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Structural Stability of Proteins

Structural Stability of Proteins. Brockwell DJ, Paci E, Zinober RC, Beddard GS, Olmsted PD, Smith DA, Perham RN, Radford SE. (2003). Pulling geometry defines the mechanical resistance of a beta-sheet protein. Nature Structural Biology , 10(9):731-7.

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Structural Stability of Proteins

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  1. Structural Stability of Proteins • Brockwell DJ, Paci E, Zinober RC, Beddard GS, Olmsted PD, Smith DA, Perham RN, Radford SE. (2003). Pulling geometry defines the mechanical resistance of a beta-sheet protein. Nature Structural Biology, 10(9):731-7. • Carrion-Vazquez, M., Li, H., Lu, H., Marszalek, P.E., Oberhauser, A.F., and Fernandez, J.M. (2003). The mechanical stability of ubiquitin is linkage dependent. Nature Structural Biology, 10(9):738-43. • Altmann, S.M., Grunberg, R.G., Lenne, P.F., Ylanne, J., Raae, A., Herbert, K., Saraste, M., Nilges, M., Heinrich Horber, J.K. (2002). Pathways and intermediates in forced unfolding of spectrin repeats. Structure, 10:1085-1096. • Best, R.B., Li, B., Steward, A., Daggett, V., and Clarke, J. (2001). Can non-mechanical proteins withstand force? Stretching barnase by atomic force microscopy and molecular dynamics simulation. Biophysical Journal, 81:2344-2356. • Paci, E. and Karplus, M. (2000). Unfolding proteins by external forces and temperature: The importance of topology and energetics. PNAS, 97(12):6521-6526. • Cieplak, M., Hoang, T.X., and Robbins, M.O. (2002). Thermal folding and mechanical unfolding pathways of protein secondary structure. Proteins, 49:104-113. Tom Ioerger

  2. Motivations: • proteins that play a structural role (resilience to physical stress) • actin/myosin, phage tail fibers, bacterial fimbrin • proteins that involve motions (transmission of forces) • protein secretory system, ATPase motor domain • DNA polymerase, helicase, ribosome • Questions: • How to quantify mechanical stability? • Dependence on secondary structure? (a-helices vs. b-sheets) • Relationship to thermodynamic stability? • Similarity of unfolding pathways? • Modeling and MD simulation? • Strengthening in protein design?

  3. Atomic Force Microscope: ubiquitin spectrin titin barnase

  4. Brockwell DJ, Paci E, Zinober RC, Beddard GS, Olmsted PD, Smith DA, Perham RN, Radford SE. (2003). Pulling geometry defines the mechanical resistance of a beta-sheet protein. Nature Structural Biology, 10(9):731-7. Fig. 1 E2lip3: 41 residues I27 (titin): 89 residues E2lip3 = lipoyl domain of dihydrolipoyl acetyltransferase subunit (E2p) of pyruvate dehydrogenase from E. coli

  5. Brockwell - Fig. 2 (I27)5 185pN, 24.2nm (I27)4:E2lip3(+) 10.0nm (I27)4:E2lip3(-) 187pN, 24.1nm (I27)2:E2lip3(-):(I27)2 Curves fit by WLC model: (worm-like chain)

  6. (I27)5 (I27)4: E2lip(+) (I27)4: E2lip(-) Brockwell - Fig. 3

  7. Unfolding Rates: ku0E2lip3(+) = 0.0076 s-1 ku0I27 = 0.0020 s-1 ku0E2lip3(+) = 3.8*ku0I27 Brockwell - Fig. 5

  8. SMD: Steered Molecular Dynamics Simulation • XPLOR or NAMD software with CHARMM force field • all-atom, implicit solvent • ends attached to harmonic spring, 1000pN/nm • pulling speeds: 108-1010nm/s (?!) • (probably ~100-10000nm/s) N-term Lys41 C-term N-term 10ns 20ns 0ns Brockwell - Fig. 6

  9. Hui Lu, Barry Isralewitz, André Krammer, Viola Vogel, and Klaus Schulten (1998). Unfolding of Titin Immunoglobulin Domains by Steered Molecular Dynamics Simulation. Biophysical Journal, 75(2):662-671. Water shells: pre-equilibrate restrain waters Steering force applied to atoms on end: f=k(vt-x) a) start state b) pre-burst c) post-burst

  10. Carrion-Vazquez, M., Li, H., Lu, H., Marszalek, P.E., Oberhauser, A.F., and Fernandez, J.M. (2003). • The mechanical stability of ubiquitin is linkage dependent. Nature Structural Biology, 10(9):738-43. Ubiquitin, 76 residues possible PDB model: 1BT0 (Rub1)

  11. Lys48-Cterm: 29 residues

  12. Unfolding kinetics: force depends on pulling speed a=a0exp(FDx/kBT) F=ln(a/a0)*(kBT)/Dx) a0=0-force unfolding rate related to pulling speed mol/s => nm/s can also get Dx by fitting Fernandez - Fig. 3

  13. Monte Carlo Simulation a) 2 state kinetic model: ku(F)=Aexp[-(DGu-FDxu)/kBT] kf(F)=Aexp[-(DGf-FDxf)/kBT] b) different trigger distances: W = F*Dx DxN-C = 0.25nm => higher force DxLys48 = 0.63nm => lower force Explaining unfolding barriers: a) both break 5 H-bonds b) both shearing c) same work to unfold WN-C = 51 pN nm WLys48 = 54 pN nm M. CARRION-VAZQUEZ, A.F. OBERHAUSER, S.B. FOWLER, P.E. MARSZALEK, S.E. BROEDEL, J. CLARKE, and J.M. FERNANDEZ (1999). Mechanical and chemical unfolding of a single protein: A comparison. PNAS, 96:3694-3699. Fernandez - Fig. 4

  14. Potential role in protein degradation by proteosomes... Fernandez - Fig. 4

  15. Best, R.B., Li, B., Steward, A., Daggett, V., and Clarke, J. (2001). Can non-mechanical • proteins withstand force? Stretching barnase by atomic force microscopy and molecular • dynamics simulation. Biophysical Journal, 81:2344-2356. barnase

  16. MD simulations show differences in pathways in forced (pulled) versus thermodynamic unfolding: • Forced unfolding retains core, unravels at ends first • Thermal unfolding is more evenly distributed throughout molecule

  17. No “key” event in unfolding for barnase • Transition states (right before burst) are highly structured and native-like • Is mechanical strength determined by fold or function? • Unfolding rates in solution are similar: • titin: ku=4.91 s-1, DG=7.5 kcal/mol • barnase: ku=3.37 s-1, DG=10.2 kcal/mol • from chemical denaturation with Gdm-HCl • Yet barnase unfolds at much lower forces: • titin: 190 pN • barnase: 70 pN • Titin needs to be mechanically strong for its function; • Barnase does not

  18. Forced unfolding of spectrin • Paci, E. and Karplus, M. (2000). Unfolding proteins by external forces and temperature: • The importance of topology and energetics. PNAS, 97(12):6521-6526. End-to-end distance (A) tertiary structure ruptures T(ps) F(pN) partially stable intermediates... In contrast, in thermal unfolding, helices tend to fray much sooner.

  19. Intermediates in the unfolding of spectrin • Altmann, S.M., Grunberg, R.G., Lenne, P.F., Ylanne, J., Raae, A., Herbert, K., Saraste, M., Nilges, M., Heinrich Horber, J.K. (2002). Pathways and intermediates in forced unfolding of spectrin repeats. Structure, 10:1085-1096. Multiple peaks over a range of elongations...

  20. Clustering of intermediates Helix B “kinks” Helix B “flips” P62A/G66A double- mutant in helix B hinge removes 15A peak Two general models of mechanical unfolding: 1) unique rupture event (force peak), followed by smooth unfolding 2) gradual unfolding through various intermediates

  21. Cieplak, M., Hoang, T.X., and Robbins, M.O. (2002). Thermal folding and • mechanical unfolding pathways of protein secondary structure. Proteins, 49:104-113. Go-like simulation: beads on a string (C-alpha atoms only) artificial force field (quadratic bond stretching, 6-12 “L-J” potential) Langevin dyanmics (solvent viscosity) On pulling, ends unravel first. Even distribution of force. Fewer native contacts stabilize ends. Timing of (i,i+4) contacts. Ends fold first too (tc). Timing of (i,16-i) contacts. Middle folds first (tc) and is pulled apart last (du). Stress focused on end bond. Conclusion: forced unfolding is NOT necessarily the opposite of the native folding pathway (at least not for a-helices).

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