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sticky:sticky [2018/03/01 13:58]
richter created
sticky:sticky [2018/03/01 14:56]
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 +Discussion on simulations to probe the molecular factors contributing to the observations in Zotter et al, JBC, 2017[[http://​www.jbc.org/​cgi/​doi/​10.1074/​jbc.M117.792119|Zotter et al, JBC, 2017]] published by [[http://​www.weizmann.ac.il/​Biomolecular_Sciences/​Schreiber/​content/​protein-protein-interactions-biophysical-characterization-biological-applications|Gideon Schreiber and colleagues]]
 +Present: RW, HXZ, NB, ANA, SKS, PF
 +Questions to address with simulations:​
 +  - Is the rate of diffusion (trans, rot) of the substrate slower in a crowded environment (like the cell) due to non-specific sticky (hydrophobic) interactions? ​ Is the diffusion rate dependent on crowder type? on crowder conc? on substrate? on other conditions (IS, pH etc).  Is slowing of the diffusion dependent on the '​sticky'​ fluorophores and not observed for the natural beta-lactam substrate?
 +  - Can SDAMM-type simulations be compared directly to expts that could be done for in vitro systems by Gideon or by Hofmann at Weizmann?
 +  - Is the bimolecular rate constant for diffusional association of the substrate to beta-lactamase slower in a crowded environment?​ Why? 
 +Aim: Tackle the first set of questions and aim to have some progress that we can discuss with Gideon at BDBDB4.
 +To do:
 +  - Generate 3D structures and force field parameters of CFF2, beta-lactam without D and A fluorophores,​ and FDG and RDG. Explore conformational space at a rather simple level (in MOE or Schrödinger)- generate e.g. for CFF2 an extended structure and a U-shaped structure that could interact with the b-lac active site.  Generate parameters for SDA (charges and radii at pH 7 (7.4)) using Gaurav'​s scripts. Examine whether reasonable models have been derived. ​ (Ariane will start this off with help from Gaurav etc)
 +  - Build SDA boxes of molecules with several substrate molecules (ca. 20?, negatively charged) and HEWL (+ve charge) or Myoglobin (small net charge) crowders at different (initially 2) protein concentrations (low and high (near cellular)). ​ Simulate each system for several microsec and compute Drot and Dtrans of substrates. Examine substrate-protein contacts ​ (and check for substrate-substrate contacts). ​
 +  - If we see trends in slower diffusion dependent on substrate and protein crowder type, do these relate to interaction strengths? ​ Test this by FMAP calculations for the excess chemical potential of putting substrate molecule in system of crowders. ​ Test this by doing calculations for a system that can be tested in vitro (discuss at BDBDB4)
 +other (just to bear in mind for now) :
 +Systems with enzyme and substrate and no crowder:
 +-Do NAM kon calculations for b-lac and different substrates and b-lac mutants: would give reference for uncrowded system
 +-Do periodic box kon/MFPT calculations for b-lac + substrate + varying crowder concs. Post analysis may miss satisfaction of reaction criteria. Would need to introduce correct monitoring of rxn criteria during simulations into sda(mm).