Structural Stability of Proteins

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)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, 101085-1096.
• 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, 812344-2356.
• 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, 49104-113.

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)4E2lip3() 10.0nm (I2
7)4E2lip3(-) 187pN, 24.1nm (I27)2E2lip3(-)(
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() 3.8ku0I27
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 fk(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)
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Lys48-Cterm 29 residues
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Unfolding kinetics force depends on pulling speed
aa0exp(FDx/kBT) Fln(a/a0)(kBT)/Dx) a00-force
unfolding rate related to pulling speed mol/s
gt nm/s can also get Dx by fitting
Fernandez - Fig. 3
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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 FDx DxN-C 0.25nm gt higher
force DxLys48 0.63nm gt 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, 963694-3699.
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,
  812344-2356.

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

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• 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 ku4.91 s-1, DG7.5 kcal/mol
• barnase ku3.37 s-1, DG10.2 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, 101085-1096.

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, 49104-113.

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,i4) 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).