“Single-molecule force spectroscopy reveals a stepwise unfolding of C. elegans giant protein kinase domains”

Dina N. Greene, Tzintzuni Garcia, R. Bryan Sutton, Kim M. Gernert,

Guy M. Benian & Andres F. Oberhauser

Supporting FIGURES

Supplemental Figure 1. Alignment of TTN1 on 1KOA. Alignment used for building the initial homology model of TTN-1. Note that the arbitrary start at 1 corresponds to twitchin (KOA) residue Y915 and TTN-1 residue E15907.
I27 domain of titin (1TIT.pdb). 6875 total atoms in simulation. Water sphere composed of 1830 water molecules with 6 Na+. Pulling speed of 0.1A/ps for a total simulated time of 1.25ns. Fixed atom = Leu1 and SMD atom = Leu 89 / Twc-Ig26 domain (1KOA.pdb). 6879 total atoms. 0.1A/ps for a total simulation time of 1.25ns. Water sphere including 5Cl- and 1781 water molecules. Fixed atom = Ala 6264, SMD atom = His6358
Ubiquitin (1UBQ.pdb). 0.5 ns total simulation time. 6670 atoms, 1813 waters. Pulling speed of 0.1A/ps. Fixed atom = Met1. SMD atom = Gly76 / C2A (1RSY.pdb). 5ns total simulation time. 0.1A/ps pulling speed 11,052 atoms total including 4 Na+ and 2 Cl-, 3025 water molecules. Fixed atom = Leu 142, SMD atom = Leu 262
Supplemental Figure 2. Constant velocity SMD simulations of the mechanical unfolding of several protein domains.

To validate the accuracy of our in silico experiments we performed SMD simulations on other protein domains, such as titin I27, ubiquitin and synaptotagmin C2A. Our SMD results are very similar to those published previously (Supplemental Figure 2).

Supplemental Figure 3 shows constant velocity SMD simulations of the mechanical unfolding of the homology model for TTN-1 twitchin kinase. The unfolding trajectory is nearly identical to that of that of twitchin kinase indicating a similar stepwise mechanical unfolding pathway, as seen in the AFM data (Fig. 5).

Supplemental Figure 3. Constant velocity SMD simulation of the mechanical unfolding of the homology model for TTN-1 twitchin kinase. Pulling speed 0.5A/ps. 2.4ns total simulation time. 33460 total atoms + 18 Na+, 9305 waters. Fixed atom= Glu 1 SMD atom = Asp 342.

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