Paramagnetic NMR for the characterization of PPIs · Dipolar coupling of the unpaired electron spin with the nuclear spins of surrounding atoms (i.e., a “through-space” effect)

Paramagnetic NMR for the characterization of PPIs

Paola Turano

NMR of Paramagnetic Metalloproteins

The hyperfine shift

dobseved = ddia + dhyperfine

Origin of dhyperfine

dhyperfine = dcontact + dpseudocontact


Contact shift: contribution to the chemical shift due to the unpaired electron spin density on the resonating nucleus

SI AH

A = hyperfine coupling constant: it is related to the spin density on the resonating nucleus.

A = m0/3S h/2p gI ge mB i ri


Ad i|i(0)|2

A is the sum of a direct delocalization mechanism and a polarization mechanism Ad + Ap

where the summation is performed on all the MO containing only one electron

Ap arises from polarization effects between electrons in singly occupied molecular orbitals and those in doubly occupied molecular orbitals.

Spin polarization The presence of an unpaired electron in an MO polarizes the paired electrons in the core spin polarization The MO containing the paired electrons is modified by the presence of the unpaired electron in another MO in such a way that the electron with the spin aligned with the unpaired electron will have a slight preference to occupy the region of space of its MO which is closer to the unpaired electron itself (see Hund’s rule). Conversely, the other electron with spin antiparallel to the unpaired elecyron will have a slight preference to occupy regions of its MO far for the unpaired electron. This mechanism accounts for the presence of spin density on nuclei when the unpaired electron occupies p or d orbitals, which have a node at the nucleus. Spin polarization has opposite sign with respect to the contribution from the spin density in the orbital containing the unpaired electron.

p

s s

Spin-polarization for the case of atomic orbitals

http://www.d.umn.edu/~pkiprof/ChemWebV2/AOs/ao2.html

Dipole-dipole interaction:

Dipolar coupling of the unpaired electron spin with the nuclear spins of surrounding atoms (i.e., a “through-space” effect).

S

I

cxx

cyy

czz

q

f

r

The origin of the pseudocontact shift

If the magnetic moment of the electron is anisotropic, the magnetic moment of the electron changes with orientation, the electron-nuclear dipolar coupling does not average to zero with molecular tumbling in solution.

+ cc

pd 2cossin

2

31cos3

12

1 22

3

pcsrhax

r

The origin of the pseudocontact shift It yields a shift that is dependent on both the electron-nuclear distance and the orientation of the electron-nucleus vector with respect to the magnetic susceptibility tensor.

Axial Totally Rhombic

positive

negative 0rh c axrh )3/2( cc

S

I

cxx

cyy

czz

q

f

r

+ cc

pd 2cossin

2

31cos3

12

1 22

3

pcsrhax

r

Surfaces with constant dpcs values:

S

I

cxx

cyy

czz

q

f

r

Metal-centered point dipole approximation = We consider that the unpaired electron is localized on the metal ion. Reasonable as long as we consider long distance effects

The origin of the pseudocontact shift

Hyperfine shift contributions SUMMARY

• Contact shift: effective on the nuclei of the paramagnetic metal ion(s) ligands.

• Pseudocontact shift: through space interaction effective on all the nuclei within a certain distance from the paramagnetic metal ion(s).

Contact shift and PPIs

kT

SSgACS

I

B

g

m

3

)1( +

Changes in contact shifs upon complex formation can only be due of the coordination sphere of the metal ion changes.

In principle good for metal-mediated PPIs

BUT Metal-trafficking pathways mainly available for diamagnetic metal ions (Cu(i), Zn(II) …

Contac shift and metal-mediated interactions

In principle, these metal mediated interactions would be particularly suitable for a characterization via paramagnetic restraints based on contact effects that would “enlighten” the resonances of the metal ligands. In practice, this approach has not yet been pursued, as the intracellular metal ion trafficking routes studied by NMR are essentially focused on diamagnetic cations, with a very deep characterization of the systems involving copper(I)

Metal transfer typically implies the formation of adducts where the metal itself acts as a bridge between proteins, by coordinating amino acids on both interacting partners.

PCS 1st example, non functional complex

ferricytochrome b5 + ferricytochrome c low spin iron(III) S=1/2

HSQC spectra of samples containing 15N-labelled cytochrome b5 in complex with unlabelled cytochrome c allowed unambiguous assessment of pseudocontact shifts relative to diamagnetic reference states.

caxpara = 2.4310-32 m3

crhpara = 1.2010-32 m3

z^z = 14°

PCS

ox ox

red ox

ox

ox

red

red

4 possible combinations; only 2 can be functionally relevant The reactive complex cannot be characterized

In redox complexes:

PCS Other drawbacks inherent to the use of intermolecular PCSs based on the paramagnetism of the heme cofactor: i) DIAMAGNETIC REFERENCE: PCSs are evaluated by subtracting the value of the diamagnetic shift from the observed shift. It is not always obvious to obtain the appropriate diamagnetic reference. Substitution of the native heme with porphyrins containing a diamagnetic metal is not easily achievable in proteins containing c-types heme or other covalently attached heme; ii) TOO SHORT RANGE EFFECT FOR LARGE COMPLEXES: Complexes between cytochromes are generally small due to the size of the two components and therefore the approach worked well. For larger systems the range of action 20 Å might not be sufficient to monitor the interface, PCS contribution may barely be distinguishable from the diamagnetic chemical shift perturbation caused by the interaction with the partner; iii) SMALL ANISOTROPY: Spin states different from S = ½ may have even smaller range of actions due to the smaller magnetic susceptibility anisotropies.

PCS: paramagnetic probes

SOLUTION: engineering the protein surfaces with paramagnetic probes giving rise to larger PCS.

Tb, Dy and Tm are the strongest anisotropic paramagnetic lanthanides, whereas Lu could serve as a diamagnetic reference.

Best performing paramagnetic tags have been developed by Ubbink and coworkers, which are called

Caged Lanthanide NMR Probes.

In these cages the lanthanide ion is tightly bound to the ligand, the tag is small and binds in a bidentate fashion to two Cys residues engineered on the protein surface: all of this ensures that the tag is rigidly anchored to the protein and the position of the paramagnetic probe on the protein surface can be defined accurately.

NMR of Large Paramagnetic Metalloproteins

Longitudinal relaxation time T1 along z:

T1 relaxation re-establishes the z-magnetization.

Transverse relaxation time T2 in the xy plane:

T2 relaxation causes the horizontal (xy) magnetization to decay.

The relaxation rate for a nucleus is the sum of contributions from all mechanisms: 1/T1 = i (1/T1)i

1/T2 = i (1/T2)i

i = all relaxation sources Main sources are: nucleus-nucleus dipolar interactions, chemical shift anisotropy, nucleus-electron interactions (=paramagnetic contribution) Paramagnetic systems will have shorter T1 and T2 values

Paramagnetic contributions to relaxation add to the diamagnetic contributions

NMR of Large Paramagnetic Metalloproteins

T1-1 = E2 tc/(1 + w0

2tc2)

T2-1 = E2 [tc + tc/(1 + w0

2tc2)]

Contact relaxation

Dipolar relaxation

Curie spin relaxation The difference in the population of the electron spin levels gives rise to a finite static magnetic moment related to <Sz>:

kT

BSSgS eBz

3

)1( 0+ m

The interaction of the nuclear spins with the static magnetic moment related to <Sz> provides a further relaxation contribution.

2262

224422

01

1

3

)3(

)1(

45

2

rI

rBeIM

rkT

SSgR

tw

tmw

p

m

+

+

Curie relaxation

R1M

R2M

For wI2tr2 >> 1, R1M

levels off at a value that is always small with respect to the dipolar contribution

++

+

2262

224422

02

1

34

)3(

)1(

45

1

rI

rr

BeIM

rkT

SSgR

tw

tt

mw

p

m

Curie relaxation

Important for high MW and high field.

Proton relaxation rates due to Solomon and Curie contributions, 5 Å, 800 MHz, no contact. No chemical exchange

Electron relaxation and nuclear relaxation

Different metal ions

Good for NMR (10-11-10-13)

• lanthanides (III) (except Gd3+)

• l.s. iron(III)

• Tetrahedral nickel(II)

• H.s. 6-coord. cobalt(II)

NMR lines broadened

beyond detection (10-8-10-9)

• chromium(III)

• copper(II)

• manganese(II)

• gadolinium(III)

Borderline (10-10-10-11) • manganese(III)

• l.s. cobalt(II)

• h.s. heme iron(III)

Free radical ts ≈ 10-7

= longer than any metal ions

CONTACT RELAXATION, an example:

heme transfer between the hemophore protein HasA and its

receptor HasR

Heme Acquisition

System(HAS) in Gram-negative bacteria

Outer membrane

Inner membrane

HasR

Hb

ABC

transporter

Periplasm

Cytoplasm

Ton

B

Hexb

B

Hexb

D

Fe3+

19 kDa

CRINEPT-TROSY

holoHasA apoHasA

Stable complex apparent molecular weigh 150 kDa

(from optimal CRINEPT delay)

HasR 98 kDa in DPC micelles

dodecylphosphocoline

Max. solubility 300 mM

Structure of HasA: a “fish biting the heme”

holoHasA (PDB 1b2v) apoHasA (PDB 1ybj)

L1

L2 L2

L1

L2

NMR fingerprint of the open and closed conformations of HasA

holoHasA

apoHasA

Caillet-Saguy, Piccioli, Turano, Izadi-Pruneyre, Delepierre, Bertini, and Lecroisey, JACS 2009

(PDB 1b2v)

(PDB 1ybj)

L1

L1

L2

L2

HoloHasA: paramagnetic effects induced by

iron(III)

33

Standard HSQC

Paramagnetic tailored HSQC

Paramagnetic resonances diagnostic for the presence of the heme

Chemical shift mapping

apoHasA in HasA-HasR

3 classes of signals: • Not affected (d < 0.25 ppm) • Disappearing from their original well-resolved position • Behavior not safely defined

CRINEPT-TROSY

holoHasA in HasA-HasR


3 classes of signals: • Not affected (d < 0.25 ppm) • Disappearing from their original well-resolved position • Behavior not safely defined

CRINEPT-TROSY

holoHasA in HasA-HasR


apoHasA in HasA-HasR

NMR SIGNATURE of HasA conformations

apoHasA apoHasA-HasR

holoHasA holoHasA-HasR

apoHasA -HasR holoHasA-HasR

apoHasA holoHasA

apoHasA holoHasA-HasR

holoHasA apoHasA-HasR

Distance matrix


Conclusions

• only three different conformations are possible for HasA in solution:

1. one for the isolated apoprotein

2. one for the isolated holoprotein (either with iron(III) or with gallium(III))

3. one for the complexed protein

• the structure of the hemophore in the complex is closer to the open conformation of the apoprotein than to the closed conformation of the holoprotein

• the surface contact area between HasA and HasR is independent of the presence of the heme, involving loop L1, loop L2, and the 2-6 strands.

• upon complex formation the heme group is transferred from holoHasA to HasR



Y75

HasA-HasR: interaction surface

HasR model structure

HasA in the apo “open” conformation


Solution NMR vs. X-ray crystallography

Loop L1 in HasA could not be modeled because of missing electron density in the X-ray structure of the complexes.

Krieg, Huché, Diederichs, Izadi-Pruneyre, Lecroisey, Wandersman, Delepelaire, Welte, PNAS 2009

Heme transfer

Paramagnetic Relaxation Enhancement

PRE

PRE, paramagnetic relaxation enhancement The dependence on r-6 can be translated in distance restrains for structure calculations that help defining the relative position of the metal ion and protein nuclei.

+++

++

+

+

2222226

2222

01

1)(1

6

1

3

)(1

1

415

2

cSI

c

cI

c

cSI

c

MH

eBI

r

SSgT

tww

t

tw

t

tww

tmg

p

m

++

+++

++

++

+

222222226

2222

01

21

6

)(1

6

1

3

)(14

1

415

1

cS

c

cSI

c

cI

c

cSI

cC

MH

eBI

r

SSgT

tw

t

tww

t

tw

t

tww

tt

mg

p

m

++

+

2262

224422

01

21

34

3

1

45

1)(

cI

cC

MH

eBI

rkT

SSgCurieT

tw

tt

mw

p

m

Dipolar

In studying interactions where at least one of the components is paramagnetic, one should consider the Curie term, which is strongly influenced by the molecular size because of its dependence upon the rotational correlation time, and therefore becomes more and more relevant upon increasing molecular dimension.

NMR structural characterization of the chromium(III)

– oxidized cyt c7 adduct

27 Cr-H relaxation restraints (+NOEs)

hemeIV

hemeI hemeIII

chromium(III)

Assfalg, Bertini, Bruschi, Michel, Turano, PNAS 2002 – PDB : 1LM2

Lys41

Lys42

Lys46

Lys50 Restraints based on 1/r-6 dependence

The encounter complex The term “encounter complex” has frequently been used in describing the pathway of protein-protein association. Different meanings in different contexts: 1) “the end-point of diffusional association”, which would be similar to what we have defined as the transient complex. 2) mechanism involving a pre-equilibrium complex followed by a reorganization 3) free-energy regions in configurational space In the paramagnetic NMR experiments based on PRE (Ubbink), the encounter complex refers to a minor, dynamic state that is in equilibrium with a dominant, stereospecific complex. The dominant complex is very similar to the X-ray structure of the pair and occupied for >70% of the time. In the encounter complex, the proteins occupy a region around the position in the dominant complex.

The encounter complex

To maintain a transient interaction, the dissociation rate constant of the complex must be high (koff 103 s-1). The association rate constants (kon) are also high and have been experimentally determined to be in the range of 107-109 M-1s-1 for electron-transfer partners.

ET proteins

Transient complexes

Transient complexes form when a high turnover is a functional requirement and their components associate and dissociate rapidly, namely with koff 103 s-1 and kon in the range of 107-109 M-1s-1. This results in dissociation constants typical for weak and ultra weak complexes and lifetimes ms. Revealing the presence of such interactions is experimentally challenging because they do not result in a sufficient amount of complexes that can be directly detected.

Caged Lanthanide NMR Probes also for PRE

Unlike the other lanthanides, gadolinium(III) has a slow electron relaxation rate, causing strong relaxation effects on surrounding nuclei. Diamagnetic reference: Lu(III) PRE has the advantage that no paramagnetic susceptibility tensor is involved and no angular dependence exists: the extent of the measured effect is only dependent upon the distance.

Unlike the other lanthanides, gadolinium(III) has a slow

electron relaxation rate, causing strong relaxation effects on

surrounding nuclei.

Paramagnetic RDCs and PPIs

Tb, Dy and Tm are the strongest anisotropic paramagnetic lanthanides, whereas Lu could serve as a diamagnetic reference.

Paramagnetic RDCs

+ )2cos(sin

2

3)1cos3(

4154

1 22

32

2

0

NHNH

para

rhNH

para

ax

NH

NH

r

h

kT

BRDC fqcqc

p

gg

p

axial and rhombic components of the anisotropic magnetic susceptibility tensor; they are the same for both PCSs and paramagnetic RDCs

Paramagnetic RDCs Under fast exchange conditions the measured values are an average, weighted by the relative populations, of those of the free and bound protein. In order to overcome the difficulties encountered in obtaining RDCs that emanate from the complex alone, a titration approach can be employed where RDCs are measured in different equilibrium mixtures of the free and bound form. While the RDCs of the free states can be measured directly, the RDCs originating from the bound state will be obtained indirectly by extrapolation of the RDCs in the different equilibrium mixtures

Applications of paramagnetic RDCs are essentially restricted to the use of paramagnetic tags similar to those suitable for PRE and PCSs, whit lanthanide ion possessing large magnetic susceptibility anisotropy.

Paramagnetic RDCs and dynamics Non-physiological complex of yeast cytochrome c with bovine adrenodoxin,: •Large paramagnetic RDCs are observed for tagged-cytochrome c, •Small RDC values are measured for the interacting adrenodoxin due to the large intermolecular dynamics

The dynamic complex of adrenodoxin and cytochrome c. Adrenodoxin is shown as a surface coloured to indicate the electrostatic potential: red for negative and blue for positive. The FeS-binding loop is shown in yellow. The distribution of cytochrome c is shown as centres of mass around adrenodoxin.

FEBS J. 2011

Suggested readings

Bashir Q, Scanu S, Ubbink M. (2011) Dynamics in electron transfer protein complexes. FEBS J. 278:1391-400. Del Conte R, Lalli D, Turano P (2013) NMR as a tool to target protein-protein interaction. In: Disruption of Protein-Protein Interfaces, Ed. Mangani S. - Springer Heidelberg New York Dordrecht London. pp.: 83-111.

Solution NMR, SS NMR … a (novel!) 3° alternative exists:

NMR of sedimented proteins

Bertini, Luchinat, Parigi, Ravera, Reif, Turano, PNAS 2011

SS NMR rotors are ultracentrifuges, creating a field of force of up a few million g at their maximum speed.

protein concentration 60 mg/ml T=290 K


For a protein sedimented at the internal rotor walls, NMR signals should be observable as if the protein were in the solid state.

1H-decoupled, CP 13C spectra of 13C,15N-apoferritin SS = black solution @ increasing MAS = red


13C-13C DARR spectra of ferritin 298 K MAS = 9 kHz

SS Sedimented


Documents

Paramagnetic NMR for the characterization of PPIs · Dipolar coupling of the unpaired electron spin with the nuclear spins of surrounding atoms (i.e., a “through-space” effect)