Performed a 50ns MD simulation (NAMD; CHARMM36FF) at various temperature ( 300, 350, 400, & 450K). for homologous protein pair one of which thermophillic and mesophillic in nature. The thermophillic structure is a experimental one (x ray crystal) and mesophillic protein is homology modelled upon a template with 75% identity. We expected the data ( RMSD, SASA, Rg, PCA, FEL) to follow the conventional trend. Usually upon heat treatment RMSD, SASA, Rg should increases at high temperatures (like 450K). Rather we found that SASA and Rg decreases for both thermophillic and mesophillic 3D structures with increasing temperatures. RMSD for mesophillic one increases significantly (RMSD of 6.0 angs at 450K), for thermophillic it rises conventionally (RMSD of 2.0 angs at 450K). Upon visual inspection of the trajectory, we found out that when the trajectory converges, both the structures shrinks in size ( as they has two conformations open and close) explaining why the SASA and Rg would decrease. But shrinked conformations attained by these structures are very high in energy (ddg, derived form Free energy landscapes).The underlying point is those shrinked conformations are so energetically expensive that it cant be called that it got stabilized and the distribution of conformation showed by PCA, that it is more extended in the case of mesophile and less for thermophile
Are you sure you interpreted the free energy landscape correctly? Can we have a look at it? By the way. 50ns is not a lot of data for drawing conclusions from FEL, how did you obtain it, from BOLZTMAN inversion?
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Norman Geist
1. Difference in the structural flexibility can be inferred from FEL, the Mesophile takes longer and rough contours to attain a stable conformation than thermophile.
2. For both the structures they attain the deepest point by lowering their Rg, which can be inferred that the proteins are getting compact as the trajectory stabilizes.
3. I used a PyMOL plugin "Geo measure" to generate FEL from the trajectories.
4. I don't know whether "BOLTZMAN inversion" has been implied for generating FEL.
Well, it's not surprising that the homology model takes 1st a bit of time to relax. The higher energy state you refer to is perhaps the starting structure. You should repeat your test for the relaxed protein state (global minimum). The thermophilic one doesn't show much movement on the landscape either, maybe because 50ns aren't enough. You also need to realize that the FEL shows no time dependence, just all "states" you have seen in their count during your MDS. You probably need more sampling, with sufficient swaps between these open and closed states to tell something about the equilibrium at different temperatures. We usually do this kind of things with replica-exchange sampling and our TIGER2hs method.
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Thank you for the suggestions Norman, I am working on them. Can you suggest me some research articles where similar dynamics have been discussed. It will be very helpful.
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Diptesh
I assume what you are ultimately interested in is the calculation of "melting points", right? To obtain these from MD simulations, REMD simulations are the method of choice, as they give you a Boltzmann-weighted ensemble of states at a range of temperatures from which melting curves can be created. I would therefore suggest to lookup "protein melting curves from MD simulations" and I'm certain you will find what you need ;)
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