NIH Resource for Macromolecular Modeling and Bioinformatics

Theoretical Biophysics Group, Beckman Institute, UIUC

A

B

C D

EF

G

Mechanical Functions of Proteins Studied by Atomic Force Microscopy

Stretching imunoglobulin and fibronectin domains of the muscle protein titin

Atomic force microscopeexperiment on three domains of titin

Moleculardynamicssimulation

Forces can be

substrates, products, signals,

catalysts of cellular processes

But to what degree can proteins and DNA sustain forces?

How do proteins need to be designed to build machines from them?

Under weak forces, the PEVK region extends(entropic spring)

Under intermediate forces, Ig domains stretch by ~10A;under strong forces, they unfold one by one, by 250A

PEVK region;

random coil

I41

titin immunoglobulin-like (Ig) domain

sarcomere

titin I-band

Titin the Longest Protein in the Human Genome

I27I1

I27I1

40 Å

2 m

15 cm

InstrumentAtomic Force Microscope

AFM Studies of Titin And FN-III Modules

Forc

e (p

N)

Extension (nm)

AFM tip

• Forced unfolding of identical modules occurs one by one v

Reviewed in Fisher et al. Nature Struct. Biol. 7:719-724 (2001)

Extension (nm)

Forc

e (p

N)

0

400

600

0 50 100 150

Titin I27 FN-III

Stretching modular proteins – Detailed View

Schematic view and typicalExtension vs. force plot

extension

Distribution of measured forcesFor step 1 and step 2

Extension occurs in two steps

titin immunoglobulin-like (Ig) domain

sarcomere

Probing the Passive Elasticity of Muscle: I27

Computational Stretching of Ig: Trajectory Animation

Marszalek et al., Nature, 402,100-103 (1999)

Krammer et al.,PNAS, 96, 1351-1356(1999)

Lu et al.,Biophys. J., 75, 662-671 (1998)

Hui Lu, Barry IsralewitzGaub et al.,Fernandez et al.

Two Force Bearing Components

Constant force: Lu and Schulten, Chem. Phys., 247 (1999) 141.

Constant velocity: Lu et al, Biophys J., 72, 1568 (1007)

Two types of SMD simulations

Reviewed in Isralewitz et al. Curr. Opinion Struct. Biol. 11:224-230 (2001)

I. Native structure(2 A of extension)

II. Mechanicalunfolding

intermediate(10 A of extension)

III. Unfolded state(250 A of extension)

three interstrand(A – B)hydrogen bonds

six interstrand(A’ – G)hydrogen bonds

force

Hui Lu, Barry Isralewitz

Quantitative ComparisonBridging the gap between SMD and AFM experiments

Steered Molecular Dynamics (SMD)

Extension curve

AFM range

Current SMD range

Target simulation range

Force-extension curve

SMD data

AFM data

Extrapolation of AFM data

Force-pulling velocity relationship

Hui Lu, Barry Isralewitz

Titin Ig Mechanical Unfolding IntermediateAFM force-extension profile

Marszalek, Lu, Li, Carrion, Oberhauser, Schulten,and Fernandez, Nature, 402, 100 (1999).

SMD simulations with constant forces

E6P

Hui Lu

determination of barrier height basedon mean first passage time

Chemical denaturing experiments(Carrion-Vazquez et al., PNAS, 1999)show a similar U = 22 kcal/mol.

NIH Resource for Macromolecular Modeling and BioinformaticsTheoretical Biophysics Group, Beckman Institute, UIUC

Lu and Schulten, Chem. Phys., 247 (1999) 141.

The key event of titin Ig unfolding can be examined as a barrier crossing process.

U(x)

U

a b

-Fx(F fixed)

a b

(F) = 2D(F) [e(F) – (F) –1]

(D) = (b – a)2/2D ~ 25 ns

(F) = [U – F(b-a)]

Sampling Titin Ig Unfolding Barrier

stationary force applied (pN)

bu

rst

time

(p

s)

400 600 800 1000 1200

1200

0

400

0

800potential barrier of

U = 20 kcal/mol

the best fit suggests a

Hui Lu

Sampling Distribution of Barrier Crossing Times

Fraction N(t) of domains that have not crossed the barrier, for linear model potential, is:

Approximated by double exponential (holds for arbitrary potential): N(t) = [t1 exp(-t/t1) – t2 exp(-t/t2)]/(t1-t2), Nadler & Schulten, JCP., 82, 151-160 (1985)

Multiple runs of I27 unfoldingwith a 750 pN stationary force

Barrier crossing times of 18 SMD simulations

Theoretical prediction of the barrier crossing times

NIH Resource for Macromolecular Modeling and BioinformaticsTheoretical Biophysics Group, Beckman Institute, UIUC

U = 20 kcal/mol

Chemical denaturing experiments(Carrion-Vazquez et al., PNAS, 1999)show a similar U = 22 kcal/mol.

Hui Lu

NIH Resource for Macromolecular Modeling and Bioinformatics

Theoretical Biophysics Group, Beckman Institute, UIUC

Rupture/Unfolding Force F0 and its Distribution

Israilev et al., Biophys. J., 72, 1568-1581 (1997)Balsera et al., Biophys. J., 73, 1281-1287 (1997)

(F0) = 1 ms time of measurement => F0 rupture/unfolding force

( )]1)(exp[)( )(00 0 −

−−Δ−−= −Δ− abFUB

eeab

TkUabFFp ββκ

ββκ

Distribution of rupture/unfolding force

= 2(F)/2Dkv

Fernandez et al., (PNAS, in press)

stationary force applied (pN)

bu

rst

time

(p

s)

400 600 800 1000 1200

1200

400

0

800potential barrier of

U = 20 kcal/mol

the best fit suggests a

Manel Balsera, Sergei Izrailev

Water-Backbone Interactions Control Unfolding

During stretching,water moleculesattack repeatedlyinterstrand hydrogen bonds,about 100 times / ns;for forces of 750 pN waterfluctuation controls unfolding!

E12

A B

A'G

Lu and Schulten, Biophys J.79: 51-65 (2000)

1000 pN

750 pN

water greases protein folding !

410 ps, 8 Ånative 585 ps, 12 Å

Time (ps)

Ext

ensi

on (

Å)

SMD-CF-750 pN

water

A A AB B B

E3 K31 K31 K31E3E3

K6 K6 K6V29 V29 V29

F8 F8F8

R27R27 R27

E9 E9 E9

Hyd

roge

n B

ond

Ene

rgy

(kca

l/m

ol)

titin I1 titin I27Mayans et al. Structure 9:331-340 (2001)

I1 I27

PEVK

2 H bonds

4 H bonds

6 A-B bonds 3 A-B bonds

time / ps

E3(H)-K31(O) E3(O)-K31(H)

K6(H)-V29(O)

K6(O)-V29(H) F8(H)-R27(O) E9(O)-R27(H)

Hydrogen bond energies /(Kcal/mol)

F

AB

GA’

I1 in 70 Å water sphere of 70 Å (18,000 atoms)

Use of programNAMD on 32Processor (1.1 GHz Athlon)Linux cluster:1 day/ns

easyslacktight

slack

Titin Domain Heterogenity:pre-stretching slack tight in I1

Mu Gao

disulfidebond

Architecture and Function of Fibronectin Modules

FN-IIIFN-IIFN-IFibronectin

FN-III7

FN-III8

FN-III9

FN-III10

• Fibronectin is a major component of extracellular matrix

• It consists of three types of modules: type I, type II, and type III

• Highly extensible

G

FB

A

10

m

Extracellular matrix

35 Å

Andre Krammer, U. Wash.

Fibronectin Matrix in Living Cell Culture

Ohashi et al. Proc. Natl. Acad. Sci USA 96:2153-2158 (1999)

100 m

Forc

e (p

N)

Extension (nm)

FN-III

Atomic force microscopy observations

RGD Loop of FnIII10

Asp80

Gly

79

Arg78

Val1 C Thr94 C

Native FnIII10

Extension of 13 Å

http://www.ks.uiuc.edu

F = 500 pN

fibronectinbinding with RGD loop tointegrin

integrin interacting with extracellular matrix

Krammer et al. Proc. Natl. Acad. Sci USA96:1351-1356 (1999)

Andre Krammer, U. Washington

Probing Unfolding Intermediates in FN-III10

FnIII10 module solvated in a water box 5560367 Å3 (126,000 atoms)

Steered Molecular Dynamics, periodic boundary conditions, NpT ensemble, Particle Mesh Edwald for full electrostatics

NAMD on Linux cluster of 32 Athlon 1.1GHz processors: 10 days/ns

Native Fibronectin III10

F

Mu Gao

ATOMIC FORCE MICROSCOPY FOR BIOLOGISTSby V J Morris, A R Kirby & A P Gunning352pp Pub. date: Dec 1999ISBN 1-86094-199-0 US$51 / £32Contents: Apparatus Basic Principles Macromolecules Interfacial Systems Ordered Macromolecules Cells, Tissue and Biominerals Other Probe Microscopes

Atomic force microscopy (AFM) is part of a range of emerging microscopic methods for biologists which offer the magnification range of both the light and electron microscope,but allow imaging under the 'natural' conditions usually associated with the lightmicroscope. To biologists AFM offers the prospect of high resolution images of biologicalmaterial, images of molecules and their interactions even under physiological conditions, and the study of molecular processes in living systems. This book provides a realisticappreciation of the advantages and limitations of the technique and the present and future potential for improving the understanding of biological systems.