1. Experimental Approach for Protein Folding C. P. Chou Department of Chemical Engineering

2. Protein folding research Static: 3-D prediction (theoretical approach) Dynamic: protein folding (misfolding and refolding) mechanism –Experimental approach: in vivo and in vitro (most studies) –Theoretical approach (in vitro): molecular dynamics, Monte carlo simulation

3. Formation of enzyme binding site through folding

4. Protein disulfide bond

5. Across the ER membrane To Golgi apparatus Storage granules Posttranslational processing for human insulin

6. Protein misfolding and refolding Most proteins except membrane proteins are soluble in in-vivo and in-vitro aqueous systems In vivo and in vitro experiment Inclusion body: protein aggregate Medical and industrial implications: loss of biological functions Including misfolding, aggregation, unexpected multimerization Misfolded proteins can be refolded to regain their biological function

7. In-vivo protein folding

9. Recombinant DNA Technology

12. S+  +C+  (preproPAC)  +C+  (proPAC)  +C  ++ pac mRNApac Gene Replication Transcription Translation Periplasm Cytoplasmic Membrane Cytoplasm Processing Outer Membrane Periplasmic inclusion bodies Penicillin Acylase (PAC)

13. Chaperon Coexpression (Preventive Approach)

14. In-vitro protein folding

15. Application Biopharmaceutical is a typical example, e.g. insulin Prevent protein misfolding (e.g. AA effect on protein folding) Refold misfolded protein to regain its biological function

16. Productive pathway Nonproductive pathway Chaotroptic agent, e.g. 5~8 M urea or protein aggregate In-vitro protein refolding

17. How long does protein folding take? µs, ms, s, min Depending on protein size, temp, etc.

18. Monitor protein folding Spectrofluorometer: fluorescent AAs, such as trp and tyr Circular dichroism (spectropolarimeter; CD): primarily for secondary structure monitoring “Stop flow system” for monitoring fast folding PAGE (SDS gel and native gel)

19. Tailspike protein (TSP) 6 tryptophan and 21 tyrosine

20. Folding and aggregation pathway of tailspike [I*] -SH -s-s- Nascent Polypeptide Chains High temp. tsf mutants Low temp. su mutants Tm = 88 o C SDS resistant

21. TSP refolding at 25°C

22. Folding intermediates of tailspike 0 1 5 10 15 20 60 120 min Multimer (O*) Tetramer (4*) Protrimer (pT) Non-prod trimer (T*) Native trimer (nT) Prod dimer (D) Non-prod dimer (D*) Monomer (M)

23. wtG244R 0 1 2 4 7 10 20 60 Tailspike refolding at 29°C A334V/G244RA334V

24. Goals Understand protein folding mechanism in in-vivo and in-vitro systems Prevent protein misfolding Refold misfolded protein