Folding of Top7-CFr
Top7 is a designed 93 residue
protein, whose topology is not found among natural proteins.
It was found to be monomeric, extremely stable, and its structure was experimentally determined
to be very close to the target of the design procedure. It was also reported that a
fragment
consisting of the 49 C-terminal residues of Top7 is efficiently
mistranslated in E.coli. This mistranslated fragment, CFr, was found to adopt an extremely
stable homo-dimeric structure. Both chains in the dimer have a structure similar to the
corresponding residues in Top7.
The PDB structure for Top7-CFr (PDB id: 2gjh)
contains 62 residues in each
chain, out of which the fragment 2—50 adopts the β–α–β–β
structure of the corresponding part of Top7. This segment has been extensively studied with
ProFASi. As usual, these were unbiased all atom parallel tempering Monte Carlo simulations
with random initial conformations for each replica. This 49 residue segment folds readily
in such simulations. The global free energy minimum
appears to be very close to the structure of one chain in the CFr dimer,
as shown in the figure below. The backbone RMSD between the chain A of the PDB entry 2gjh and
the centre of the native free energy minimum is about 2 Å.
The following animation shows one instance of this fragment folding in a parallel
tempering MC simulation with ProFASi. The reason why this should be interesting is that
our all-atom simulations are not small perturbations about a given structure. Neither does
the force field make use of any information about the native structure. The movie
covers a short segment of the Markov chain generated in the simulation just before the
protein folds.
One instance of CFr folding in a parallel tempering MC
simulation in ProFASi. Each frame in this movie is separated by 236000 Monte Carlo updates
from the previous frame.
Although there is a large number of MC updates between consecutive frames in the above
animation, it is possible to see that there appears to be a preference for a helix and
the C-terminal hairpin. The N-terminal β-strand joins the hairpin only at the last
stage of folding. It is also possible to see that this N-terminal strand was often
folded into an extension of the native helix. These observations hold for a
large number of folding events seen in our simulations. This is an indication that the
most likely path to the native state found by our Markov chain MC simulations passed
through an unexpected non-native extension of the helix. Using simulations
of fragments corresponding to different secondary structure elements,
we proposed a folding
mechanism, which makes energetic sense in our model. The figure below (right),
sketches this mechanism (which we call the "caching
mechanism") using snapshots from the simulation.
Left: Structure of the Top7-CFr dimer (gray) and the
free energy minimum structure (coloured) for a monomer at 274 K as seen in ProFASi simulations.
Right: A sequence of events leading up to the folded structure
in MC simulations. This figure illustrates what we call the "caching mechanism". The N-terminal
(blue) strand, which is in contact with the C-terminal strand in the native state, spends its
time as a non-native extension of the native α-helix until the native C-terminal β-hairpin
forms. The native state is then formed by unfolding of the non-native helix extension and
attachment of the released N-terminal residues as the third strand of the β-sheet.
The caching mechanism is a folding scenario involving the transient folding of a short
segment of a protein chain into
a non-native secondary structure element, until the native environment of the segment forms.
In the case of CFr folding in our model, it depends on the chameleon like behaviour of the
N-terminal segment, which prefers
a helical form when there is only a helix in its neighbourhood, and a β-strand form
in presence of the β-hairpin. This mechanism appears spontaneously in the simulations.
The following smoothed out version of the above animation, by eliminating the rapid
fluctuations makes the caching mechanism easier to see.
A smooth version of the movie above. It is obtained by taking key frames from the last part of
the previous movie
and creating intermediate "morph" snapshots with Pymol. The morph snapshots do not preserve
the geometrical constraint of the model, and do not have any physical basis. But since they
get rid of a lot of random jiggles of the Monte Carlo evolution, the movie is easier on the
eye, and brings out the proposed caching mechanism more clearly.
References
The results discussed in this page have been published in the following articles.
- Simulation of Top7-CFr: A Transient Helix Extension Guides Folding, Sandipan Mohanty, Jan H. Meinke,
Olav Zimmermann, Ulrich H.E. Hansmann,
Proc. Natl. Acad. Sci. USA 105(23) 8004
- Caching of a Chameleon Segment Facilitates Folding of a Protein with End-to-End β-sheet,
Sandipan Mohanty and Ulrich H.E. Hansmann,
(2008) J. Phys. Chem. B 112(47)15134
- An Effective All Atom Potential for Proteins, Anders Irbäck, Simon Mitternacht and
Sandipan Mohanty,
(2009)
PMC Biophysics 2:2