there is no way one can determine protein stability (that is, Gibbs free energy difference between the unfolded and folded states) based on the high-resolution structure of the folded state. First, even a small fraction of an Angstom shift in the atomic positions of certain groups can change G of the folded state. Same is true for the unfolded state whose structure you don't know. You cannot derive protein stability but have to have to measure it, alas.

there is no way one can determine protein stability (that is, Gibbs free energy difference between the unfolded and folded states) based on the high-resolution structure of the folded state. First, even a small fraction of an Angstom shift in the atomic positions of certain groups can change G of the folded state. Same is true for the unfolded state whose structure you don't know. You cannot derive protein stability but have to have to measure it, alas.

The FoldX algorithm (http://foldx.crg.es/) can calculate a quantitative estimation of the importance of the interactions contributing to the stability of proteins and protein complexes. This can be useful if you want to compare WT and mutant-enzymes.

Using molecular mechanics, the *relative* free energy between conformational basins of interest can be estimated; there is a large amount of different methods available such as the confinement approach, or non-equilibrium approaches, etc. It is important that the equilibrium ensemble of those conformational basins are sampled properly; a single structure is not acceptable; I agree with Olga.

Dear Paulo,

the algoritm of FoldX takes several aspects into account (solvation, unfolded state. entropic contributions etc.), you can check the respective publications. It is a parameterized approach, trained by real protein variants, so for an ab initio appraoch using molecular dynamics calculations check e.g. the publications by the Van Gunsteren Group. C.D. Christ, A.E. Mark, W.F. van Gunsteren

Basic ingredients of free energy calculations: a review

J. Comput. Chem. 31 (2010) 1569-1582, DOI: 10.1002/jcc.21450

Have Fun,

Marco

If your protein is multi-domain which unfolds irreversibly, then Gibbs Free energy has no meaning. Its only applicable on small proteins which unfold reversibly. In case you can not access expensive eqipment such as DSC, CD, fluorimeter etc and your protein unfolds reversibly I can suggest TRANSVERSE UREA GRADIENT PAGE (TUG-PAGE) which can determine DG using electrophoresis equipment. See my reference:Siddiqui, KS., Francisci, D. and Thomas, T (2007) Analysis of unfolding-refolding patterns and stabilities of proteins by transverse urea gradient gel electrophoresis (TUG-GE). In: Protein Structures. Methods in Protein Structure and Stability Analysis. (eds. Uversky VN. & Permyakov AE) Nova Science Publishers, Inc., Hauppauge, NY, USA (ISBN: 1-60021-704-4) pp 147-158.

Agree with Olga & Khawar. Only DSC/CD can be used to calculate dG between two equilibrium states.

Using molecular mechanics, the *relative* free energy between conformational proteins. the gibbs free energy defference between unfolded and folded states and there is no way could determine protein stability.

Yes, you can calculate the Gibbs free Energy between folded and unfolded state of a protein by using experimental or simulation techniques. The best experimental technique is DSC (differential scanning calorimetry) because using this approach you can calculate Gibbis free energy (derived from enthalpy, entropy and deltaCp) also if the unfolding is irreversible (see you my paper published on J. Phys Chem.,1995, 99,14864-14870 and Biophys. Chem., 1997, 69, 43-51.) . If you prefer the simulation way, use the Steered Molecular Dynamics simulations (see you New J. Chem., 2007, 31, 901–905).

Conformational changes of macromolecules often are described in terms of the so-called collective variables (CV), which in simple molecules can be a single coordinate or a dihedral angle. Collective variables give insight and help in understanding the mechanisms by which a molecular system explores phase space. The computation of the free energy landscape of a molecular system is parametrized by CVs. A non-exhaustive list of important methods is Umbrella Sampling and the Weighted Histogram Analysis Method, the Adaptive Biasing Force method (ABF), Metadynamics, and non-equilibrium work methods. Unfortunately, the success of these methods hinges upon a good choice of CV, far from obvious in large macromolecules.

You can see among others the work of Tribello (Parrinello's group), “Using sketch- map coordinates to analyze and bias molecular dynamics simulations,” PNAS 109, 5196–5201 (2012).

you can use molecular dynamics simulation to do this. you should perform molecular simulation under NpT ensemble condition to calculate Gibbs Free Energy. In doing so, I suggest you can use Gromacs, CHARMM, and NAMD program packages.