studying structure and dynamics of protein complexes by...
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Christian Ader, Gitta Angerstein, Manuel Etzkorn, Itzam de Gortari, Henrike Heise, AshutoshKumar, Henrik Müller, Robert Schneider, Karsten Seidel, Marc Baldus
Solid-state NMR groupMax Planck Institute for Biophysical Chemistry
37077 Göttingen, Germany
Studying structure and dynamics of protein complexes by Studying structure and dynamics of protein complexes by solidsolid--state NMR spectroscopystate NMR spectroscopy
TheThe EBSA EBSA prizeprize lecturelecture
JulyJuly 18th 200718th 2007
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Schematic representation of a crowded cell.Dobson Nature 2004
Molecular Model of an Average synaptic vesicle Jahn et al., Cell 2006
Life Life cancan bebe crowdedcrowded……
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"magnetic resonance imaging"
“solution-state NMR"
Magnetic resonanceMagnetic resonance
H20,etc
“high-resolution NMR"
θ
μmagn
z
yx
Interactions on the molecular level
B0
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“solution-state NMR"
HighHigh--resolution NMRresolution NMR
H20,etc
Solution-state NMR
Radio frequencyschemes
hardwarehardware
InteractionsInteractions areare relativelyrelatively weakweak duedue to to motionmotion
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Solid-state NMR
“solution-state NMR"
Magnetic resonance: solution vs. solidMagnetic resonance: solution vs. solid--state NMRstate NMR
H20,etc
Solution-state NMR
Radio frequencyschemes
hardwarehardware
InteractionsInteractions areare relativelyrelatively weakweak
Interaction Interaction strengthstrength increasesincreases
Radio frequencyschemes
hardwarehardware
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ssNMRssNMR: Interactions are stronger and anisotropic: Interactions are stronger and anisotropic
J12μ1
μ2θr12μ1
μ2θ
μ1
θ
μ1
θ
Dipolar coupling Scalar coupling
Chemical shift quadrupolar coupling
σxx
σyy
σzz
σzz
σyy
σxx
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ssNMRssNMR MethodsMethods: : StructuralStructural parametersparameters
1. Sample 1. Sample preparationpreparation 2. 2. ChemicalChemical ShiftShift assignmentassignment 3: 3: StructuralStructural parametersparameters
S. Luca, H. Heise, M. Baldus, Acc. Chem. Res. 2003, 36, 858-865. .
MASMAS
Radio frequencyschemes
hardwarehardware
1H ssNMR exhibits limited resolution
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ssNMRssNMR MethodsMethods: : StructuralStructural parametersparameters
C
H
H
NH
C
N
CH
1. Sample 1. Sample preparationpreparation 2. 2. ChemicalChemical ShiftShift assignmentsassignments & & StructuralStructural parametersparameters
C
S. Luca, H. Heise, M. Baldus, Acc. Chem. Res. 2003, 36, 858-865. .
MASMAS
Radio frequencyschemes
hardwarehardware
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ssNMRssNMR MethodsMethods: : StructuralStructural parametersparameters
1. Sample 1. Sample preparationpreparation
3D 3D structurestructure
2. 2. ChemicalChemical ShiftShift assignmentsassignments & & StructuralStructural parametersparameters
S. Luca, H. Heise, M. Baldus, Acc. Chem. Res. 2003, 36, 858-865.
MASMAS
Radio frequencyschemes
hardwarehardware
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ns μs ms s
spin-spin interactions
ssNMRssNMR: : StructureStructure and and DynamicsDynamics
RigidMobile
…
M. Baldus, J.Biomol. NMR. 2007, in press.
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ns μs ms s
spin-spin interactions
RigidMobile
ssNMRssNMR: : StructureStructure and and DynamicsDynamics
… ……
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Molecular complexes investigated by solidMolecular complexes investigated by solid--state NMR spectroscopystate NMR spectroscopy
• For a large range of molecular sizes and correlation times
• Proteoliposomes• Powders• frozen solutions• microcrystals• gels• precipitates• aggregates• etc.
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Molecular complexes investigated by solidMolecular complexes investigated by solid--state NMR spectroscopystate NMR spectroscopy
• For a large range of molecular sizes and correlation times
• Proteoliposomes• Powders• frozen solutions• microcrystals• gels• precipitates• aggregates• etc.
•• In a functional system
Baldus M, Curr. Opin. Struct. Biol. 2006, 16, 618-623.
-100 –50 0 50 100Membrane potential (mV)
1.00.80.60.40.20.0
G/G
max
0
2
4
6
8
0
• For a large range of molecular sizes and correlation times
• Proteoliposomes• Powders• frozen solutions• microcrystals• gels• precipitates• aggregates• etc.
Proteoliposomes Microcrystals Precipitates
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OutlineOutline
Protein Folding & Aggregation
Ligand – Membrane Protein interactions
Membrane Protein complexes
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Native protein Intermediate Fibril
Protein Protein foldingfolding & & aggregationaggregation
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αα--synucleinsynuclein (AS)(AS)
MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEEQQVVTNVGGTNVGGAVVTAVVTGVGVTAVAQTAVAQKTKTVVEGEGAGSIAAATGFVAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA
α-synuclein fibrils are found in brains of patients with Parkinson disease.(intracellular inclusions in dopaminergic neurons)
140 aa
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ssNMRssNMR methodsmethods forfor αα synucleinsynuclein fibrilsfibrils and and beyondbeyond
ββ--strandstrand corecore Mobile C Mobile C terminusterminus
disordereddisordered monomermonomer
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Heise, H.; Hoyer, W.; Becker, S.; Andronesi, O. C.; Riedel, D.; Baldus, M. PNAS 2005, 102, 15871-15876.
Solid-state NMR
EM
AS: Correlation between molecular structure and fibril morphology
Form A
Form B
Form A
Form B
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Native protein Intermediate Fibril
Protein Protein aggregationaggregation and and fibrilfibril formationformation
Crhα synuclein
α-synuclein
protein tau
polyglutamine
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CharacterizeCharacterize foldingfolding intermediateintermediate byby 2D 2D ssNMRssNMR
precipitateCatabolite repression Histidine-containing phosphorcarrier protein (Crh)
Precipitate vs.Micro Xtals
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CharacterizeCharacterize foldingfolding intermediateintermediate byby 2D 2D ssNMRssNMR
?269 K 282 K
aggregate
precipitateCatabolite repression Histidine-containing phosphorcarrier protein (Crh)
Domain Domain swappedswapped dimerdimer
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A
Unfolded intermediateB
C
A
RefoldingRefolding accordingaccording to to timetime--resolvedresolved 2D 2D ssNMRssNMR
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A
B
C
“Unfolded“ intermediate
+ +
A
B
RefoldingRefolding accordingaccording to to timetime--resolvedresolved 2D 2D ssNMRssNMR
269 K 282 K
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A
“Unfolded“ intermediate
B
C
+ +
A
B
C
RefoldingRefolding accordingaccording to to timetime--resolvedresolved 2D 2D ssNMRssNMR
269 K 282 K
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2D 2D ssNMRssNMR datadata areare sensitive to sensitive to aggregationaggregation kineticskinetics
k1,k2 optimal
Etzkorn, M., Böckmann, A., Penin, F., Riedel, D., and Baldus, M. (2007) J. Amer.Chem. Soc., 129, 169-175.
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OutlineOutline
Protein Folding & Aggregation
Ligand – Membrane Protein interactions
Membrane Protein complexes
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LigandLigand –– membranemembrane proteinprotein interactionsinteractions byby ssNMRssNMR
Neurotensin
Neurotension receptor (GPCR)
Phospholamban
Ca-ATPase
Kaliotoxin
KcsA-Kv1.3
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Psychosis, schizophreniaParkinsons disease
Neurotensin (NT)
ELYENKPR8R9P10Y11I12L13
Sub-nanomolar bindingaffinity of NT and NT(8-13) to
NTS-1
Rat Neurotensin receptor NTS-1
Vincent, J. P.; Mazella, J.; Kitabgi, P. Trends Pharmacol. Sci. 1999, 20, 302.
TheThe NeurotensinNeurotensin –– NTSNTS--1 System1 System
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13C/15N
13C/15N
ELYENKPR8R9P10Y11I12L13
neurotensin
Lipid reconstituted NTRDetergent solubilized NTR
NeurotensinNeurotensin boundbound to a to a GG--proteinprotein coupledcoupled receptorreceptor
U[13C,15N] NT(8-13) 10 μg – 22 μg
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NeurotensinNeurotensin boundbound to a to a GG--proteinprotein coupledcoupled receptorreceptor
13C/15N
13C/15N
ELYENKPR8R9P10Y11I12L13
neurotensin
Luca, S., White, J.F., Sohal, A.K., Filipov, D.V., van Boom, J.H., Grisshammer, R., and Baldus, M., 2003, PNAS,100, 10706
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Arg 7/8 Pro 9 Tyr 10 Ile 11
−180 −120 −60 0 60 120 180−180
−120
−60
0
60
120
180
−180 −120 −60 0 60 120 180−180
−120
−60
0
60
120
180
−180 −120 −60 0 60 120 180−180
−120
−60
0
60
120
180
−180 −120 −60 0 60 120 180−180
−120
−60
0
60
120
180
−180 −120 −60 0 60 120 180−180
−120
−60
0
60
120
180
−180 −120 −60 0 60 120 180−180
−120
−60
0
60
120
180
−180 −120 −60 0 60 120 180−180
−120
−60
0
60
120
180
−180 −120 −60 0 60 120 180−180
−120
−60
0
60
120
180
−180 −120 −60 0 60 120 180−180
−120
−60
0
60
120
180
−180 −120 −60 0 60 120 180−180
−120
−60
0
60
120
180
−180 −120 −60 0 60 120 180−180
−120
−60
0
60
120
180
−180 −120 −60 0 60 120 180−180
−120
−60
0
60
120
180
ΔG (H2O->Lipid) kcal/mol0.81 0.45 –0.94 –0.31
Heise, H., Luca, S., de Groot, B. L., Grubmueller, H. & Baldus, M. (2005) Biophys. J. 89, 2113-2120.
ConformationalConformational disorderdisorder of of NeurotensinNeurotensin
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ψ (Pro) 146 ± 15o
KD [nM] 0.27 12 16000
Bittermann, H.; Einsiedel, J.; Hubner, J.; Gmeiner, P. J. Med. Chem. 2004, 47, 5587-5590.
A ssNMR-structure / affinity relationship !
Arg
Arg
Ile Leu
Arg
Arg
Ile Leu
~ 120 o ~ -120 o
4,4-spirolactam
ComparisonComparison: Binding : Binding affinitiesaffinities of NT and of NT and rigidizedrigidized NTNT
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LigandLigand –– membranemembrane proteinprotein interactionsinteractions byby ssNMRssNMR
Luca, S. et al., 2003, PNAS,100, 10706.
Heise et al, 2005 Biophys. J. 89, 2113.
Neurotensin
Neurotension receptor (GPCR)
Phospholamban
Ca-ATPase
Kaliotoxin
KcsA-Kv1.3
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VoltageVoltage--gatedgated ionion channelschannels
Yu FH, Catterall WA: The VGL-Chanome: A Protein Superfamily Specialized for Electrical Signaling and Ionic Homeostasis. Sci. STKE 2004, 2004:re15-
HowHow isis a a channelchannel blockedblocked ??
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A. Lange, S. Luca, M. Baldus, J.Am.Chem.Soc. 2002, 124, 9704-9705.Lange, K. Seidel, L. Verdier, S. Luca, M. Baldus, J.Am.Chem.Soc. 2003, 125, 12640-12648.K. Seidel, M. Etzkorn, C. Griesinger, A. Sebald, M. Baldus, J.Phys.Chem. A., 2005, 109, 2436-2442.
Relative Relative fractionfraction of of longlong--rangerange correlationscorrelations
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CC vs. CHHC 2D
I4CD1C33CA
ObtainingObtaining thethe 3D 3D ssNMRssNMR structurestructure of KTXof KTX
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Lange A, Becker S, Seidel K, Pongs O, Baldus M. 2005. Angew. Chem.-Int. Edit. 44:2089.M. Gairi, R. Romi, I. Fernandez, H. Rochat, M. F. Martin-Eauclaire, J. Van Rietschoten, M. Pons, E. Giralt, 1997. J. Pept. Sci., 3: 314.
CC vs CHHC on KTX PDB: 1XSWBackbone RMSD: 0.8 ÅBackbone RMSD (residues 4-38) between solid KTX and KTX in solution:1.9 Å
ObtainingObtaining thethe 3D 3D ssNMRssNMR structurestructure of KTXof KTX
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Free vs. Free vs. ChannelChannel--boundbound UU--[[1313C,C,1515N] KTXN] KTX
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Free vs. Free vs. KTXKTX--boundbound U[U[1313C,C,1515N] KcsAN] KcsA--Kv1.3Kv1.3
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Eriksson, M. A. L.; Roux, B. Biophys. J. 2002, 83, 2595-2609.
Lock – Key interaction
Toxin Toxin –– Ion Ion channelchannel complexcomplex accordingaccording to MDto MD
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Lange, A.; Giller, K.; Hornig, S.; Martin-Eauclaire, M. F.; Pongs, O.; Becker, S.; Baldus, M. Nature (2006) 440, 959-962.
Toxin Toxin –– Ion Ion channelchannel complexcomplex accordingaccording to to ssNMRssNMR
4. Selectivity filter changes conformation
4.
3. Turret not directly involved in binding interface
3.
1. Toxin inserts deeper into pore
2. Toxin structure altered.
1.2.
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ChannelChannel: : IntrinsicIntrinsic conformationalconformational flexibilityflexibility
-100
-50
0
50
100
150
φ [ο]
-100
-50
0
50
100
76 77 78 79
ψ [ο]
1K4C
1K4D
1R3I1R3J
1R3K
1R3L
2BOB
2BOC
Residue number
Lange A, Giller K, Pongs O, Becker S, Baldus M (2006),J. Recept. Signal. Transduct. Res., 26, 379-393
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OutlineOutline
Protein Aggregation
Ligand – Membrane Protein interactions
Membrane Protein complexes
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sRII
HtrII
HowHow cancan oneone receptorreceptor exertexert twotwo different different functionsfunctions ??
sRII
CytoplasmicCytoplasmic sideside
extracellularextracellular sideside
Bogomolni, R. A., Stoeckenius, W., Szundi, I., Perozo, E., Olson, K. D., and Spudich, J. L. (1994) PNAS 91, 10188-10192Schmies, G., Engelhard, M., Wood, P. G., Nagel, G., and Bamberg, E. (2001) PNAS 98, 1555-1559Sudo, Y., Iwamoto, M., Shimono, K., Sumi, M., and Kamo, N. (2001) Biophys. J. 80, 916-922Sudo, Y., and Spudich, J. L. (2006) PNAS 103, 16129-16134
TransportTransport SignallingSignalling
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Ion channels photo-sensors
bR hR sR HtrI HtrII
bacteriorhodopsin halorhodopsin Sensory rhodopsin I
sRII
Sensory rhodopsin II
Gordeliy, V. I. et al., Nature 2002, 419, 484-487., Nature 2006, 440, 115-119
SensorySensory rhodopsinrhodopsin II II belongsbelongs to to thethe familyfamily of of RetinalRetinal proteinsproteins
CytoplasmicCytoplasmic sideside
extracellularextracellular sideside
E. Bordignon, J. P. Klare, M. Doebber, A. A. Wegener, S. Martell, M. Engelhard, H.-J. Steinhoff, J. Biol. Chem. 2005, 280, 38767-38775.
adapted from: Y. Sudo, M. Yamabi, S. Kato, C. Hasegawa, M. Iwamoto, K. Shimono, N. Kamo, J. Mol. Biol. 2006, 357, 1274-1282.
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SRII proteoliposomes: ssNMR assignments
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U[13C,15N\(V,L,F,Y)] NpSRII
Static protein residues, SRII proteoliposomes
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Dynamic protein residues, SRII proteoliposomes
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Water exposed protein residues, SRII proteoliposomes
[1] Etzkorn, M., Matell, S., Andronesi, O.C., Seidel, K., Engelhard, M., and Baldus, M. (2007):, Angew. Chem.-Int. Edit., 46, 459-462.
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SummarySummary
Solid-state NMR can be applied to protein complexes undera variety of experimental conditions
Protein folding and aggregation can be studied at atomicresolution and in real time
Molecular plasticity plays an important role in high-affinityligand binding, complexation events and proteinfunctionality in membranes
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Christian AderGitta AngersteinManuel EtzkornDr. Itzam de GortariDr. Henrike HeiseDr. Ashutosh KumarDr. Henrik MüllerRobert Schneider Karsten Seidel
Dr. Sorin Luca (NIH)Lars Sonnenberg (TU Munich)Dr. Colan Hughes (Cardiff)Dr. Ovidiu C. Andronesi (Harvard)Dr. Adam Lange (ETH Zurich)
Acknowledgements Acknowledgements Hamburg:O. PongsS. Hornig
M. Von BergenE. Mandelkow
NIH:R. Grisshammer
CNRS Lyon: A. Böckmann
CNRS Marseille: M.F. Martin-Eauclaire
Edmonton:H.S. Young
Berkeley:D. Trauner
Göttingen:MPIBPC:S. BeckerC. Griesinger
K. GillerV. Klaukien
T. CarlomagnoM. Zweckstetter
W. HoyerT. Jovin
D. Riedel
B. De GrootH. Grubmueller
MPI Dortmund:M. EngelhardS. Martell
Mainz:G. UndenH. Kneuper
MPGDFGVW StiftungHumboldt StiftungFCIEU