Recent advances in biomolecular NMR - EMBL …...Recent Advances in Biomolecular NMR •Protonless...
Transcript of Recent advances in biomolecular NMR - EMBL …...Recent Advances in Biomolecular NMR •Protonless...
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Recent Advances in
Biomolecular NMR
Lucia Banci
CERM – University of Florence
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Recent Advances in Biomolecular NMR
•Protonless NMR for the characterization of Unfolded proteins,
Large protein assemblies, Paramagnetic
systems
•In cell NMR For studying biomolecules in a cellular context
•Combination of Solution and Solid State
NMR For characterization of dynamic proteins and
large aggregates
•Mechanistic Systems Biology To describe and understand biological
processes at molecular level
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Why protonless NMR?
1H
13C
15N Inverse (i.e. through 1H) detection
of heteronuclei was a major
advanchement!!
Properties of 1H (high gH, ..)
high 1H sensitivity
/ large dipolar interactions
/ efficient relax processes
(large and paramagnetic molecules)
relatively low chemical shift dispersion
(unfolded systems)
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13C direct detection
…with the increase in
spectrometers sensitivity,
(high B0, cryo!)
direct detection of heteronuclei
(low nuclei)
becomes accessible
Isotopic enrichment necessary anyway
13C direct detection
is a complementary tool
1H
13C
15N
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DDn(13C)
13C direct detection – unique properties
Different spins, different properties! 1H
13C
Dd(13C)
Dd(1H)
DDn(1H)
Dd(13C)
Dd(1H)
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13C direct detection, protonless NMR
A complementary tool for
challenging systems
- paramagnetic proteins
- very large proteins
- parts of proteins affected by
exchange processes
- unfolded systems
- high salt concentrations
1H
13C
15N
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C´ direct detection – The experiments
Set of exclusively heteronuclear experiments based on C´ and
Ca detection for sequence specific assignment of a protein
More complete information automation
Solution & solid state NMR common/complementary
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Intrinsically disordered proteins - IDPs!
Folded Aggregated
One of the powerful applications of 13C direct
detection NMR
By I. Felli & R. Pierattelli
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Synuclein 140 AA
IDP
Cu(I)Zn(II)SOD 153 AA
Well folded
... Reduction in 1H chemical shifts
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CON of intrinsically unfolded a-synyclein
All residues assigned
(N,C´,Ca,Cb)
Bermel W., Bertini I., Felli I.C., Lee Y.M., Luchinat C., Pierattelli R., J. Am. Chem. Soc., 2006, 128, 3918-3919
Prolines are visible
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Intrinsically unfolded a-synyclein
Bermel W., Bertini I., Felli I.C., Lee Y.M., Luchinat C., Pierattelli R., J. Am. Chem. Soc., 2006, 128, 3918-3919
3D CBCACON-IPAP
3D COCON-IPAP
Strips from the 3D COCON-IPAP
Sequence specific
assignment
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Interphase Prophase Prometaphase Metaphase Anaphase Telophase Cytokinesis
Metaphase Anaphase
Securin
inhibitor of
separase
Securin
Intrinsically disordered protein (IDP!)
202 AA (>10% PROs)
Securin – Intrinsically disordered protein
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Intrinsically unfolded human securin
PRO (N)
22 corr obs
GLY (N)
11 corr obs
Observed well resolved peaks:
HSQC: 122
68% of the expected
60% of the whole protein
CON: 165
82% of the expected
82% of the whole protein
GLY (N)
9 corr obs
Securin – 202 AA, 24 PRO
Csizmok V., Felli I., Tompa P., Banci L., Bertini I., J. Am. Chem. Soc., 2009, 130, 16873-16879
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Intrinsically unfolded human securin
193, out of the 201 expected, spin patterns are identified
(96%) in CBCACON-IPAP.
Correlations observed:
Cai,C´i,Ni+1
Cbi, C´i,Ni+1
Csizmok V., Felli I., Tompa P., Banci L., Bertini I., J. Am. Chem. Soc., 2009, 130, 16873-16879
Securin – 202 AA, 24 PRO
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a-helical secondary structure propensity for the stretch D150-F159 Csizmok V., Felli I., Tompa P., Banci L., Bertini I., J. Am. Chem. Soc., 2009, 130, 16873-16879
Assignment and chemical shift analysis of securin
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D150-F159,
E113-S127 and W174-L178
Csizmok V., Felli I., Tompa P., Banci L., Bertini I., J. Am. Chem. Soc., 2009, 130, 16873-16879
Human securin - other NMR observables
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13C direct detection – increasing dimensions
nD?
High resolution
necessary for
IDPs only
possible through
reduced
sampling
strategies
& longitudinal
relaxation
enhancement
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13C direct detection – 4Ds
4D (HN-flip)NCACON - 1d, 12h, 0.036 % 4 scans per increment, 0.7 s d1, 2000 points, max evolution: 40 ms (15N), 24 ms (13C), 24 ms (15N)
C’(i)-N(i+1)
Cα(i)-N(i+1) C
α(i)-N(i)
Bermel W., Bertini I., Felli I.C., Gonnelli L., Koźmiński W., Piai A., Pierattelli R., Stanek J., J. Biomol. NMR, 2012, 53, 293-301.
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13C direct detection – 4D HCBCACON
4D HCBCACON – 1d, 4h, 0.053 % 4 scans per increment, 1.1 s d1, 1200 points, max evolution: 30 ms (15N), 6 ms (13C), 10 ms (1H)
4D HCCCON - 1d, 19h, 0.015 % 4 scans per increment, 1.1 s d1, 1800 points, max evolution: 28 ms (15N), 12 ms (13C), 20 ms (1H)
C’(i)-N(i+1) Hα,β(i)-C
α,β(i) H
ali(i)-C
ali(i)
Bermel W., Bertini I., Felli I.C., Gonnelli L., Koźmiński W., Piai A., Pierattelli R., Stanek J., J. Biomol. NMR, 2012, 53, 293-301.
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In-cell NMR spectroscopy
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• In-cell NMR allows the characterization of
biomolecules inside living cells.
• It relies on high resolution NMR
experiments to obtain information at
atomic resolution on biomolecule
structure, folding and interactions.
• It has a high biological relevance, as the
biomolecules are monitored in a cellular
environment.
In-cell NMR spectroscopy
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• Different cells have been and are used: bacteria, oocytes
and mammalian cells. Different techniques are exploited
to obtain high protein concentration: overexpression and
injection/insertion.
• Prokaryotic cells are more commonly used. Indeed, the
bacterial cytoplasm is a good model of the eukaryotic
one, in terms of molecular crowding, pH and redox
potential.
• Eukaryotic cells have been used to monitor protein
interactions with specific cellular components, such as
kinases. They have the machineries and chaperones for
the correct maturation of eukaryotic proteins.
Different living organisms
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Proteins are produced in bacteria and then inserted in mammalian
cells strains (e.g. CHO, HeLa) or in Xenopus laevis oocytes.
Protein insertion in eukaryotic cells
For mammalian cell cultures, protein
insertion is achieved by using cell-
penetrating peptides to deliver a fusion
protein, or porins to permeabilize the
cells.
To insert the protein into X. laevis oocytes
the microinjection technique is used.
Fig. From: D.S. Burz, A. Shekhtman, Nature, 458 (2009).
P. Selenko, G. Wagner, J Struct Biol, 158 (2007). K. Inomata et al, Nature, 458 (2009).
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Virtually any solution NMR pulse sequence can be used for
in-cell NMR experiments, BUT:
• The low sensitivity of NMR requires high protein
concentration, not always obtained;
• The viability of the cell sample is limited to few hours;
NMR pulse sequences
Therefore fast and sensitive experiments are often needed:
• Fast pulsing experiments: 2D SOFAST-HMQC, 3D
BEST-triple resonance experiments;
• Sparsed sampling experiments: non-uniform
sampling, projection spectroscopy.
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The 1H-15N SOFAST-HMQC(1) is often used for in-cell NMR.
It is the fast equivalent of the 1H-15N HMQC.
The selective 1H pulse excites only the amide protons,
allowing faster longitudinal relaxation between the scans:
shorter interscan delays.
The pulse can be set at the Ernst angle α (120° instead of
90°), to maximize sensitivity.
SOFAST-HMQC
Schanda,P., Kupce,E., and Brutscher,B., J. Biomol. NMR 33, 199-211 (2005).
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A functional process analyzed in living cells
with atomic details:
Maturation of
Cu,Zn-SOD1
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Nucleus Ctr
Regulators SOD CCS Cu(I)
MT
Cu(II) Cu(I)
A Cu transport process in human cells
No free copper ions in the cytoplasm
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Present in cytoplasm, mitochondrial IMS, nucleus, peroxisomes
Superoxide Dismutase
It catalyzes the dismutation of superoxide anion through reduction and oxidation of the copper ion
(2O2- + 2H+ O2+ H2O2)
A conserved SS bond
Quaternary Structure -Dimeric protein
Two metal ions per subunit
Zn
Cu
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hSOD1 maturation
monomeric apo hSOD1SH-SH
Copper binding
C57
C146
Zinc
binding
Disulfide bond formation
dimeric (Cu2,Zn2) hSOD1SS
Zn
Cu
SS bond
These post translational modifications affect the fold properties and monomer/dimer equilibrium
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Domain III Disordered
Domain I Atx1-like
Domain II SOD1-like
It dimerizes through the SOD1-like domain
CCS – the chaperon required for SOD1 in vivo maturation
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How is SOD1 in the cytoplasm? Maturation of SOD1 in human cells followed by in-cell NMR
Banci, L., Barbieri, L., Bertini, I., Cantini, F., Luchinat, E., PlosONE 2011
Banci, L., Barbieri, L., Bertini, I., Luchinat, E., Zhao, Y., Aricescu A.R., submitted, 2012
Zinc uptake is very selective in intact cells at variance with cell lysates and in vitro
Blue: Zn(II) added
Red: no metal added
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15N-Cys labelling: cysteine redox state
All cysteines of E,Zn-SOD1 are reduced.
1H
15N Cys 146
Cys
111
Cys 6
E,Zn-SOD1SH-SH
E,Zn-SOD1SH-SH
Banci, L., Barbieri, L., Bertini, I., Luchinat, E., Aricescu A.R., submitted, 2012
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hCCS is needed to incorporate copper.
When CCS is coexpressed, more than 50%
of total SOD1 binds copper
Cu uptake is a much more complex
Cu,Zn-SOD1
histidines
1H
10 uM CuSO4 added
100 uM CuSO4 added
Banci, L., Barbieri, L., Bertini, I., Luchinat, E., Zhao, Y., Aricescu A.R., submitted, 2012
His protons monitor the metallation state
• Only ~25% of total SOD1 binds copper
• Partial cysteine oxidation
Copper is added as 2+ but in
cells it is bound as 1+
Cys 146
Cys 111
Cys 6
SH S-S
Cys 57
1H
15N
Copper addition to
cells induces a
mixture of species
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SOD1 and its cysteine redox state
With both CCS and copper,
SOD1 disulfide is
completely oxidized!
With CCS co-expressed with SOD1, in absence of
copper, the SOD1 disulfide bond is partially oxidized!
Cys 146
Cys 111
Cys 6
SH-SH S-S
1H
15N
Cys 57
Human cells
Banci, L., Barbieri, L., Bertini, I., Luchinat, E., Zhao, Y., Aricescu A.R., submitted, 2012
Immature forms of SOD1 are
structurally unstable
They give rise to fibrils
SS NMR
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HS SH
SH SH
Zn(II)
S S
S S SH SH
Cu(II)
Zn(II)
Cu(I)
Summarizing SOD1
maturation steps in
human cells
E,E-SOD1SH
E,Zn-SOD1SH
E,Zn-SOD1S-S
Cu(I),Zn-SOD1S-S HS SH
S S
S S
Banci, L., Barbieri, L., Bertini, I., Luchinat, E., Zhao, Y., Aricescu A.R., submitted, 2012
HS SH
HS SH
HS SH
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All domains of hCCS are needed for hSOD1 maturation
Domain II SODI like
Domain III
One subunit of human SODI
Banci, Bertini, Cantini, Kozyreva, Massagni, Palumaa, Rubino, Zovo, PNAS, 2012
Domain I Atx-like
protein-protein recognition
Copper transfer
disulphide bond
formation
E,Zn-hSOD1SH + Cu(I)-hCCSD1,2
Cu,Zn SS form
Cu,Zn-hSOD1SH + hCCSD2,3
E,Zn SHSH form
Cu,Zn SHSH form
E,Zn-hSOD1SH
The steps of the process
are characterized
combining NMR and Mass
Spec measurements
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In cell and in vitro NMR
provide detailed info on
biological pathways in a
Mechanistic Systems
Biology frame within their
cellular context
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• Solution NMR assignment serves as the starting points of SSNMR data
assignment
• SSNMR assignment can help to resolve some uncertainties in solution NMR
assignment of partially unfolded species
A Cross-talk between Solid-State (SS) and Solution NMR
Apo SOD1 – a partially disordered molecule
Hints on the
structures of
fibril-ready
states
Average Structural
Differences between
solid-state and
solution (b propensity)
Apo dimer
chemical shifts
(13C,15N) + X-ray
structure Pintacuda et al
Angew. Chem. 2007
Banci et al, Proc. Natl. Acad. Sci.
2009
Banci et al, Biochemistry 2003
Banci et al
Eur. J. Biochem. 2002
Solid-state (crystals/microcrystals)
Solution-state
Cu, Zn dimer
chemical shifts (13C, 15N)
Apo
dimer/monomer
chemical shifts (1H, 13C, 15N)
Cu, Zn dimer
chemical shifts (13C, 15N)
aid for
assigning
loops
starting
point of
assignment
TALOS
+
comparison
comparison
aid for
assignment
TALOS
+
Banci L., et al, J. Am. Chem. Soc., 2011, 133, 345–349
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NMR spectra of ApoSOD1 in Microcrystals and Solution
• Similarity in spectral patterns permits the integrative analysis of both
SSNMR and solution NMR data
Red : 13C-13C 2D TOCSY
spectrum of apoSOD1 in
solution
Blue : 13C-13C 2D DARR
spectrum of apoSOD1 in
microcrystals
d2 (13C) /ppm
d1 (
13C
) /p
pm
Banci L., et al, J. Am. Chem. Soc., 2011, 133, 345–349
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Hot Spots for ApoSOD1 Amyloidosis
Loop IV
Loop VII
A/L (crystals) -> B (solution)
• In solution loops IV and VII gain transiently high β-propensity
• SSNMR and solution NMR are complementary methods
• SSNMR facilitates the use of solution NMR data for understanding
the mechanism of amyloidosis at residue specific level
Banci L., et al, J. Am. Chem. Soc., 2011, 133, 345–349
B (crystal) -> A/L (solution)
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Mechanistic Systems
Biology
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Mechanistic Systems Biology
Complex living systems should be studied in
their integral state
Functional processes need to be described based on
the 3D structural and dynamic interactions of the
various players.
…A system-wide perspective requires the
identification of all the players in the studied process
and within the “system” under analysis
Proteins must be framed within their cellular context