mechanical functions of proteins studied by atomic force microscopy
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Mechanical Functions of Proteins Studied by Atomic Force Microscopy. Molecular dynamics simulation. C. D. F. E. G. Stretching imunoglobulin and fibronectin domains of the muscle protein titin. Atomic force microscope experiment on three domains of titin. B. A. - PowerPoint PPT PresentationTRANSCRIPT
NIH Resource for Macromolecular Modeling and Bioinformatics
Theoretical Biophysics Group, Beckman Institute, UIUC
A
B
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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.