Recent Advances in Biomolecular NMR Lucia Banci CERM University of - - PowerPoint PPT Presentation

Recent Advances in Biomolecular NMR

Lucia Banci CERM – University of Florence

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

Why protonless NMR?

1H 13C 15N

Inverse (i.e. through 1H) detection

• f 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)

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

DDn(13C)

13C direct detection – unique properties

Different spins, different properties!

1H 13C

Dd(13C) Dd(1H) DDn(1H) Dd(13C) Dd(1H)

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

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

Intrinsically disordered proteins - IDPs!

Folded Aggregated

One of the powerful applications of 13C direct detection NMR

By I. Felli & R. Pierattelli

Synuclein 140 AA IDP Cu(I)Zn(II)SOD 153 AA Well folded

... Reduction in 1H chemical shifts

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

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

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

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

Intrinsically unfolded human securin

193, out of the 201 expected, spin patterns are identified (96%) in CBCACON-IPAP. Correlations observed: Ca

i,C´i,Ni+1

Cb

i, 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

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

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

13C direct detection – increasing dimensions

nD?

High resolution necessary for IDPs only possible through reduced sampling strategies & longitudinal relaxation enhancement

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) Cα (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.

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)-Cα,β (i) Hali (i)-Cali (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.

In-cell NMR spectroscopy

• 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

• 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

• ne, 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

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).

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.

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).

A functional process analyzed in living cells with atomic details: Maturation of Cu,Zn-SOD1

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

Present in cytoplasm, mitochondrial IMS, nucleus, peroxisomes

Superoxide Dismutase

It catalyzes the dismutation of superoxide anion through reduction and

• xidation of the copper ion

(2O2

• + 2H+  O2+ H2O2)‏

A conserved SS bond Quaternary Structure -Dimeric protein Two metal ions per subunit Zn Cu

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

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

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

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

hCCS is needed to incorporate copper. When CCS is coexpressed, more than 50%

• f 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

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

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

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

In cell and in vitro NMR provide detailed info on biological pathways in a Mechanistic Systems Biology frame within their cellular context

• 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

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) /ppm Banci L., et al, J. Am. Chem. Soc., 2011, 133, 345–349

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)

Mechanistic Systems Biology

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