Lecture 2

Single-Molecule Methods

Advantages of single-molecule experiments

Observe heterogeneity: static (differences between molecules)dynamic (history of a single molecule)

Observe single molecules in vitro and in vivo, in real time

Measure positions with nanometre precision

Measure separations with Ångström precision

No need to synchronize population to observe dynamic behaviour

Can study transient events

Can apply force to molecules

Very small sample sizes

Particle trackingDirect observation of rotation of F1 ATPase, Part 2

120° periodicity reflects symmetry of F1.

90°, 30° substeps reveal mechanistic detail.

40nm gold bead greatly reduces drag

bead position measured by dark field microscopy

Yasuda, R. et al., Nature 410, 898 (2001)

Optical transitions between molecular electronic energy levels

Ab

so

rba

nc

e

Flu

ore

sc

en

ce

em

iss

ion

Cy3

wavelength nm

440 490 540 590 640 690

emission filter

excitation filter or laser

absorbed photon

emitted photon (radiative decay)

non-radiative decay

Fluorescence microscopy

Total Internal Reflection Fluorescence (TIRF) microscopy

glass cover slip

objective lens

transmitted rayevanescent field

reflected ray(total internal

reflection)

immersion fluid

specimen in water

ray incident on glass-water interface beyond

critical angle

n1

n2

22

222;22

yxair

ykxiki kkknkeee yx rk

21

211;11

yxairykxkii kkknkee yx rk

12

1

2

1 n

n

k

k

x

y

Fili, N. et al. Nucl. Acids Res. 38, (2010)

Helicase activity measured by TIRF microscopy

helicase unwinds immobilized duplex

fluorophore binds to single-stranded

DNA

pauses and bursts of activity

Each spot corresponds to a single helicase molecule. Intensity increases as the helicase unwinds its DNA substrate.

Single-molecule enzymatic dynamics

Lu, H.P. et al. Science 1998;282:1877 (1998)

Single molecules of cholesterol oxidase immobilized in an agarose gel

The enzyme is fluorescent during part of the catalytic cycle, so individual catalytic turnovers can be monitored. The enzyme operates stochastically, but displays static and dynamic inhomogeneity.

Static inhomogeneity: histogram of on-times for a single molecule fits a single exponential, giving a well-defined rate constant ,BUT different molecules have different rate constants.

Dynamic inhomogeneity: autocorrelation function of on-times reveals memory effects with correlation time ~1s.

on off

deviation of mth on-time from mean

fluorescent labelhand over hand?

inchworm?

asymmetric half steps

23nm 52nm 74nm

full step hand over hand

Microscopy with 1 nm resolution: stepping of myosin V

A Yildiz et al. Science 300, 2061(2003)

Flors, C. et al. ChemPhysChem 10, 2201 (2009)

Super-resolution imaging using switchable fluorophores

1 μm 1 μm

reconstructed super-resolution image

wide field image

DNA

Fluorescence Resonance Energy Transfer (FRET)

(Förster Resonance Energy Transfer)

61

1

oRrE

Energy transfer efficiency:

excitation of donor

radiative decay of donor energy transfer to acceptor

radiative decay of acceptor

donor acceptorr

Following dynamics and function of single molecules by FRET

X Zhuang et al. Science 296,1473 (2002)

substrate S

docking interface

cleavage site

acceptor

donor

Optical tweezers

principle of operation

Power stroke of myosin II

time

disp

lace

men

t

power stroke 5 nm

Veigel, C. et al, Nature 398, 530 (1999)

Force exerted by kinesin

Feedback maintains constant bead position in trap constant force

8nm steps

stall force

Visscher, K. et al, Nature 400, 184 (1999)

Atomic Force Microscopy (AFM)

High-resolution AFM images of native membrane proteins. (a) Ion-driven rotors from spinach chloroplast(b) I. tartaricus FoF1-ATP synthase(c) Native photosynthetic membrane from R. photometricum.(d) Perfringolysin O pore complexes. (e) Dimeric bovine rhodopsin. (f) Extracellular surface of gap junction hemichannels.

Muller, D.J. & Engel, A. Nature Protocols 2, 2191 (2007)

quadrant photodiode

cantilever

tip

Alb

erts

Mol

ecul

ar B

iolo

gy o

f th

e C

ell

4e©

200

2 G

arla

nd S

cien

ce

Single-molecule mechanics of titin by AFM

Stepping of myosin V by high-speed AFM

37 nmKodera, N. et al. Nature 468, 72 (2010)

Other important single-molecule techniques include:

patch clamp

super-resolution optical microscopies with active control of fluorophore emission

fluorescence polarization

magnetic tweezers

imaging by cryoelectron microscopy, electron tomography