Presentation is loading. Please wait.

Presentation is loading. Please wait.

Structural Stability of Proteins Tom Ioerger Brockwell DJ, Paci E, Zinober RC, Beddard GS, Olmsted PD, Smith DA, Perham RN, Radford SE. (2003). Pulling.

Similar presentations


Presentation on theme: "Structural Stability of Proteins Tom Ioerger Brockwell DJ, Paci E, Zinober RC, Beddard GS, Olmsted PD, Smith DA, Perham RN, Radford SE. (2003). Pulling."— Presentation transcript:

1 Structural Stability of Proteins Tom Ioerger 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): 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): 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: 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: Paci, E. and Karplus, M. (2000). Unfolding proteins by external forces and temperature: The importance of topology and energetics. PNAS, 97(12): Cieplak, M., Hoang, T.X., and Robbins, M.O. (2002). Thermal folding and mechanical unfolding pathways of protein secondary structure. Proteins, 49:

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? (  -helices vs.  -sheets) –Relationship to thermodynamic stability? –Similarity of unfolding pathways? –Modeling and MD simulation? –Strengthening in protein design?

3 Atomic Force Microscope: ubiquitin titin barnase spectrin

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): Fig. 1 E2lip3 = lipoyl domain of dihydrolipoyl acetyltransferase subunit (E2p) of pyruvate dehydrogenase from E. coli E2lip3: 41 residues I27 (titin): 89 residues

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

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

7 Brockwell - Fig. 5 Unfolding Rates: k u 0 E2lip3(+) = s -1 k u 0 I27 = s -1 k u 0 E2lip3(+) = 3.8*k u 0 I27

8 XPLOR or NAMD software with CHARMM force field all-atom, implicit solvent ends attached to harmonic spring, 1000pN/nm pulling speeds: nm/s (?!) (probably ~ nm/s) Brockwell - Fig. 6 Lys41 N-term C-term 0ns 10ns20ns SMD: Steered Molecular Dynamics Simulation

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): 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): Ubiquitin, 76 residues possible PDB model: 1BT0 (Rub1)

11 Lys48-Cterm: 29 residues

12 Unfolding kinetics: force depends on pulling speed Fernandez - Fig. 3 a=a 0 exp(F  x/k B T) F=ln(a/a 0 )*(k B T)/  x) a 0 =0-force unfolding rate related to pulling speed mol/s => nm/s can also get  x by fitting

13 Fernandez - Fig. 4 Explaining unfolding barriers: a) both break 5 H-bonds b) both shearing c) same work to unfold W N-C = 51 pN nm W Lys48 = 54 pN nm Monte Carlo Simulation a) 2 state kinetic model: k u (F)=Aexp[-(  G u -F  x u )/k B T] k f (F)=Aexp[-(  G f -F  x f )/k B T] b) different trigger distances: W = F*  x  x N-C = 0.25nm => higher force  x Lys48 = 0.63nm => lower force 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:

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

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: 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: k u =4.91 s -1,  G=7.5 kcal/mol barnase: k u =3.37 s -1,  G=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 Paci, E. and Karplus, M. (2000). Unfolding proteins by external forces and temperature: The importance of topology and energetics. PNAS, 97(12): Forced unfolding of spectrin T(ps) F(pN) End-to-end distance (A) tertiary structure ruptures 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: Multiple peaks over a range of elongations...

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

21 Cieplak, M., Hoang, T.X., and Robbins, M.O. (2002). Thermal folding and mechanical unfolding pathways of protein secondary structure. Proteins, 49: 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) Conclusion: forced unfolding is NOT necessarily the opposite of the native folding pathway (at least not for  -helices). 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.


Download ppt "Structural Stability of Proteins Tom Ioerger Brockwell DJ, Paci E, Zinober RC, Beddard GS, Olmsted PD, Smith DA, Perham RN, Radford SE. (2003). Pulling."

Similar presentations


Ads by Google