1. 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)

2. Neutral protease

3. Goals Increase neutral protease stability Don’t alter specificity Understand how it works (Use many neutral proteases)

4. The assay - 1

5. The assay - 2

6. The assay - 3 Representative ?

7. Domains

8. Domains

9. Cavity between domains

11. Cavities everywhere

12. Helix capping

13. Loop transplantations

15. Surface ‘packing’

17. Other methods Proline in loop Pester a water out Cysteine bridge Surface salt bridge Buried hydrogen bond

18. 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

19. Model building by mutagenesis

23. Mutations should add up

24. But, they don’t ….

25. Position dependent effect

26. It’s a protease!

27. Local unfolding

28. 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.

29. One weak loop

30. Thousand weak loops

31. Two weak loops?

32. Make two weak loops!

33. Two weak loops

34. Weak loop protection

35. 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…

36. 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.