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Folding Mechanisms and Intermediates for Aggregation-Prone Native Structures

Folding Mechanisms and Intermediates for Aggregation-Prone Native Structures. Patricia L. Clark Department of Chemistry & Biochemistry University of Notre Dame, Notre Dame, Indiana Workshop on Biomolecules - Bedlewo, Poland May 14, 2004. The protein folding problem:.

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Folding Mechanisms and Intermediates for Aggregation-Prone Native Structures

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  1. Folding Mechanisms and Intermediates for Aggregation-Prone Native Structures Patricia L. Clark Department of Chemistry & Biochemistry University of Notre Dame, Notre Dame, Indiana Workshop on Biomolecules - Bedlewo, Poland May 14, 2004

  2. The protein folding problem: fold or aggregate? ? misfolded, aggregated state ? native state i ensemble of denatured states

  3. The folding of small globular single-domain proteins • Common proteins: • 100-250 amino acids • Single structural domain • Rich in -helix structure • Monomeric • Common folding themes: • Fast folding kinetics (sec-sec) • Few (if any) folding intermediates besides ‘molten globule’ • Negligible competition from off-pathway aggregation HEWL RNaseA

  4. Funnels for protein folding: energy landscapes Folding funnel diagrams capture many of the features observed for the folding pathways of small, monomeric, single domain, helix-rich proteins

  5. Benefits and caveats of energy landscapes/funnels: • Folding funnels make it clear why proteins fold: • - Energy difference between the unfolded ensemble • and the native state • Folding funnels have shifted focus to fast folding rates: • - What is the barrier for folding? • - What is the ‘speed limit’ for folding? • But what about proteins that: • (i) fold slowly, and/or • (ii) are prone to aggregation? • - How does this affect the energy landscape?

  6. A folding funnel for many proteins in solution:

  7. What kinds of proteins are prone to aggregation? • Topology effects: Contact order? (D. Baker, U. Washington) • Kinetic effects: Long-lived folding intermediates? Plaxco et al. (1998) JMB277:985

  8. Non-local contacts = High contact order A A A B B B contacts between residues in the primary sequence: NEARBY FAR APART B A ordering many more residues at once = selecting from more conformational states -> How is aggregation avoidance encoded?

  9. Protein folding in the cell: • E. coli: • 200-400 mg/ml total protein • [nascent chains] = 30-50 M • ribosomes > 1/4 cell weight • chain synthesis ~ 20 aa/sec --> How are partially folded conformations protected from aggregation in this environment? David Goodsell: http://www.scripps.edu/pub/goodsell/illustration/public/

  10. How do high CO structures form co-translationally? A B in vitro: in vivo: ribosome B A A • What conformations does A adopt • before B appears? • How much native structure can be • formed co-translationally? ordering many more residues at once = selecting from more conformational states -> How is aggregation avoidance encoded?

  11. Bordetella pertussis P.69 pertactin Cross-section of 7 central rungs (residues 140-357) • 60 kDa, single domain -helix • All parallel -sheet: no local contacts • Average rung-to-rung contact distance: 34 amino acids • No Cys, cofactors, etc. • C-terminal 59 residues disordered in structure; can be • deleted with no effect on folding or stability

  12. Spacefilling model of pertactin backbone structure • Long loops are clustered on one face of structure • -helix backbone is remarkably regular

  13. Pertactin far-UV CD spectra, thermal denaturation • Three-state thermal unfolding • Partially folded state populated at 70ºC • 1.5 uM pertactin in 50 mM phosphate pH 8.8 Mirco Junker

  14. Pertactin tryptophan fluorescence spectra: N and D N D • Seven tryptophan residues (some solvent exposed) in native • -helix structure, plus one in C-terminus • 0.5 uM pertactin in 50 mM TRIS pH 8.8, 25ºC

  15. Pertactin unfolding/refolding: Reversibility? • Each sample incubated for 2 hr at room temperature • Unfolding and refolding titrations do not overlay • No aggregation …microscopic reversibility? Mirco Junker

  16. Pertactin refolding IS reversible, but very slow: • Similar results with urea, and when monitored by CD • GH2O = 46 kJ/mol (N-I) and 55 kJ/mol (I-D) • Partially folded structure forms extremely slowly • Origin of slow folding? Mirco Junker

  17. Models for the partially folded structure • Trp fluorescence is halfway between N and D spectra • Half folded, Half unfolded… • Or: Half-folded? Half folded/Half unfolded? Half-folded?

  18. Testing the models: limited proteolytic digestion • Native pertactin: • Protease K resistant • Eventually degraded to • 37 & 29 kDa fragments • Partially folded state • in 1.4 M GdnHCl: • Less protease K resistant • Degraded to 29 kDa • fragment • Stepwise: rung by rung? Kelli Whiteman

  19. MALDI-TOF mass spectrum of intact fragment • Proteinase K-resistant fragment: • harsher digestion results in 21 kDa band by SDS-PAGE, MALDI Kay Finn & Elizabeth Klimek

  20. MALDI-TOF: Tryptic digest of 21 kDa band • Trypsin digestion, followed by MALDI-TOF: • no fragments larger than 4 kDa • several peaks map to unique fragments Kay Finn & Elizabeth Klimek

  21. Identifying the partially folded structure N C • Mapping tryptic peptides onto the pertactin native structure: • RGD/PRR loop = red/blue (residues 226-262) • fragments cover residues 351-388, 395-435, 438-475, 480-509 Mirco Junker & Kay Finn

  22. Mapping pertactin slow folding/unfolding kinetics • What occurs prior to 2 hr? How long does unfolding take? • How many events between 2 hr and 3 weeks? • How protect chromophores from bleaching/degradation? Mirco Junker

  23. Unfolding is extremely slow at high [GdnHCl] Black = 200 hr unfolding Black = 100 hr unfolding Black = 10 hr unfolding Black = 1 hr unfolding Black = 2 hr unfolding Black = 3 hr unfolding Black = 4 hr unfolding 30 min unfolding Diamonds = 30 min unfolding • Unfolding takes ~100 hr to complete • Slowest step represents unraveling of partially folded state • What creates high energy barrier for unfolding? Mirco Junker

  24. Spacefilling models of pertactin backbone structure • -helix backbone is remarkably regular • Long loops are clustered on one face of structure

  25. Refolding is even slower! Black = 216 hr refolding Black = 312 hr refolding Black = 10 hr refolding Black = 4 hr refolding Black = 2 hr refolding 30 min refolding Diamonds = 30 min refolding Black = 24 hr refolding Black = 76 hr refolding • Refolding occurs over >200 hr • 0.5 M: fast events en route to native structure: HØ collapse? • 1.5 M: slow folding: conformational search? Chris Schuster & Katie O’Sullivan

  26. Pertactin slow refolding kinetics: Unfolded • Refolding at 1.5 M GdnHCl; monitored by Trp fluor. emission • Multiple slow components Chris Schuster

  27. Pertactin slow refolding kinetics: Unfolded • Refolding at 0.5 M GdnHCl; monitored by Trp fluor. emission • Fast and slow components Chris Schuster

  28. Pertactin slow refolding kinetics: • Refolding at 0.5 M GdnHCl; monitored by Trp fluor. emission • Fast and slow components Mirco Junker

  29. Slow formation of the partially folded structure: Large conformational search to form the native -helix ? Fast formation of trapped, non-native structure ? OR:

  30. A folding funnel for many proteins in dilute solution:

  31. Summary & Future directions • Pertactin folding/unfolding is reversible, but equilibrium • established very slowly • --> Large energy barrier to form partially folded state • --> A ‘template’ for -helix rungs? • --> Selecting between energetically similar folded and • misfolded states? • Slow step at intermediate concentrations involves forming • structure in C-terminal half of -helix • --> What parallel -sheet elements initiate folding? • --> What rungs are more stable than others? Why? • What cellular components regulate pertactin folding in vivo?

  32. Acknowledgements Thomas Clarke Neil Isaacs, U. Glasgow Michael Evans Mirco Junker Andre Palmer, ND Krastyu Ugrinov Chris Schuster Katie O’Sullivan Bill Boggess Elizabeth Klimek ND Mass Spec Facility Kelli Whiteman Kay Finn NSF • AHA Clare Boothe Luce Program, Henry Luce Foundation University of Notre Dame

  33. Pertactin partially folded state is monomeric • Static light scattering detection of gel filtration eluate: Kay Finn

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