BMB November 17, Single Molecule Biophysics (I)

Transcription

1 BMB November 17, Single Molecule Biophysics (I)

2 Goals 1. Understand the information SM experiments can provide 2. Be acquainted with different SM approaches 3. Learn to interpret SM results

3 Text books Methods in Molecular Biophysics - chapter F4 Single molecule detection - chapter F5 single molecule manipulation Useful Readings - Joo et al., Annu. Rev. Biochem 77: 51 (2008) Advances in single-molecule fluorescence methods - Cecconi et al., Proc. Int. School Enrico Fermi, Societa Italiana di Fisica: 145 (2007) Studying protein folding with laser tweezers - Engel & Gaub, Annu. Rev. Biochem 77: 127 (2008) Structure and Mechanics of membrane proteins

4 Why Single Molecules? 1. Overcome Sample Heterogeneity

5 Why Single Molecules? 2. Observe intermediates that do not accumulate G

6 Why Single Molecules? 3. Dissect Multiple Pathways Molecules reaction

7 Why Single Molecules? 4. Overcome Loss of molecular synchrony Molecules

8 Challenges in 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

9 Earliest Single Molecule Studies: Patch Clamp A glass pipette makes contact with a small area of membrane containing a single channel Mild suction creates a tight seal with membrane allows control of membrane potential to study the voltage dependence of the channel Bert Sakmann & Erwin Neher, 1981

10 Different Patch Clamp Configurations

11 Single Channel Studies of AchR QX-222 open QX222 +QX222 closed Charnet et al, Neuron 2: 87 (1990)

12 Characterize the kinetics of channel opening p i = Ae (t t 0 )/τ τ = 1/F dwell time, blocked Charnet et al, Neuron 2: 87 (1990)

13 Pushing the limit of Patch Clamp Techniques Reduce stray capacitance of micropipettes thick wall, hard borosilicate or quartz faster detectors with reduced noise => <3 µs time resolution membranes with higher resistance reconstituted liposomes and vesicles instead of spheroplast

14 Detecting channel substates at µs resolution MscS channel recording at 3 µs resolution open 2/3 open Kinetic analysis closed Voltage-gating response equilibrium analysis Shapovalov & Lester, J. Gen. Physiol. 124: 151 (2004)

15 New Advances in Single Molecule Techniques Atomic Force Microscopy Single Molecule Manipulation - optical traps and tweezers Single Molecule Fluorescence Single Molecule Imaging (STORM, PALM)

16 Atomic Force Microscopy Deflections resulting from tip-sample forces are detected: - van der Waals - capillary - electrostatic - magnetic - solvation Feed-back mechanism adjusts tip-to-sample distance to maintain a constant force

17 Tips in AFM The ideal AFM tip has a small R c and Φ 0. Mostly carbon nanonubules

18 Imaging Modes in AFM Static Mode: tip deflection is the feedback signal - very high resolution - Lateral shearing forces pushes sample off - Needs low stiffness cantilevers

19 Imaging Modes in AFM Dynamic Mode: cantilever is externally oscillated - changes in oscillation amplitude, phase or frequency due to tip-sample interactions provide the feedback signal - stiff cantilevers provide stability close to the surface - minimizes damage and displacement of the sample

20 AFM Images Single molecules of poly(2-vinylpyridine) recorded by tapping mode AFM Roiter & Minko, JACS 127: (2005)

21 AFM allows Proteins to be Imaged in Native Membrane Bacterhodopsin trimer on purple membrane Low force High force Muller et al, Biochim. Biophys. Acta 1460: 27 (2000)

22 Probe conformational dynamics of membrane proteins with ultrafast AFM Lipid bilayers containing SecYEG channel Gary et al, PNAS 2013: 16868

23 Probe conformational dynamics of membrane proteins with fast AFM Gary et al, PNAS 2013: 16868

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

25 Single Molecule Manipulations AFM can be used to mechanically manipulate single molecules and study unfolding; However, force constants are nn/nm with RMS force noise of 1 15 pn. Suited to monitor events under high force. Low force lazer tweezers: RMS noise < 1 pn. Allow studies of molecular events occurring under low forces. Examples: - protein / RNA folding / unfolding - molecular motors - transcription / translation

26 Principle of Optical tweezers Attracts dielectric particles in a highly focused laser beam. Strong electric field gradient across beam waist.

27 Principle of Optical tweezers Balance between momentum from reflected and refracted rays traps bead downstream of the light focus.

28 Dual-beam Optical Trap Scattering forces by reflected rays are canceled; Bead is trapped at the light focus.

29 Light-momentum Force Sensor Offset distance measured by PSD is converted into momentum flux of the tapping beam W - light intensity; F = (W/c) ( x/r L ) R L - focal length of the lens bead position is proportional to applied force for displacement up to ~200 nm => measures both displacement and force

30 Basic Optical Tweezer Setup

31 Different Geometries of Optical Tweezers DNA/RNA/protein folding motors

32 RNase H Folding: Bulk studies An intermediate (I) forms within the instrument deadtime (<12 ms) protected from H/D exchange in I Questions: - Is I a distinct state, or a redistribution of the unfolded protein? - Is I on or off the pathway? - Is formation of I obligatory for RNase H folding?

33 Single Molecule Study: RNase H Folding Experimental setup Cecconi et al, Science 309: 2057 (2005)

34 DNA handles do not affect RNase H s Global Structure Folding Enzymatic activity Cecconi et al, Science 309: 2057 (2005)

35 Force-Extension Curves for Individual RNase H Molecules Unfolds in a two-state manner (N U) at ~ 19 pn Refolding occurs through an intermediate (I) at ~ 5.5 pn I further refolds to N at lower foces Cecconi et al, Science 309: 2057 (2005)

36 Force-Extension Curves for Individual RNase H Molecules Unfolds in a two-state manner (N U) at ~ 19 pn Refolding occurs through an intermediate (I) at ~ 5.5 pn I further refolds to N at lower foces I can be further unfolded in the next cycle at ~5.5 pn Cecconi et al, Science 309: 2057 (2005)

37 Direct Observation of Reversible Folding / Unfolding of I at low pulling speed Molecules hop between I and U states at forces close to 5.5 pn Cecconi et al, Science 309: 2057 (2005)

38 I directly Folds into the Native State I is an obligatory intermediate during folding Cecconi et al, Science 309: 2057 (2005)

39 Equilibrium analysis of I U transition I U distance unfolding free energy at F = 0 x = 11 ± 2 nm G = 4 ± 1 kcal/mol; agrees with bulk studies (3.6 kcal/mol) Cecconi et al, Science 309: 2057 (2005)

40 Analysis of the kinetics of I unfolding k I U = 9.1 s -1 k U I = 3.3 s -1 Instrument contribution I distance x I U = 5 ± 1 nm x U I = 6 ± 2 nm unfolding rate at F = 0 I is a pliable structure that can deform substantially Cecconi et al, Science 309: 2057 (2005)

41 Free Energy Landscape of RNase H Cecconi et al, Science 309: 2057 (2005)

42 DNA Packaging Motor in phage Φ29 Three layers: gp10: dodecamer (seal) Prohead-RNA: pentamer (o-ring) gp16: pentamer (motor) Prohead-RNA gp10: connector

43 Study DNA packaging by Φ29 motor with optical trap Smith et al, Nature 413:478 (2001)

44 Follow the dynamics of DNA packaging: Constant Feedback Mode Smith et al, Nature 413:478 (2001)

45 Highly efficient DNA packaging by Φ29 motor > 5 µm DNA can be packaged by a single motor highly processive motor takes ~5.5 min to package the entire Φ29 genome Smith et al, Nature 413:478 (2001)

46 DNA packaging against internal pressure Packaging slows down when the genome is > 50% filled Rate decrease results from internal pressure buildup in the prohead Smith et al, Nature 413:478 (2001)

47 Measure force during packaging: No-feedback Mode Smith et al, Nature 413:478 (2001)

48 Motor can work against 57 pn external force The most powerful motor studied to date Smith et al, Nature 413:478 (2001)

49 Internal pressure builds up as the genome is packaged Internal force reaches 50 pn when the whole genome is packaged can be used to initiate DNA injection during infection Smith et al, Nature 413:478 (2001)

50 Pauses and Slips during packaging Pauses: ATP binding? Slips: dissociation and re-binding Smith et al, Nature 413:478 (2001)

51 Dual beam laser traps allow more sensitive detection Moffitt et al, Nature 457:446 (2009)

52 Observation of discrete steps with ATP-dependent pauses pause mean step size ~ 10 bp pauses: ATP-binding bursts: power stroke burst Moffitt et al, Nature 457:446 (2009)