Proteins, vol. 59, p. 534-544 (2005).
|The two end-states: Ras p21 is a crucial signaling-protein involved in regulating cell reproduction. Upon hydrolysis of the bound GTP, the protein undergoes a transition from its GTP-bound form ("reactant", in yellow) to its GDP-bound form ("product", in red). The predominantly mobile regions are the switch I and switch II elements. The gray region is very similar in the two states, and thus can be held fixed during the computation of the path.||Methods to identify MEPs rely on the local optimization of an initially guess for the path. With most initial guesses, these optimization techniques tend to get stuck in local solutions, producing paths with unrealistically high energy barriers. Such paths typically include unphysical transition-states, like the interpenetration of bonds (here of an Arg and a Thr side-chain, top panel), or the passage of a water molecule through an aromatic ring (bottom panel).|
To obtain an initial guess that can be optimized to a MEP with low barriers, we used a combined interpolation technique, interpolating the coordinates of backbone atoms in Cartesian and the coordinates of side-chain atoms in internal coordinates. In addition, the side-chain bonds are shrunken in the initial path, resulting in miniature side-chains which are unlikely to produce clashes or interpenetrations in the initial path. These unnaturally short bonds expand to their normal size in the course of CPR's optimization procedures.
After the first CPR refinement, the MEP involves no structural deformations, but its rate-limiting energy barrier is still high (~100 kcal/mol).
To reduce this, CPR is run again between local minima adjacent to high barriers. The new path sections thus produced replaced the old ones. Following this strategy, a MEP with a rate-limiting barrier of less than 40 kcal/mol is found. Repeating this procedure yields a path with a rate-limiting barrier of only 24 kcal/mol. Enthalpic barriers in the order of 20-40 kcal/mole can be compensated by entropic effects, thus it is possible that a realistic pathway has been identified. The final path has more than 100 transition-states (saddle-points), due to the large number of local rearrangements that are necessary for side-chain re-orientations and for backbone re-folding, each of which involves at least one saddle-point.
Showing the motion of the backbone, as it unfolds and then
the two Switch regions.
The reactant (green) and product (red) structures are shown throughout the movie to get a feeling of the structural progress (yellow). One can distinguish phases of the transition: A) Unfolding of the Helix in Switch II, B) conformational change of Switch I, C) conformational change of Switch II. The Mg++ is shown as green sphere, the GDP as balls&sticks.
Download the movie , 1.7Mb
Showing the motion of the side-chains, which one after the
undergo their own re-orientation.
Side-chains are colored according to their instantaneous energy: brighter colors correspond to higher energies (Dark blue: energy <= 0. White: energy >= 30).
The energy assigned to each residue at each frame is computed by its self-energy plus half the interaction energy with all other residues (i.e. these energies add up to the total potential energy). The difference between this energy in a given frame and in the end-states gives the instantaneous energy.
Download the movie , 3.5Mb.
Same movie, but very fast, 2Mb.
Go to Home of S. Fischer