Structural Stability of ProteinsTom 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):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.

• 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?

Atomic Force Microscope:

ubiquitin

titin

barnase

spectrin

•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 = lipoyl domain of dihydrolipoyl acetyltransferase subunit (E2p)of pyruvate dehydrogenase from E. coli

E2lip3: 41 residuesI27 (titin): 89 residues

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

L

x

Lxp

TkxF B

4

1

)/1(4

1)(

2

Brockwell - Fig. 3

(I27)5

(I27)4:E2lip(+)

(I27)4:E2lip(-)

Brockwell - Fig. 5

Unfolding Rates: ku

0E2lip3(+) = 0.0076 s-1

ku0

I27 = 0.0020 s-1

ku0

E2lip3(+) = 3.8*ku0

I27

• 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)

Brockwell - Fig. 6

Lys41N-term

N-term C-term

0ns 10ns 20ns

SMD: Steered Molecular Dynamics Simulation

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 stateb) pre-burstc) post-burst

• 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 residuespossible PDB model: 1BT0 (Rub1)

Lys48-Cterm: 29 residues

Unfolding kinetics: force depends on pulling speed

Fernandez - Fig. 3

a=a0exp(Fx/kBT)F=ln(a/a0)*(kBT)/x)a0=0-force unfolding rate related to pulling speed mol/s => nm/scan also get x by fitting

Fernandez - Fig. 4

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

Monte Carlo Simulation a) 2 state kinetic model:

ku(F)=Aexp[-(Gu-Fxu)/kBT]kf(F)=Aexp[-(Gf-Fxf)/kBT]

b) different trigger distances:W = F*xxN-C = 0.25nm => higher forcexLys48 = 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, 96:3694-3699.

Fernandez - Fig. 4

Potential role in protein degradation by proteosomes...

•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

MD simulations show differences in pathways inforced (pulled) versus thermodynamic unfolding:

•Forced unfolding retains core, unravels at ends first•Thermal unfolding is more evenly distributed throughout molecule

•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, G=7.5 kcal/mol•barnase: ku=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

•Paci, E. and Karplus, M. (2000). Unfolding proteins by external forces and temperature: The importance of topology and energetics. PNAS, 97(12):6521-6526.

Forced unfolding of spectrin

T(ps) F(pN)

End-to-enddistance (A)

tertiary structureruptures

partially stableintermediates...

In contrast, in thermal unfolding, helices tend to fray much sooner.

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...

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 hingeremoves 15A peak

Clustering of intermediates

•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)

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) andis pulled apart last (du).

Stress focused on endbond.