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Biophysik der Moleküle

kl. Phys. HS Mo 14-16 u. Do 9-10

Vorlesung Rädler WS 2010

Tutorials: Do 10-11 od. Do 11-12 od. Do 18-19 (Fr 9-10)

http://www.softmatter.physik.uni-muenchen.de/tiki-index.php?page=Biophysik_MolekueleWS10

Erich Sackmann &

Rudi Merkel

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Rob PhilipKane KondrevJulie Theriot

Websitehttp://www.garlandscience.com/textbooks/0815341636.asp

- Jonathon Howard

„Mechanics of Motor Proteins and the Cytoskeleton“

Literatur

Bruce Alberts et al.:Molecular Biologyof the Cell (MBoC)

Helmut Pfützner

Roland Glaser

Rodney Cotterill

links : Auf den Folien und auf der Vorlesungsseitewww.softmatter.physik.uni-muenchen.dewww.schwerpunkt-biophysik.physik.lmu.de

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Rädler/ WS 2010

Inhalt Biophysik der Moleküle

Proteine! Struktur und Dynamik! Funktion als Enzyme! Molekulardynamik Rechnung!

Mechanik der Biomoleküle! Reversible Entfaltung! Bindungen unter Last

Life at low Reynods numbers! Brown‘sche Motoren

Muskel! Molekulare Mechanismen ! ! !

Molekulare Motoren! Turbinen! ! ATP Synthase! ! Flagellenmotor ! Linearmotoren! ! Myosin! ! Kinesin! Cilienmotor

Zellmotilität und Form! Physik der Biopolymere! ! Struktur! ! Dynamik! ! Regulation! Zytoskelett! ! Interm. Filament ! ! Miktotubuli! ! Aktin !

From the living organism down to molecules

movie H.Berg lab

From the living organism down to molecules

cartoon by Goodsell

From the living organism down to molecules

a) Kohlenstoff

b) Zucker

c) ATP

d) Chlorophyll

e) tRNA

f) Antikörper

g) Ribosome

h) Poliovirus

i) Myosin

j) DNA

k) F-actin

l) Enzyme

m) Pyruvat dehydrogenase

Goodsell, 1993

Nucleic

Acids

Proteins

Lipids

Sugars

There are four classes of macromolecules

Macromolecules exhibit a

hierachy of structures

storage of genetic information

cellular

building blocks

& machinery

e.g.

cytoskeleton

enzymes

pores

ECM

Inventory by numbers

Ribosome: Translating Code into Function

MOVIE : „Inner Life“ shows biomolecules at work

http://multimedia.mcb.harvard.edu/media.html

(be aware : animations use artistic freedom)

Speed:

30 na/s

Fidelity:

105

RNA Polymerase II Complex

See Patrick Cramer et al. Genzentrum LMU

_ o=EE

(!

= 4g c

o r !. = 6

ä e3 =

G

ol

- 2 ! 3

E + !5ö f t t ( u--tr

u

_o

WWWWWWWWWWWI o-ls

- f f iäx i r l lum b io log ica l t imesca le ; age o f Ear th , 4 b i l l i on years = l0 l7 s

<- d ivers i f i ca t ion o f metazoans, 600 mi l l ion years = 2 x l0 l6 s

- d ivers i f i ca t ion o f humans and ch impanzees, 6 mi l l ion years = 2 x l0 la s

- sequo ia l i fespan, 3000 years = l0 l I s

<- Calapagos tortoise l i fespan, I 50 years = 5 x l0e s

+ human embryon ic s tem ce l l l i ne doub l ing t ime, 72 h = 3 x l0s s

- mayf ly adu l t l i fespan, I day = 9 x l0a s

<J E. co l i doub l ing t ime, 20 min = I .2 x | 0

j s

\ uns tab le p ro te in ha l f - l i fe , 5 min = 300 s

- lysozyme turnover rate, = 0.5 s-l

- carbonic anhydrase turnover rate, = 600,000 s-'

<- side chain rotat ion, = 500 ps

- H-bond rear rangements in water , = l0 ps

o

G L

_ > : : ,6 0qJ - .,;

o a- Y -

o '

r,,

- coVäl!ht bond vibrat ion in water, = I fs

typical

biological

timescales

See Grubmüller et al. MPI Göttingen

MD Calculation on Water Transport through ecoli Aquaporin

Stochastic Transport!

MD Calculation on Water Transport through ecoli Aquaporin

See Grubmüller et al. MPI Göttingen

See Joe Howard et al. MPI Dresden

Manfred Schliwa et al. LMU

Intracellular Traffic over Long Distances

Axon 10 µm

Kinesin ‘Walking’

S.M. Block, Cell 93, 5 (1998)

Cytoskeleton: a dynamic polymer network

Cell Motility

Fish Keratinicyte

Dis

tance [nm

]

Actin helical

repeat: 36

nm

Force Feedback

Dis

pla

cem

en

t [n

m]

266

228

190

152

114

76

38

0

-38

-76

-114

0.80.60.40.20.0

Time[s]

Single Molecule

Optical Force

Clamp

Matthias Rief et al.

Bead with

MyosinV on Actin

Filament

Where is the physics ?

• measurements

• quantitative models

• concepts

Biophysical models the same object (DNA) depending on the problem/context

use approximations / idealization

212 Chapter 6. Information, entropy, temperature [[Draft March 8, 2002]]

that numerous biological processes like cell division and protein synthesis depend on the abilityof the cell to unfold RNA (as well as to unfold proteins and DNA), and that such unfoldinginvolves mechanical forces, which one might be able to reproduce using biophysical techniques.In the general strategy utilized by cells, an enzyme converts chemical energy (for example, fromATP) into mechanical work, which is then used to restructure the polymer. To investigate howRNA might respond to mechanical forces, we needed to find a way to grab the ends of individualmolecules of RNA, and then to pull on them and watch them buckle, twist and unfold under thee!ect of the applied external force.

We used an optical tweezer apparatus, which allows very small objects, like polystyrene beadswith a diameter of ! 3 µm, to be manipulated using light (Figure 6.10). Though the beads are

actuatorbead

trap bead

laser trap

actuator bead

trap bead

handle

RNAmolecule

actuator

Figure 6.10: (Cartoon.) Optical tweezer apparatus. A piezo-electric actuator controls the position of the bottom

bead. The top bead is captured in an optical trap formed by two opposing lasers, and the force exterted on the

polymer connecting the two beads is measured from the change in momentum of light that exits the dual beam trap.

Molecules are stretched by moving the bottom bead vertically. The end-to-end length of the molecule is obtained as

the di!erence of the position of the bottom bead and the top bead. Inset: The RNA molecule of interest is coupled

to the two beads via molecular handles. The handles end in chemical groups that can be stuck to complementary

groups on the bead. Compared to the diameter of the beads (! 3000 nm), the RNA is tiny (! 20 nm).

transparent, they do bend incoming light rays. This transfers some of the light’s momentum toeach bead, which accordingly experiences a force. A pair of opposed lasers, aimed at a commonfocus, can thus be used to hold the beads in prescribed locations. Since the RNA is too small tobe trapped by itself, we attached it to molecular “handles” made of DNA, which were chemicallymodified to stick to specially prepared polystyrene beads (Figure 6.10, inset). As sketched in theinset, the RNA sequence we studied has the ability to fold back on itself, forming a “hairpin”structure (see Figure 3.19 on page 88).

When we pulled on the RNA via the handles, we saw the force initially increase smoothly withextension (Figure 6.11a, black curve), just as it did when we pulled on the handles alone: The DNAhandles behaved much like a spring (a phenomenon to be discussed in Chapter 9). Then, suddenly,at f = 14.5 pN there was small discontinuity in the force-extension curve (points labeled “a” and“b”). The change in length (! 20 nm) of that event was consistent with the known length of thepart of the RNA that could form a hairpin. When we reduced the force, the hairpin refolded andthe handles contracted.

ExperimentsDown to the single molecule level

the coil - globule transition

light scattering force spectroscopy

Brownian Motor

Concepts

multiple

manifestations

of the simple

harmonic oscillator

in biophysics