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Experimental Approach for Protein Folding

Experimental Approach for Protein Folding

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Experimental Approach for Protein Folding

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

  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

  8. RecombinantDNA Technology

  9. Penicillin Acylase (PAC) Periplasmic inclusion bodies Outer Membrane a+C Processing Processing a+C+b (proPAC) Periplasm a+b b Cytoplasmic Membrane Cytoplasm Replication Transcription Translation S+a+C+b (preproPAC) pac mRNA pac Gene

  10. Chaperon Coexpression(Preventive Approach)

  11. In-vitro protein folding

  12. 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

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

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

  15. 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)

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

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

  18. TSP refolding at 25°C

  19. 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)

  20. Tailspike refolding at 29°C 0 1 2 4 7 10 20 60 0 1 2 4 7 10 20 60 wt G244R A334V A334V/G244R

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