1 Paramagnetic Paramagnetic NMR: a NMR: a versatile versatile tool tool in in structural biology structural biology Claudio Luchinat Claudio Luchinat CERM CERM University of Florence University of Florence 800 800 700b 700b 600 600 850ss 850ss 700 700 400 400 500 500 Bio Bio-labs labs Library Library 700ss 700ss 900 900 GENEXPRESS, CRYST, CISM GENEXPRESS, CRYST, CISM Department Department of of Chemistry Chemistry ( offices offices, , bio bio-labs labs, , relaxometer relaxometer, instruments instruments..) ..) Workshop Workshop Conference Conference room room 600b 600b The Magnetic Resonance Center in Florence The Magnetic Resonance Center in Florence Computer room Computer room Electron/ Electron/ nuclear relaxation nuclear relaxation (Relaxometry) (Relaxometry) Drug discovery Drug discovery Structural proteomics Structural proteomics Metabolomics Metabolomics Protein structure determination Protein structure determination Methodological advancements Methodological advancements in NMR in NMR Solid Solid state NMR state NMR Bioinformatics and Bioinformatics and computational biology computational biology
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Paramagnetic NMR: a versatile tool in structural biology · Paramagnetic NMR: a versatile tool in structural biology Claudio Luchinat CERM University of Florence 800 700b 600 850ss
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Paramagnetic Paramagnetic NMR: a NMR: a versatile versatile tool tool in in
structural biologystructural biology
Claudio LuchinatClaudio Luchinat
CERMCERMUniversity of FlorenceUniversity of Florence
800800700b700b
600600
850ss850ss
700700
400400
500500
BioBio--labslabs
LibraryLibrary
700ss700ss
900900
GENEXPRESS, CRYST, CISMGENEXPRESS, CRYST, CISMDepartment Department of of ChemistryChemistry((officesoffices, , biobio--labslabs, , relaxometerrelaxometer,, instrumentsinstruments..)..)
WorkshopWorkshop
ConferenceConference roomroom
600b600b
The Magnetic Resonance Center in FlorenceThe Magnetic Resonance Center in Florence
Protein structure determinationProtein structure determinationMethodological advancementsMethodological advancements in NMRin NMRSolidSolid state NMRstate NMRBioinformatics andBioinformatics and computational biologycomputational biology
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EUEU--NMRNMR –– EuropeanEuropean Network ofNetwork of Research Infrastructures for Research Infrastructures for providingproviding Access andAccess and Technological AdvancementsTechnological Advancements inin biobio--NMRNMR
Contact shiftContact shift restraintsrestraints:: unpairedunpaired electronelectron spinspin density density on heme nuclei on heme nuclei isis a function ofa function of axialaxial ligandligand orientationorientation
8-CH3 5-CH3 1-CH3 3-CH3
φ = 0° φ = 30° φ = 45°
M80A cyano-cytochrome c
I. I. BertiniBertini, C. Luchinat, G. , C. Luchinat, G. ParigiParigi, F.A. Walker, , F.A. Walker, JBICJBIC 19991999
The solution structure is found significantly different from the solid state structure due to a different relative position of the two CaM domains
Even larger differences are being observed in other CaM-
peptide complexes
X-ray structure
NMR structure
NN
CC
A“true” solid state structure “true” solution structure
X-ray structure NMR structure
Indetermination ofthe X-ray structure
Indeterminationof the NMR structure
“true” solid state structure “true” solution structure
X-ray structure NMR structure
indetermination of the“NMR-corrected” X-ray structure
“NMR-corrected”X-ray structure
B
Can the Can the accuracyaccuracy of aof a protein structureprotein structure in in solution be extendedsolution be extended??
1515
A challenge for A challenge for paramagnetismparamagnetism--based restraints: based restraints: proteins with partially independent domainsproteins with partially independent domains
N-terminal
C-terminal
Barbato, G., Ikura, M., Kay, L.E., Pastor, R.W. & Bax, A Biochemistry(1992)
Baber, J. L., Szabo, A. & Tjandra, N. J. Am. Chem. Soc. (2001)
Small 15N-1H NOEsfor residues 78-81
The case of CaThe case of Ca44CalmodulinCalmodulin
A paramagnetic ion is attached to the N-terminal domain, causing its partial orientation in the magnetic field
The Florence strategyThe Florence strategy
The C-terminal domain MAY experience some induced orientation
Structure Structure of calmodulin of calmodulin interactinginteracting withwith the the intrinsicallyintrinsically unfoldedunfolded proteinprotein αα--synucleinsynuclein
A
N CThe The structure structure of of each domain is very each domain is very similar to isolated calciumsimilar to isolated calcium calmodulin calmodulin
Chemical shift changes indicate that an interaction occurs
Refined using PCS
Refined using RDC
larger spreadinglarger spreading of Cof C--terminal rdcterminal rdc thanthan in thein theisolated proteinisolated protein
PCS and RDC can provide the relative position of twoproteins in a complex.
If the adduct is not rigid, PCS and RDC are averaged on the values relative to all experienced conformations
Ln3+
RDC provide information on the orientations with respect to the metal tensor
PCS provide information on the positions with respect to the metal tensor
Assessing the usefulness of LanthanideAssessing the usefulness of Lanthanide--Binding Tags Binding Tags (LBT) to investigate protein(LBT) to investigate protein--protein interactionsprotein interactions
A tag able to bind paramagnetic metal ions can be exploited to obtain structural and dynamic informationA LBT tag has been fused with the copper chaperoneHAH1Theoretically, RDC are scaled down by mobility but still fully usable. PCS are scaled down and not fully usable
HAH1
LBT Ln3+
HAH1 MNK1
Er3+
HAH1 MNK1
Dy3+
MNK1? The PCS obtained by inserting Tm3+, Dy3+, Er3+, and Tb3+
on LBT have been tested for their ability to monitor the interaction of HAH1 with MNK1
2020
Flexibility in LBTFlexibility in LBT--HAH1 makes the solution not univocalHAH1 makes the solution not univocal
Only calculations starting with one of the two HAH1-G1LBT conformations resulted in a structure of the adduct that is similar to that expected:flexibility hampers the use of PCS
Second generation tags have been conceived to reduce flexibility:Su, Huber, Dixon, Otting, ChemBioChem, 2006, 7, 1599Martin, Hähnke, Nitz, Wöhnert, Silvaggi, Allen, Schwalbe, Imperiali, JACS, 2007, 129, 7106Keizers, Desreux, Overhand, Ubbink, JACS, 2007, 129, 9292…
HAH1 MNK1
- Solid state NMR structures are difficult to obtain because protons cannot be routinely observed and NOE-like cross peak intensities are not quantitatively related to distances
-- Paramagnetic proteins may provide distinct advantages:Paramagnetic proteins may provide distinct advantages:in SS NMR Curie relaxation is absent (no rotation!)PCS should be equally well observable…
• insoluble proteins• fibrils• membrane proteins
Solid state NMR of paramagnetic proteinsSolid state NMR of paramagnetic proteins
Why SS NMR of Proteins?Why SS NMR of Proteins?
Why SS NMR of Why SS NMR of paramagneticparamagnetic Proteins?Proteins?
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ZnCatalytic-active domain of the
Matrix MetalloProteinase 12
159 AA (17.6 kDa)Zn(II)-containing proteinZn(II) replaceable with Co(II)(Bertini et al., PNAS, 2005)
44 AA (28%) helices27 AA (17%) ß-strands
Easily crystallisable
A test case: Zinc(II)A test case: Zinc(II) and and Cobalt(II)Cobalt(II) MMPMMP--1212
SolidSolid--state Protein Arrangement state Protein Arrangement Determined with InterDetermined with Inter--Molecular SS PCSMolecular SS PCS
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