Molecular Dynamics Simulation Reveals a Surface Salt Bridge Forming a Kinetic Trap in Unfolding of Truncated Staphylococcal Nuclease.

Andreea D. Gruia, Stefan Fischer*, Jeremy C. Smith

Proteins, vol. 50, 507-515 (2003).

We performed molecular dynamics (MD) simulations to study the unfolding behavior of a truncated form of Staphylococcal nuclease (SNase, see Figure) missing the last 13 C-terminal residues. This construct is known experimentally to be compact, but disordered under physiological conditions. The simulations reveal that a salt bridge between Arg 105 and Glu 135 is kinetically trapping the protein in a wild-type conformation and thereby hindering its unfolding. In a 1 ns-simulation performed at 300K the salt bridge does not break, contributing to maintaining the truncated SNase in a native-like conformation. Similar behavior is observed in a 550 ps-trajectory performed at 400K, a 250 ps-trajectory at 450K and six 200ps simulations at 430K.

Forced unfolding.
Unfolding of the protein was forced in simulations at 300K by gradually increasing the radius of gyration of the protein. The simulations reveal persistence of the salt bridge until a radius of gyration of ~ 17.5 A, indicating that the barrier for breaking this interaction is significant.  This was confirmed by a potential of mean-force (PMF) calculation, which reveals that the free-energy barrier is 7 kcal/mol at 300K.

Movie 1. Persistence of the salt-bridge during forced unfolding.
Shows a 500 ps-trajectory of SNase (shown in magenta) during which a constraint was imposed on the radius of gyration of the protein, which  was increased by 1 A every 100 ps (from the initial value of 14.8 A of folded SNase to a final value of 20, using a force constant of 50 pN).  The side-chains of Arg 105 and Glu 135 are shown.  For the purpose of comparison, the crystal structure of native SNase is shown in green.
Download the movie , ~2.5Mb

Weakening of the salt-bridge.
When the potential function is selectively altered so that the net charges of Arg 105 and Glu 135 is removed (but keeping local bond-dipoles unchanged), the salt bridge breaks rapidly in simulations performed at 300K and 430K and the protein starts to unfold.

Movie 2.  Breaking of neutralized salt-bridge at 300K.
Shows a 1 ns-trajectory performed with neutralized charges on Arg 105 and Glu 135. Coloring scheme is the same as Movie 1. Breaking of the salt bridge occurs spontaneously after ~250 ps and is followed by disorganization of the last turn of helix alpha3 situated at the C-terminal end of the protein, whereas the rest of the protein remains close to the native w.t. structure.
Download the movie , ~3.7Mb

Movie 3. Same as Movie 2, but close-up view of the salt bridge.
Download the movie , ~3.7Mb

Movie 4. Breaking of neutralized salt-bridge at 430K.
Shows a 200 ps-trajectory, of which the first 80 ps represent heating from 300K to 430K.  The charges are neutralized at the end of the heating procedure, leading to the immediate breaking of the salt bridge.
Download the movie , ~2Mb

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