[gmx-users] Umbrella Sampling - Experiment Question

Douglas Grahame dgrahame at uoguelph.ca
Thu Aug 27 17:56:21 CEST 2015

Hi there, I'm thinking about running an Umbrella sampling of a protein and before I start spending a ton of time on running/figuring out how to run the simulations I was wondering if I could get some feedback on my experimental design aka I want to find out if what I want to do makes sense.

Basic setup is this; I have an ~350 amino acid protein that contains a distinct N and a distinct C terminal lobe connected by a large beta sheet between each lobe. Although the protein is a whole singular unit naming convention and the morphology/structure of the protein provide a clear center point to pull from in the beta sheet connecting them. We know from wet lab experiments that one lobe is far less stable than the other so in order to try and find out why I've generated a series of mutants in the wet lab for the unstable lobe only and I am currently testing said mutants using chemical unfolding experiments to try and determine if the mutants have increased or decreased the stability of the enzyme. I have run previous experiments using standard MD simulations to try and simulate the unfolding but I have to have my protein unfold at high temperature or in a timely manner (over 100ns) and in a fashion that makes sense/matches the wet lab data. Unfortunately I do not have any structural information on the denatured states only the initial state. I have checked the energy, pressure, and temperature equilibration steps and yes they are all in acceptable limits and do come to stable baseline.

Okay so to my actual questions! If I conduct an Umbrella experiment for my control protein and my mutants in which I pull at the COM as defined as the midpoint between the N and C terminal lobes can I utilize the PMF difference between the mutant and control to examine any increases or decreases in protein stability due to the mutation. Aka I'm hoping I can use umbrella sampling as a corollary to my unfolding experiments and if so does anyone have any tips/advice they can throw my way! If not are there any suggestions on things I should be looking into? 

Thanks so much for your help and sorry for asking a vague question as I don't have too many local resources to turn to for modelling help. Cheers,

-Doug Grahame


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