protein folding protein folding diseases protein ... 02-14-03.pdf · protein folding protein...
TRANSCRIPT
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Proteins?
Protein function
Protein folding
Protein folding diseases
Protein interactions
Macromolecular assemblies
The end product of Genes
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Protein Unfolding
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N
H
C
R O
CC
O
N
C
DOD
D+
DOD
N
D
C
R O
CC
O
N
C
HDOD+
Acid Catalysis
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OD-
N
H
C
R O
CC
O
N
C
DOD
N
D
C
R O
CC
O
N
C
HOD
N
H
C
R O
CC
O
N
C
DOD
D+
DOD
N
D
C
R O
CC
O
N
C
HDOD+
Acid Catalysis
Base Catalysis
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0 2 4 6 8-4
-2
0
2
4
Log[
k(se
c-1)
]
pH
Amide Proton Exchange is pH dependent
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EX1 mechanism: kch > kcl
Folded Open hydrogen exchangekcl
kop kch
N H N D
EX2 mechanism: kch < kcl
kex = kop
kex = K(op/cl) ·kch
“pH dependent”
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Cytochrome C
Cooperative Unfolding
Englander, et. al. 2002
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Cooperative Unfolding
Cytochrome CEnglander, et. al. 2002
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Cytochrome C Folding Pathway
Hoang, et. al. 2002
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Multi-state vs Two State Protein Unfolding
Englander, et. al. 2002
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NMR H/D exchange
Individual amide proton exchange rates
Sensitive to subtle protein dynamics
Used extensively to study protein folding
Problems
Need pure sample
Need high concentrations
Only small proteins
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Mass Spec H/D exchange?David Smith, (Zhang et al 1993)
Sample need not be pure
Low sample concentrations
Large proteins
Macromolecular complexes
Problems
Digestion coverage
Exchange rate is averaged over the whole peptide
Buffer intolerance
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Mass spectrometry
Protein
Pepsin digestion (Optional)
•Exchange with D20•Quench with low pH over time
•Fragment size 10-20 residues
•Assignment of peptides•Quantifying exchange
H/D Exchange Experimental Protocol
0 2 4 6 8-4
-2
0
2
4
Log[
k(se
c-1)
]
pH
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Protein stability“pulsed labeling H/D exchange”
[GdmCL]
0 1 2 3 4
0
20
40
60
80
100
Perce
ntage
excha
nge
[GdmCl]
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MALDI Analysis of Pulse labeled proteins“SUPREX”
SUPREX; Stability of Unpurified Proteins from Rates of H/D Exchange
•Unpurified proteins
•Rapid analysis
•High throughput
•Protein stability in the cell
Ghaemmaghami, et. al. 2000
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SUPREX
4-oxalocrotonate tautomerase
Suprex =
CD =
Powell, et. al. 2000
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Maltose binding protein
SUPREX analysis of mutations and protein stability
Proteinstability
Ghaemmaghami, et. al. 2000
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10 mM =56 mM =
Trp repressor (TrpR)- L-tryptophan =+ L-tryptophan =
Association detected by SUPREX
Powell, et. al. 2000
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Protein Stability in Cells
Ghaemmaghami, et. al. 2001
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MALDI Analysis of Pulse labeled proteins“SUPREX”
•Unpurified proteins
•Rapid analysis
•High throughput
•Protein stability in the cell
•?
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Continous H/D Exchange
Protein folding
Protein interfaces
Quantitatively determine rates
?
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Mass spectrometry
CA Tubes or Dimers
Pepsin digestion
•Exchange with D20•Quench with low pH over time
•Fragment size 10-20 residues
•Assignment of peptides•Quantifying exchange
H/D Exchange Experimental Protocol
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Identification of protein interaction interfaces
DD
DD D
DD
D
DDH
H
HH H
HH
H
HH
Surface amideprotons
D2O
Protein Surface amide protonsreplaced withdeuterons
DD
DD D
DD
D
DD
HH
HH H
HH
H
HHH
H
HH D
DH
H
DH
H2O
Deuteriums trapped
Deuterium foot print
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Protonated
Deuterated
Off exchange (10 min)
+ ATP
+ ATP & PKI
Detection of PKI Interaction with PKA
Mandell et al. 1998
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PKI, PKA, and ATP
ATP
PKA
PKI
Mandell et al. 1998
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Epitope mapping of a monoclonal antibodyagainst thrombin by H/D-exchange
mAB epitope
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Novel Approaches for UnderstandingVirus Assembly and Dynamics.
Lanman et al. 2003
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Image Reconstructions of Procapsid and Mature Virion
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N-terminal domain21 kDa
C-terminal domain25 kDa
175 204
HINGE
The Coat Protein Subunit has a Two Domain Structure
Lanman, et. al. 1999
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2
2
4
3
4
5
3
51
1
1 2 21
Monomer Mutant Dimer
Procapsid Expanded Lattice
N C
Unfolded Subunit
A domain-shuffling model for capsid expansion
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The Model Predicts Trapping of Deuterium during Expansion
Transition State Expanded Procapsid
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M ES XPS
M PS ES X
M PS XES M ES XPS M ES XPS
M ES XPS
M ES XPS
M ES XPS
M PS ES X
expanded shell protection
deuterium retention after expansion
procapsid shell protection
monomer protection
M ES XPS
M PS ES X
M PS XES M ES XPS M ES XPS
M ES XPS
M ES XPS
M ES XPS
MALNEGQIVT LAVDEIIETI SAITPMAQKA KKYTPPAASM QRSSNTIWMP VEQESPTQEG 60
WDLTDKATGL LELNVAVNMG EPDNDFFQLR ADDLRDETAY RRRIQSAARK LANNVELKVA 120
NMAAEMGSLV ITSPDAIGTN TADAWNFVAD AEEIMFSREL NRDMGTSYFF NPQDYKKAGY 180
DLTKRDIFGR IPEEAYRDGT IQRQVAGFDD VLRSPKLPVL TKSTATGITV SGAQSFKPVA 240
WQLDNDGNKV NVDNRFATVT LSATTGMKRG DKISFAGVKF LGQMAKNVLA QDATFSVVRV 300
VDGTHVEITP KPVALDDVSL SPEQRAYANV NTSLADAMAV NILNVKDART NVFWADDAIR 360
IVSQPIPANH ELFAGMKTTS FSIPDVGLNG IFATQGDIST L SGLCRIALW YGVNATRPEA 420
IGVGLPGQTA 430
M PS ES X
expanded shell protection
deuterium retention after expansion
procapsid shell protection
monomer protection
Changes in Exchange Protection during Assembly & Maturation
Tuma, et. al. 2000
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TM
Immature Mature
SU
Gag
MA
CA
RNA
NC
MA CA p2 NC p1 p6
Gag
HIV Assembly & Maturation
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Thin-Section EM Analysis of Assembly Products
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CA is Comprised of Distinct N- and C- Terminal Structural Domains
N-domain
C-domain
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CA Cylinders are Based on a Hexamer Lattice
From Li et al, Nature 407:409 (2000)
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Hypothesis: The CA that does not form dimers, because of a mutation atthe dimer interface should form hexamers at high NaCl concentrations
+NaCl
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+NaCl
CA does not form hexamers without C-domain (dimer) interaction.
Hypothesis: The CA that does not form dimers, because of a mutation atthe dimer interface should form hexamers at high NaCl concentrations
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H/D exchange analyzed with FT-MS
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Mass spectrometry
CA Tubes or Dimers
Pepsin digestion
•Exchange with D20•Quench with low pH over time
•Fragment size 10-20 residues
•Assignment of peptides•Quantifying exchange
H/D Exchange Experimental Protocol
![Page 41: Protein folding Protein folding diseases Protein ... 02-14-03.pdf · Protein folding Protein folding diseases Protein interactions Macromolecular assemblies The end product of Genes](https://reader030.vdocuments.pub/reader030/viewer/2022040121/5eace5c48a23d376e77efc86/html5/thumbnails/41.jpg)
2 3
56
1 4
Waste
Sample injection
C18 column
Pump AH2O, 0.05% Formic acid
Pump BACN, 0.05% Formic acid
ESI-MS Analysis of Peptides
MS
Sample loop
Ice bath (4 C)
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Time
Ab
un
dan
ce
Elution of Digest from C4 Column
~1 min
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m/z1 4001 2001 000 800 600
Representative FT-MS Scan
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m/z 752 751 750 749 748 747 746 745
A
B
C
A = (744.842 * 2) Da
B = (746.390 * 2) Da
C = (749.386 * 1) Da
Expanded View of 744-752 m/z Region
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744.842 within 1 ppm746.390 within 0.5 ppm
With High Resolution Peptides of Similar m/zcan be Analyzed
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1PIVQNLQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF SALSEGATPQ DLNTMLNTVG
!61GHQAAMQMLK ETINEEAAEW DRLHPVHAGP IAPGQMREPR GSDIAGTTST LQEQIGWMTH
121NPPIPVGEIY KRWIILGLNK IVRMYSPTSI LDIRQGPKEP FRDYVDRFYK TLRAEQASQE!!181VKNWMTETLL VQNANPDCKT ILKALGPGAT LEEMMTACQG VGGPGHKARV L
Peptides Covering 95% of the Sequence have Been Assigned
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m/z 752 751 750 749 748 747 746 745
A
B
C
A = (744.842 * 2) Da
B = (746.390 * 2) Da
C = (749.386 * 1) Da
Expanded View of 744-752 m/z Region
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m/z 752 751 750 749 748 747 746 745
40 mDa
Extremely High Resolution is Achievable with FT-MS
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m/z 752 751 750 749 748 747 746 745
The Related Peaks are Analyzed toDetermine the Distribution
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Isotopic distributions during H/D exchange
0 min
0.5 min
2 h
746 748 750m/z
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0 min
0.5 min
2 h
Centroid = 745.5
Centroid = 748
Centroid = 749
746 748 750m/z
The Centroids of the Distribution are Calculated
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Deuterium Incorporation at the Dimer Interface
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68 h
0 sec
30 sec
2 min
1 h
4 h
849843 846m/z
H/D Exchange Period
Deuterium Incorporation Causes a Mass Increase
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68 h
0 sec
30 sec
2 min
1 h
4 h
849843 846m/z
H/D Exchange Period
Assembled CA Unassembled CA
Deuterium Incorporation Causes a Mass Increase
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68 h
0 sec
30 sec
2 min
1 h
4 h
849843 846m/z
H/D Exchange Period
Assembled CA Unassembled CA
Deuterium Incorporation Causes a Mass Increase
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1 10 100 1000
1490
1491
1492
1493
1494
1495
1496
1497
1 10 100 10002530
2532
2534
2536
2538
2540
2542
1 10 100 1000
1524
1525
1526
1527
1528
1529
1530
1 10 100 1000
2136
2138
2140
2142
2144
2146
Time (min)
Hexamer1488 Monomer1488 F I Hex1488Sd mon1488Sd ### ### ### ###
Time (min)
Cen
troi
d of
Mas
s
hexamer2525 Monomer2525 mon2525fit Hex2525fit
Time (min)
Cen
troi
d of
mas
s
Hexamer1521 Monomer1521 Mon1521fit Hex1521fit
Hexamer2134 Monomer2134 Hex2134fit Hex2134fit3 Hex2134Sd
The Dimer Interface is Protected Upon Assembly
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1 10 100 1000
1267
1268
1269
1270
1271
1272
Cen
troi
d of
Mas
s
Time (min)
Helix II Shows Protection Upon Assembly
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Helices VI and VII Show Little Protection
1 10 100 10001933
1934
1935
1936
1937
1938
1939
1940Data: Dmean1932mon_MeanModel: 3expHD Chi^2 = 0.04466R^2 = 0.99146 A 1.45506 ±0.42438B 1.619 ±0.41511C 5.59556 ±0.32398k1 0.08275 ±0.04485k2 0.00548 ±0.00358k3 0.00015 ±0.00002
Cen
troi
d of
Mas
s
Time (min)
H1932mean M1932mean
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1 10 100 1000
1490
1491
1492
1493
1494
1495
1496
1497
1498
Cen
troi
d of
Mas
s
Time (min)
The Bottom of Helix III becomes Protected
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1 10 100 1000
1490
1491
1492
1493
1494
1495
1496
1497
1 10 100 10002530
2532
2534
2536
2538
2540
2542
1 10 100 1000
1524
1525
1526
1527
1528
1529
1530
1 10 100 1000
2136
2138
2140
2142
2144
2146
Time (min)
Hexamer1488 Monomer1488 F I Hex1488Sd mon1488Sd ### ### ### ###
Time (min)
Cen
troi
d of
Mas
s
hexamer2525 Monomer2525 mon2525fit Hex2525fit
Time (min)
Cen
troi
d of
mas
s
Hexamer1521 Monomer1521 Mon1521fit Hex1521fit
Hexamer2134 Monomer2134 Hex2134fit Hex2134fit3 Hex2134Sd
The Dimer Interface is Protected Upon Assembly
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Helices VI and VII Show Little Protection
1 10 100 10001933
1934
1935
1936
1937
1938
1939
1940Data: Dmean1932mon_MeanModel: 3expHD Chi^2 = 0.04466R^2 = 0.99146 A 1.45506 ±0.42438B 1.619 ±0.41511C 5.59556 ±0.32398k1 0.08275 ±0.04485k2 0.00548 ±0.00358k3 0.00015 ±0.00002
Cen
troi
d of
Mas
s
Time (min)
H1932mean M1932mean
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Changes in Exchange Rates due to CA Assembly
Lanman, et. al. 2003
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SEC separation of cross-linked CA monomer and dimer
CAdst101901:jkl_UV1_280nm CAdst101901:jkl_UV1_280nm@01,COPY
0
100
200
300
400
mAU
0 20 40 60 80 100 ml
CA Monomer
CA Dimer
CA Monomer
CA Dimer
80 10020 40 600
Abso
rban
ce a
t 280
nm
Elution volume (ml)
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Mass spectra of the cross-linked species
1000 1500 2000
0 10 20 30 40 50 60
0
50
100
150
200
250
10000 15000 20000
0 10 20 30 40 50 60
0
50
100
150
200
250
m/z
Ion
coun
t
Time (min)
m/z
Time (min)
10+11+
12+
13+
9+
8+
14439
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Lys 182
Lys 70
Lysine 70 was cross-linked to Lysine 182
31 to 131 = 10948.9 171 to 199 = 3375.9 +DST = 114.0
Expected mass = 14438.8
Observed mass = 14439
Lys70 cross-linked to 182
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12B
3A
A’
B’
An N-domain to C-domain Interaction in CA Assembly
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C:C
N:C
N:N
Three Sites of Interaction During Assembly
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Advantages of Hydrogen/Deuterium Exchange
•Small quantities required (10-12 mole)
•Needn’t be pure
•No symmetry constraints
•Can provide time resolved or dynamic information
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The future of H/D exchangeMulti protein macromolecular complexes
Cooperativity in large proteins or macromolecular complexes
Exchange rates for individual amide protons
Protein dynamics during motor motions