Transcript Hysteresis in the Folding/Unfolding of a Monomeric Single

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:
fold or aggregate?
?
i
?
native state
ensemble of
denatured states
misfolded, aggregated state
The folding of small globular single-domain proteins
Common proteins:
• 100-250 amino acids
• Single structural domain
• Rich in -helix structure
• Monomeric
HEWL
Common folding themes:
• Fast folding kinetics (sec-sec)
RNaseA
• Few (if any) folding intermediates besides ‘molten globule’
• Negligible competition from off-pathway aggregation
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?
A folding funnel for many proteins in solution:
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:
NEARBY
A
B
B
A
FAR APART
B
A
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
--> How are partially folded
conformations protected
from aggregation in this
environment?
David Goodsell: http://www.scripps.edu/pub/goodsell/illustration/public/
How do high CO structures form co-translationally?
in vitro:
in vivo:
ribosome
B
A
A
A
B
• 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?
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
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
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
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
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
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
Unfolding is extremely slow at high [GdnHCl]
Fluorescence
Intensity
(a.u.)
FluorescenceIntensity
Intensity(a.u.)
(a.u.)
Fluorescence
Intensity
(a.u.)
140
140
140
140
130
130
130
130
1min
hr
2
Black
Black
= 30
=200
100
3
hr unfolding
unfolding
Black
=10
4
hr
unfolding
120
120
120
120
Diamonds = 30 min unfolding
110
110
110
110
100
100
100
100
90
90
90
90
90
80
80
80
80
80
70
70
70
70
70
60
60
60
60
50
50
50
50
50
40
40
40
40
40
0.50
0.50
0.50
0.50
0.50
0.75
0.75
0.75
0.75
1.00
1.00
1.00
1.00
1.25
1.25
1.25
1.50
1.50
1.50
1.75
1.75
1.75
2.00
2.00
2.00
2.25
2.25
2.25
2.50
2.50
2.50
2.75
2.75
2.75
2.75
3.00
3.00
3.00
3.00
[GdnHCl](M)
(M)
[GdnHCl]
[GdnHCl]
(M)
• Unfolding takes ~100 hr to complete
• Slowest step represents unraveling of partially folded state
• What creates high energy barrier for unfolding?
Mirco Junker
Spacefilling models of pertactin backbone structure
• -helix backbone is remarkably regular
• Long loops are clustered on one face of structure
Refolding is even slower!
140
140
140
140
Black
Black
Black
=216
30
24
=102
4min
hr
hrrefolding
refolding
refolding
Black
76
hr
refolding
Black
===312
hr
Fluorescence
Intensity
(a.u.)
FluorescenceIntensity
Intensity(a.u.)
(a.u.)
Fluorescence
Fluorescence
130
130
130
130
Diamonds = 30 min refolding
120
120
120
120
110
110
110
110
100
100
100
100
90
90
90
90
80
80
80
80
80
70
70
70
70
70
60
60
60
60
60
50
50
50
50
50
40
40
40
40
40
0.50
0.50
0.50
0.50
0.50
0.75
0.75
0.75
0.75
1.00
1.00
1.00
1.00
1.25
1.25
1.25
1.50
1.50
1.50
1.75
1.75
1.75
1.75
2.00
2.00
2.00
2.00
2.25
2.25
2.25
2.25
2.50
2.50
2.50
2.50
2.75
2.75
2.75
2.75
2.75
3.00
3.00
3.00
3.00
3.00
[GdnHCl]
(M)
[GdnHCl](M)
(M)
[GdnHCl]
(M)
• 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
Pertactin slow refolding kinetics:
Fluorescence Intensity (a.u.)
90
85
80
75
70
65
60
55
50
Unfolded
45
40
0
20
40
60
80
100
120
140
160
180
200
Time (hr)
• Refolding at 1.5 M GdnHCl; monitored by Trp fluor. emission
• Multiple slow components
Chris Schuster
Pertactin slow refolding kinetics:
Fluorescence Intensity (a.u.)
140
120
100
80
60
Unfolded
40
0
20
40
60
80
100
120
140
160
180
200
Time (hr)
• Refolding at 0.5 M GdnHCl; monitored by Trp fluor. emission
• Fast and slow components
Chris Schuster
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
Michael Evans
Mirco Junker
Krastyu Ugrinov
Chris Schuster
Katie O’Sullivan
Elizabeth Klimek
Kelli Whiteman
Kay Finn
Neil Isaacs, U. Glasgow
Andre Palmer, ND
Bill Boggess
ND Mass Spec Facility
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