Presentation on theme: "Folding Mechanisms and Intermediates for Aggregation-Prone Native Structures Patricia L. Clark Department of Chemistry & Biochemistry University of Notre."— Presentation transcript:
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: ensemble of denatured states native state ? fold or aggregate? ? misfolded, aggregated state i
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
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
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?
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) JMB 277:985
Non-local contacts = High contact order contacts between residues in the primary sequence: NEARBYFAR APART A B B A A B A B ordering many more residues at once = selecting from more conformational states -> How is aggregation avoidance encoded?
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 David Goodsell: http://www.scripps.edu/pub/goodsell/illustration/public/ --> How are partially folded conformations protected from aggregation in this environment?
How do high CO structures form co-translationally? in vitro: B A A B in vivo: A What conformations does A adopt before B appears? How much native structure can be formed co-translationally? ribosome ordering many more residues at once = selecting from more conformational states -> How is aggregation avoidance encoded?
Bordetella pertussis P.69 pertactin 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 Cross-section of 7 central rungs (residues 140-357)
Spacefilling model of pertactin backbone structure Long loops are clustered on one face of structure -helix backbone is remarkably regular
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
Pertactin tryptophan fluorescence spectra: N and 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 N D
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
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
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?
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
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
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
Identifying the partially folded structure 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 NC
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
30 min unfolding Unfolding is extremely slow at high [GdnHCl] Mirco Junker Black = 1 hr unfolding Black = 2 hr unfolding Black = 3 hr unfolding Black = 4 hr unfolding Black = 10 hr unfolding Black = 100 hr unfolding Unfolding takes ~100 hr to complete Slowest step represents unraveling of partially folded state What creates high energy barrier for unfolding? Black = 200 hr unfolding Diamonds = 30 min unfolding
Spacefilling models of pertactin backbone structure -helix backbone is remarkably regular Long loops are clustered on one face of structure
Refolding is even slower! Chris Schuster & Katie O’Sullivan 30 min refolding Black = 2 hr refolding Black = 4 hr refolding Black = 10 hr refolding Black = 24 hr refoldingBlack = 76 hr refolding Black = 216 hr refolding Black = 312 hr refolding Diamonds = 30 min 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?
Pertactin slow refolding kinetics: Refolding at 1.5 M GdnHCl; monitored by Trp fluor. emission Multiple slow components Chris Schuster Unfolded
Pertactin slow refolding kinetics: Refolding at 0.5 M GdnHCl; monitored by Trp fluor. emission Fast and slow components Chris Schuster Unfolded
Pertactin slow refolding kinetics: Refolding at 0.5 M GdnHCl; monitored by Trp fluor. emission Fast and slow components Mirco Junker
Slow formation of the partially folded structure: Large conformational search to form the native -helix ? Fast formation of trapped, non-native structure ? OR:
A folding funnel for many proteins in dilute solution:
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?
Acknowledgements Thomas Clarke Neil Isaacs, U. Glasgow Michael Evans Mirco Junker Andre Palmer, ND Krastyu Ugrinov Chris Schuster Katie O’Sullivan Bill Boggess Elizabeth KlimekND Mass Spec Facility Kelli Whiteman Kay Finn NSF AHA Clare Boothe Luce Program, Henry Luce Foundation University of Notre Dame
Pertactin partially folded state is monomeric Static light scattering detection of gel filtration eluate: Kay Finn