recent advances in biomolecular nmr - embl …...recent advances in biomolecular nmr •protonless...

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

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)

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:

Cai,C´i,Ni+1

Cbi, C´i,Ni+1


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


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) 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.

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.

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

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

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 oxidation 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%

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


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


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


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

) /p

pm


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


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

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