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

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 I27 I1 I27 I1 40 Å 2  m

15 cm Instrument Atomic Force Microscope

AFM Studies of Titin And FN-III Modules Force (pN) Extension (nm) AFM tip Forced unfolding of identical modules occurs one by one v Reviewed in Fisher et al. Nature Struct. Biol. 7: (2001) Extension (nm) Force (pN) Titin I27FN-III

Stretching modular proteins – Detailed View Schematic view and typical Extension vs. force plot extension Distribution of measured forces For 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, (1999) Krammer et al., PNAS, 96, (1999) Lu et al., Biophys. J., 75, (1998) Hui Lu, Barry Isralewitz Gaub 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: (2001) I. Native structure (2 A of extension) II. Mechanical unfolding 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 Comparison Bridging 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 Intermediate AFM 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 based on 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 Bioinformatics Theoretical 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U a b -Fx (F fixed) ab  (F) = 2  D  (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) burst time (ps) potential 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) = [t 1 exp(-t/t 1 ) – t 2 exp(-t/t 2 )]/(t 1 -t 2 ), Nadler & Schulten, JCP., 82, (1985) Multiple runs of I27 unfolding with 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 Bioinformatics Theoretical 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 F 0 and its Distribution Israilev et al., Biophys. J., 72, (1997) Balsera et al., Biophys. J., 73, (1997)  (F 0 ) = 1 ms time of measurement => F 0 rupture/unfolding force Distribution of rupture/unfolding force  =  2 (F)/2  D kv Fernandez et al., (PNAS, in press) stationary force applied (pN) burst time (ps) potential barrier of  U = 20 kcal/mol the best fit suggests a Manel Balsera, Sergei Izrailev

Water-Backbone Interactions Control Unfolding During stretching, water molecules attack repeatedly interstrand hydrogen bonds, about 100 times / ns; for forces of 750 pN water fluctuation controls unfolding! E12 A B A' G Lu and Schulten, Biophys J.79: (2000) 1000 pN 750 pN water greases protein folding !

410 ps, 8 Ånative585 ps, 12 Å Time (ps) Extension (Å) SMD-CF-750 pN water AAABB B E3K31 E3 K6 V29 F8 R27 E9 Hydrogen Bond Energy (kcal/mol) titin I 1 titin I 27 Mayans et al. Structure 9: (2001) I1I1 I 27 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 A B G A’ I 1 in 70 Å water sphere of 70 Å (18,000 atoms) Use of program NAMD on 32 Processor (1.1 GHz Athlon) Linux cluster: 1 day/ns easy slack tight slack Titin Domain Heterogenity: pre-stretching slack tight in I1 Mu Gao disulfide bond

Architecture and Function of Fibronectin Modules FN-IIIFN-IIFN-IFibronectin FN-III 7 FN-III 8 FN-III 9 FN-III 10 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 F B 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: (1999) 100  m Force (pN) Extension (nm) FN-III Atomic force microscopy observations

RGD Loop of FnIII 10 Asp 80 Gly 79 Arg 78 Val 1 C  Thr 94 C  Native FnIII 10 Extension of 13 Å F = 500 pN fibronectin binding with RGD loop to integrin integrin interacting with extracellular matrix Krammer et al. Proc. Natl. Acad. Sci USA 96: (1999) Andre Krammer, U. Washington

Probing Unfolding Intermediates in FN-III 10 FnIII 10 module solvated in a water box 55  60  367 Å 3 (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 III 10 F Mu Gao

ATOMIC FORCE MICROSCOPY FOR BIOLOGISTS by V J Morris, A R Kirby & A P Gunning 352pp Pub. date: Dec 1999 ISBN US$51 / £32 Contents: 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 light microscope. To biologists AFM offers the prospect of high resolution images of biological material, images of molecules and their interactions even under physiological conditions, and the study of molecular processes in living systems. This book provides a realistic appreciation of the advantages and limitations of the technique and the present and future potential for improving the understanding of biological systems.