Mechanical Proteins. Stretching imunoglobulin and fibronectin. domains of the muscle protein titin. Adhesion Proteins of the Immune System

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Elmer Whitehead
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Mechanical Proteins F C D B A domains of

1 Mechanical Proteins F C D B A domains of the muscle protein titin E Stretching imunoglobulin and fibronectin G NIH Resource for Macromolecular Modeling and Bioinformatics Theoretical Biophysics Group, Beckman Institute, UIUC Adhesion Proteins of the Immune System

2 Immunoglobulin Domains

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

4 Contracting and Relaxing Muscle Needs ATP!

(entropic spring) titin immunoglobulin-like (Ig) domain 40 Å Under intermediate

5 Titin the Longest Protein in the Human Genome titin I-band sarcomere 2 µm I1 PEVK region; random coil I27 I41 Under weak forces, the PEVK region extends (entropic spring) titin immunoglobulin-like (Ig) domain 40 Å Under intermediate forces, Ig domains stretch by ~10A; under strong forces, they unfold one by one, by 250A

Probing the Passive Elasticity of Muscle: I27 Marszalek et al., Nature, 402,100-103 (1999) Krammer et al., PNAS, 96, 1351-1356 (1999) Lu et al., Biophys. J.

6 Probing the Passive Elasticity of Muscle: I27 Marszalek et al., Nature, 402, (1999) Krammer et al., PNAS, 96, (1999) Lu et al., Biophys. J., 75, (1998) sarcomere Computational Stretching of Ig: Trajectory Animation titin immunoglobulin-like (Ig) domain Gaub et al., Fernandez et al. Hui Lu, Barry Isralewitz

Quantitative Comparison Bridging the gap between SMD and AFM experiments Steered Molecular Dynamics (SMD) Force-extension curve Force-pulling

7 Quantitative Comparison Bridging the gap between SMD and AFM experiments Steered Molecular Dynamics (SMD) Force-extension curve Force-pulling velocity relationship AFM range Current SMD range Target simulation range SMD data AFM data Extrapolation of AFM data Extension curve Hui Lu, Barry Isralewitz

8 Stretching modular proteins Detailed View Extension occurs in two steps extension Schematic view and typical Extension vs. force plot Distribution of measured forces For step 1 and step 2

Two Force Bearing Components Two types of SMD simulations I. Native structure (2 A of extension) II. Mechanical unfolding intermediate (10 A of extension) III.

9 Two Force Bearing Components Two types of SMD simulations I. Native structure (2 A of extension) II. Mechanical unfolding intermediate (10 A of extension) III. Unfolded state (250 A of extension) Constant velocity: Lu et al, Biophys J., 72, 1568 (1007) three force interstrand (A B) hydrogen bonds Hui Lu, Barry Isralewitz six interstrand (A G) hydrogen bonds Constant force: Lu and Schulten, Chem. Phys., 247 (1999) 141. Reviewed in Isralewitz et al. Curr. Opinion Struct. Biol. 11: (2001)

about 100 times / ns; for forces of 750 pn water fluctuation controls

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

Titin Domain Heterogenity: Comparing Oxidized and Reduced I1 titin I 1 I 27 PEVK oxidized reduced Stretching I 1 with constant velocity titin I 1 Mayans et al.

13 Titin Domain Heterogenity: Comparing Oxidized and Reduced I1 titin I 1 I 27 PEVK oxidized reduced Stretching I 1 with constant velocity titin I 1 Mayans et al. Structure 9: (2001) B A A G I 1 in 70 Å water sphere of 70 Å (18,000 atoms) F disulfide bond titin I27 Use of program NAMD on 32 Processor (1.1 GHz Athlon) Linux cluster: 1 day/ns Mu Gao

14 Titin Domain Heterogenity: I 1 I 27 pre-stretching slack tight in I1 Stretching with constant force of 750 pn titin I 1 Mayans et al. Structure 9: (2001) Hydrogen bond energies /(Kcal/mol) I 1 in 70 Å water sphere of 70 Å (18,000 atoms) B A A E3(H)-K31(O) G F PEVK 6 A-B bonds tight slack disulfide bond E3(O)-K31(H) time / ps titin I27 3 A-B bonds easy slack K6(H)-V29(O) Use of program NAMD on 32 Processor (1.1 GHz Athlon) Linux cluster: 1 day/ns K6(O)-V29(H) F8(H)-R27(O) E9(O)-R27(H) Mu Gao

15 Fibronectins

Architecture and Function of Fibronectin

FN-I FN-II FN-III 10 µm FN-III 7 FN-III 9

16 Architecture and Function of Fibronectin Modules Extracellular matrix Fibronectin FN-I FN-II FN-III 10 µm FN-III 7 FN-III 9 FN-III 8 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 A B 35 Å F G Andre Krammer V. Vogel, U. Wash.

17 Fibronectin Matrix in Living Cell Culture Force (pn) FN-III Extension (nm) 100 µ m Atomic force microscopy observations Ohashi et al. Proc. Natl. Acad. Sci USA 96: (1999) Andre Krammer V. Vogel, U. Wash.

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

Probing Unfolding Intermediates in FN-III 10 F FnIII 10 module solvated in a water box 55 60 367 Å 3 (126,000 atoms) Steered Molecular Dynamics, periodic boundary conditions, NpT ensemble,

19 Probing Unfolding Intermediates in FN-III 10 F FnIII 10 module solvated in a water box 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 Mu Gao, U. Illinois A. Krammer, D. Craig, V. Vogel, U. Wash.

20 Probing Unfolding Intermediates in FN-III 10 Specific stretching and unfolding pathway for constant force (500 pn) stretching: scenario with A rupturing first Complete stretching and unfolding pathway: after straightening and partial A- B separation, A,, or A+G or G rupture first

21 Stretching FN-III 1 : Pronounced Intermediate SMD simulations (150,000 atoms) M. Gao, D. Craig, O. Lequin, I. D. Campbell, V. Vogel, and K. Schulten. Structure and functional significance of mechanically unfolded fibronectin type III1 intermediates. Proc. Natl. Acad. Sci. USA, 100: , AFM experiments Force (pn) 281±9 Å 204±16 Å 87±16 Å Extension (Å) NMR structure

22 Stretching FN-III 1 : Pronounced Intermediate SMD simulations (150,000 atoms) M. Gao, D. Craig, O. Lequin, I. D. Campbell, V. Vogel, and K. Schulten. Structure and functional significance of mechanically unfolded fibronectin type III1 intermediates. Proc. Natl. Acad. Sci. USA, 100: , AFM experiments Force (pn) 281±9 Å 204±16 Å 87±16 Å Extension (Å) NMR structure

23 Stretching FN-III 1 : Pronounced Intermediate SMD simulations (150,000 atoms) M. Gao, D. Craig, O. Lequin, I. D. Campbell, V. Vogel, and K. Schulten. Structure and functional significance of mechanically unfolded fibronectin type III1 intermediates. Proc. Natl. Acad. Sci. USA, 100: , AFM experiments Force (pn) 281±9 Å 204±16 Å 87±16 Å Extension (Å) NMR structure

24 Adhesion Proteins of the Immune System

(1999) Cell, 97, 791 90,000 atoms NAMD simulation on Linux cluster 120 ps

25 Strengthening the Immune System CD2 and CD58 contain Ig domains 0.05 Å / ps slow Model Based on Crystal Structure: Wang et al. (1999) Cell, 97, ,000 atoms NAMD simulation on Linux cluster 120 ps so far Å / ps pulling velocity Marcos Bayas, D. Leckband, K. Schulten, submitted 1 Å / ps

26 Separation at 0.05 Å /ns Pulling : salt bridges important (Aralunandam et al 1993, 1994) for adhesion are broken last Marcos Bayas, D. Leckband, K. Schulten, Biophys. J. 84: , 2003

27 Behavior of Non-Mechanical Proteins

Force-induced Unfolding of Other Domains C2 domain of synaptotagmin I (all sheet protein) no initial force peak much less resistance to external forces than Ig and FnIII during unfolding hydrogen

28 Force-induced Unfolding of Other Domains C2 domain of synaptotagmin I (all sheet protein) no initial force peak much less resistance to external forces than Ig and FnIII during unfolding hydrogen bonds not required to be broken in clusters Lu and Schulten, Proteins, 35, (1999) NIH Resource for Macromolecular Modeling and Bioinformatics Theoretical Biophysics Group, Beckman Institute, UIUC

29 Classification of β Sandwich Domains Class I a: strong resistance Class I b: less resistance Class I c: little resistance Lu and Schulten, Proteins, 35, (1999) NIH Resource for Macromolecular Modeling and Bioinformatics Theoretical Biophysics Group, Beckman Institute, UIUC

Force-induced unfolding of alpha-helical protein Cytochrome C6 (all helix protein) no initial force peak much less resistance to external forces than Ig and FnIII during unfolding hydrogen bonds

30 Force-induced unfolding of alpha-helical protein Cytochrome C6 (all helix protein) no initial force peak much less resistance to external forces than Ig and FnIII during unfolding hydrogen bonds not required to be broken in clusters Lu and Schulten, Proteins, 35, (1999) NIH Resource for Macromolecular Modeling and Bioinformatics Theoretical Biophysics Group, Beckman Institute, UIUC

31 Ubiquitin Fatemeh Araghi, Timothy Isgro, Marcos Sotomayor

32 Monoubiquitylation versus multi-ubiquitylation

33 Structure-Function Relationship Proteasome Degradation

34 First SMD Simulation First peak when the first beta strand is stretched out SMD simulation, with constant velocity Box of water 70x240x70 A ~81K atoms smd velocity 0.4 A/ps smd spring constant 7 kcal/mol A^2

35 Ubiquitin Unfolding I Force (pn) cv-0.02 Å/ps E64 Q2 S65 F4 L67 K6 L69 water N-term fixed 0 Å, 1 ps Q2 F4 extension (Å) K6 E64 S65 L67 L69 14 Å, 700ps Q2 F4 K6 E64 15 Å, 813ps S65 L67 L69 Mu Gao

36 Ubiquitin Unfolding II Extension (Å) cf-500pn K48 K48 fixed F45 I44 L50 H68 V70 L43 R42 R72 Q40 F45 K48 F45 H68 I44 H68 L43 V70 R42 V70 R72 Q40 R72 0 Å, 1 ps 9 Å, 60 ps L50 Time (ps) K48 L50 I44 L43 R42 Q40 17 Å, 320 ps R72 H68 V70 F45 Mu Gao K48 I44 L43 Q40 R42 L50 19 Å, 340 ps

37 Pulling Dimer SMD (v=0.4 A/ps k=7 kcal/mol A^2) constant P Two monomers separate.

Mechanical Proteins. Stretching imunoglobulin and fibronectin. domains of the muscle protein titin. Adhesion Proteins of the Immune System

Mechanical Proteins. Stretching imunoglobulin and fibronectin. domains of the muscle protein titin. Adhesion Proteins of the Immune System Mechanical Proteins F C D B A domains of the muscle protein titin E Stretching imunoglobulin and fibronectin G NIH Resource for Macromolecular Modeling and Bioinformatics Theoretical Biophysics Group,