of illuminating light after entry into confinement - reduces observation volume to L Levene et al, Science 299, 682 (2003); Uemura et al, Nature 464, 1012 (2010)