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Protein stability. The use of homology modelling (or structure determination for that matter). Or, to teach you that a (good) bioinformatician knows a bit about everything. Or, to teach you that experiments occasionally are useful.
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Protein stability The use of homology modelling (or structure determination for that matter). Or, to teach you that a (good) bioinformatician knows a bit about everything. Or, to teach you that experiments occasionally are useful. (And sorry, all work is 10 years old, or older, including the pictures)
Goals Increase neutral protease stability Don’t alter specificity Understand how it works (Use many neutral proteases)
The assay - 3 Representative ?
Other methods Proline in loop Pester a water out Cysteine bridge Surface salt bridge Buried hydrogen bond
Model problems The models weren’t at all times overly trivial to build. We therefore also designed mutants to improve the model, so that the model could improve the mutants
Local unfolding The enough=enough effect: once a loop is stable, further mutations in that loop don’t help you any more. Mutations should give big effects in the weakest loop.
Conclusions Homology model is good enough for stability engineering. Precision is hardly ever needed, and when it is needed, even an X-ray structure isn’t precise enough yet. Most stabilizing mutations are at the surface, and there, model errors aren’t a problem…
Acknowledgements V.G.H.Eijsink, B.v.d.Burg, G.Venema, B.Stulp, J.R.v.d.Zee, H.J.C.Berendsen, B.Hazes, B.W.Dijkstra, O.R.Veltman, B.v.d.Vinne, F.Hardy, F.Frigerio, W.Aukema, J.Mansfeld, R.Ulbrich-Hofmann, A.d.Kreij.