Site-directed Spin Labeling (SDSL) and Electron ... · Electron Paramagnetic Resonance (EPR)...

Site-directed Spin Labeling (SDSL)and

Electron Paramagnetic Resonance (EPR)Spectroscopy

An Introduction

1

Johann P. Klare

Department of Physics, University of Osnabrück, 49069 Osnabrück, Germany

2

Outline

EPR Spectroscopy – A short introduction

What is a spin label?

Spin labeling of proteins: the „classical“ approach

EPR-Tools: What can we learn from spin labeled biomolecules

Spin-Spin Distance Determination

Spin Labeling Techniques

A general protocol for spin labeling using cysteine-specific reagents

3

Electron Paramagnetic Resonance (EPR)

or

Electron Spin Resonance (ESR)

EPR is the resonance spectroscopy of molecular systems with an unpaired electron.

What is it ?

4

Does my system have unpaired electrons?...

Organic Radicals in proteins

amino acid radicals (tyrosine...) protein bound cofactor radicals

(semiquinones, flavines)

cytochrome c oxidase

myoglobin

Metal centres in proteins /protein complexes Cu, Mn, Ni, Co, Mo, Fe hemes, FeS clusters

cytochrome bc1 complex

Short-lived radicals / Reactive Oxygen Species (ROS) Spin Traps / Spin Probes

O2-O2-O2-

++

5

Why Spin labelling?

(Most) proteins and nucleic acids don’t have unpaired electrons ! no EPR ?!

...but...

we can introduce the wanted unpaired electron into almost any system under investigation

and we can do this also (almost) wherever we want

Site-directed spin labeling (SDSL) Site-directed spin labeling (SDSL)

6

Does my system have unpaired electrons?...

Organic Radicals in proteins

amino acid radicals (tyrosine...) protein bound cofactor radicals

(semiquinones, flavines)

cytochrome c oxidase

myoglobin

Metal centres in proteins /protein complexes Cu, Mn, Ni, Co, Mo, Fe hemes, FeS clusters

cytochrome bc1 complex

Spin labels attached to proteins

or nucleic acids

Short-lived radicals / Reactive Oxygen Species (ROS) Spin Traps / Spin Probes

O2-O2-O2-

++

7

The Electron Spin

Elementary particles such as an electron (and also atomic nuclei !) are characterized by an intrinsic angular momentum: The Spin S!

→ they behave like spinning tops

Elementary particles are quantum particles – the rules of quantum mechanics apply !

The Spin can be in two states, which differ in the orientation of the angular momentum.

The Spin makes the electron behave like a tiny magnet !The Spin makes the electron behave like a tiny magnet !

mS = ½ & mS = - ½mS = ½ & mS = - ½

8

The Electron Spin in a Magnetic Field: The Concept of Magnetic Resonance

undisturbed electron in an external magnetic field B

Zeeman Effect or Electron-Zeeman Effect Zeeman Effect or Electron-Zeeman Effect

without a magnetic field, the two spin states of the electron are degenerate they have the same energy

if we place the electron in a magnetic field, the two spin states will have different energies (the energy levels are separated by DE).

9

The Resonance Condition

First: What does resonance mean ?

(Microwave) radiation is used for the transition of molecules from one state to the other:

lower state → higher state (absorption)higher state → lower state (stimulated emission)

Normally, we have more molecules (n0) in the (energetically) lower state (ground state) than in

the higher state (n1) (excited state) according to the Boltzmann distribution

Resonance results in net absorption of radiation. Resonance results in net absorption of radiation.

n1 = n0 ∙ exp(-DE/kT) k = Boltzmann constant

(1,381 · 10−23 J/K)

The Resonance condition for a two-level system in EPR is

hn = g b Bhn = g b B

Energy of the radiation

Energy difference between the twomolecular states produced by B.

Frequency n (or often angular frequency w = 2p∙n), for which the resonance condition is fulfilled:

Larmor FrequencyFrequency n (or often angular frequency w = 2p∙n), for which the resonance condition is fulfilled:

Larmor Frequency

10

We can explain our first EPR spectrum !


3280 3300 3320 3340 3360 3380

-40000

-20000

0

20000

40000

EP

R s

ign

al

Field / Gauss

DPPH

DPPH (Diphenylpikrylhydrazyl)A common standard for field calibration in EPR → g = 2.0036+/-0.0002

In most EPR machines the 1st

derivative of the absorptionspectrum is recorded !

(→ Field Modulation)

n 9.5 GHz (X band)

11

...most EPR spectra have more then one line...

another submolecular magnet comes into play:

the nucleus with its nuclear spin I Multiplicity:

Metal Isotope Spin Multiplicity

Mn 55 5/2 6

Fe54,56,57,58

1/2 (2%) 2

Co 59 7/2 8

Cu 63, 65 3/2 4

Mo92,94,95,96,97,98,100

5/2 6

Biological transition metal ions

Ligand Isotope Spin Multiplicity

H 1, 2 1/2, 1 (0.02%) 2, 3

C 12, 13 1/2 (1.1%) 2

N 14, 15 1, 1/2 3, 2

O 16,17,18 5/2 6

P 31 1/2 2

Cl 35,27 3/2 4

Ligand atoms

MI = 2I + 1MI = 2I + 1

Multiplicity = number of EPR lines

12

Types of Interactions

EPR spectra can reflect many different magnetic interactions, giving rise to (sometimes complicated) multi-line spectra.

...but (good news)...

We can describe and analyse most of the spectra just by considering

pairwise interactions between magnets of three types:

1. electron spins S2. nuclear spins I3. laboratory magnets B

We can describe and analyse most of the spectra just by considering

pairwise interactions between magnets of three types:

1. electron spins S2. nuclear spins I3. laboratory magnets B

...and (even better news)...

This results in five basic types of interactions, from which two are usuallyso weak, that we can ignore them !

13


electron spins Snuclear spins Ilaboratory magnets B


Interaction Phenomenon Example

S x B Zeeman Interaction Basic EPR Pattern

S x I Hyperfine InteractionsMetal hyperfine (e.g. Cu (I=3/2))

“Ligand” hyperfine (1H (I=½), 14N (I=1))

S x SZero-Field Interactions

Dipolar Interactions

I x I Quadrupole Interaction Mn (I = 5/2)

I x BNuclear Zeeman

InteractionDouble Resonance Spectra (ENDOR)

and NMR !!

14

Interaction of the Electron Spin with Nuclear Spins: Hyperfine Interaction S x IS x I

The electron experiences not just one,but MI different “types” of nuclei (withdifferent local magnetic fields!).

Each “type” causes its own shift in the EPR resonance line

Hyperfine interaction for a Nitroxide radical (S = ½, I = 1)

A – Hyperfine constantA – Hyperfine constant

Multiplicity:

MI = 2I + 1MI = 2I + 1

Splitting into MI EPR lines ! Splitting into MI EPR lines !

15




Interaction Phenomenon Example

S x B Zeeman Interaction Basic EPR Pattern

S x I Hyperfine InteractionsMetal hyperfine (e.g. Cu (I=3/2))

“Ligand” hyperfine (1H (I=½), 14N (I=1))

S x SZero-Field Interactions

Dipolar Interactions

I x I Quadrupole Interaction Mn (I = 5/2)

I x BNuclear Zeeman

InteractionDouble Resonance Spectra (ENDOR)

and NMR !!

16

S x SS x SDipolar InteractionsDipolar Interactions

A not so simple equation (the Hamiltonian describing the dipole-dipole interaction)...

...with a simple message:

The dipolar interaction between two electrons is proportional to r -3,r being the distance between the two electrons.The dipolar interaction between two electrons is proportional to r -3,r being the distance between the two electrons.

Quantification of the dipolar interaction allows us to determine the distance between two paramagnetic centers!Quantification of the dipolar interaction allows us to determine the distance between two paramagnetic centers!

S1 and S2: spin operators for electrons 1 and 2.

^ ^

...what is dipolar interaction?

...how to quantify ? ...

17


...what is dipolar interaction ?

angular frequency w(at a fixed B field position)


w = 2p∙n

ħw = g b Bħw = g b B

resonance frequency of spin 1without dipolar interaction

18



dipolar interaction with parallel spin 2 B field at spin 1 is decreased lower resonance frequency

resonance frequency of spin 1with dipolar interaction withparallel aligned spin 2



w = 2p∙n


19



resonance frequency of spin 1with dipolar interaction withantiparallel aligned spin 2

dipolar interaction with antiparallel spin 2 B field at spin 1 is increased higher resonance frequency



w = 2p∙n


20



dipolar frequency wdddipolar frequency wdd

wdd (r) r-3wdd (r) r-3



w = 2p∙n


21

Outline

EPR Spectroscopy – A short introduction


Spin labeling of proteins: the „classical“ approach

EPR-Tools: What can we learn from spin labeled biomolecules



• Proteins

• Nucleic Acids


22


...a stable chemical compound which possesses an unpaired electron (i.e. it is a stable radical) and a specific reactive group to be bound to (bio)molecules....a stable chemical compound which possesses an unpaired electron (i.e. it is a stable radical) and a specific reactive group to be bound to (bio)molecules.

The vast majority of spin labels are nitroxides, where the unpaired electron is locatedat an –NO group, which is usually part of a heterocyclic ring.

A little bit of history:Spin labels were first synthesized in the laboratory of H. M. McConnell in 1965.The first spin labeling studies have been performed using thiol-specific functional groups to label natively occuringcysteines in proteins (e.g. in hemoglobin).Site-directed spin labeling (SDSL) was pioneered in the laboratory of Dr. W.L. Hubbell in the late 80’s/early 90’s.

Functional groups contained within the spin label allow them to be attached to the moleculeunder investigation and determine their specificity.

e.g.: Protein thiol groups specifically react with the functional groups methanethiosulfonate, maleimide, and iodoacetamide,

creating a covalent bond with cysteine.

23

Site-directed spin labeling of proteins: the „classical“ approach

24

1. Spin Label Mobility

2. Accessibilitytowards paramagneticquencher molecules(NiEDDA, CrOx, O2)

→ Discrimination betweenlipid bilayer, aqueous phaseand protein interior

5. Spin-Spin Distancedetermination(~ 10 – 70 Å)

4. Transient StructuralChanges

Information about Structure and Dynamics

SDSL-EPR-Tools: Overview

3. Polarityof the SL Microenvironment

25

Spin Label Mobility

The (RT) EPR spectral shape is sensitive to the reorientationalmotion of the nitroxide side chain (partial motional averaging of the anisotropic components of the g- and hyperfine tensors).

For spin-labeled sites exposed to the bulk water, the nitroxidemobility is slightyl restricted.

rotational correlation times in the ns range. small line widths of the center lines, DH0

small apparent hyperfine splittings.

Restricted mobility of the spin label side chain(interaction with neighboring side chains or backbone atoms)

line widths and the apparent hyperfine splittingsare increased

26

Spin Label Mobility

Klare, J.P., Steinhoff, H.-J., Photosynth. Res. 102, 377-390 (2009)

The correlation between the inverse linewidth DH0-1 and the inverse of the spectral

second moment H2-1 allows a general classification of the region where the spinlabel is located.

The spectral second moment H2 quantifiesthe breadth of the EPR spectrum.

27

Spin Label Mobility - Identification/analysis of multiple components

Arrhenius plot(logarithm of the rotational correlation times of the twocomponents vs. 1/T)

van’t Hoff plot(natural logarithm of theratio of the two spectralcomponents vs. 1/T)

Example: Analysis of a temperature and ionic strength dependent equilibrium between a compact and a dynamic form of a protein domain.

Doebber et al, J.Biol.Chem. .283, 28691-28701 (2008)

The cw EPR spectra show two components (1 and 2)with different rotational correlation times tc.The cw EPR spectra show two components (1 and 2)with different rotational correlation times tc.

28

0 1 2 3 4 5 6 7

O2

N2

Am

plit

ud

e ce

ntr

al li

ne

CrOx

lipid

0 1 2 3 4 5 6 7

O2

N2

Am

plit

ud

e ce

ntr

al li

ne

CrOx

water

0 1 2 3 4 5 6 7

O2

N2

Am

plit

ud

e ce

ntr

al li

ne

CrOx

2/1mWP

protein

Klare, J.P., Steinhoff, H.-J., Photosynth. Res. 102, 377-390 (2009)

P1/2 [Relaxing agent]- P1/2 [N2] [Relaxing agent]

[Relaxing agent]

P1/2

Accessibility for Paramagnetic Quencher Molecules

29

Polarity of the SL Microenvironment

Polarity and proticity of the spin label microenvironmentare reflected in

the hyperfine component Azz

the g tensor component gxx

A polar environment shifts

Azz to higher values gxx to lower values

polarity

Azz can be readily obtained from X band spectra.

To obtain gxx we need to goto W band (at least).

30

Assignment of secondary structures – Mobility, Accessibility, Polarity

Mobility Analysis

Polarity Analysis

Accessibility Analysis

324

320

310

300294

313

PutP

A Na+/prolinesymporter

31

Monitoring Transient Structural Changes

Klare, J.P., Steinhoff, H.-J., and Engelhard, M. (2004) FEBS Lett. 564 , 219

F

GTM2

Conformational changes in an archaebacterial photoreceptor/-transducer protein complex upon light excitation.

Position 159: change of the EPR signal due to transient mobilisation upon outward motion of helix FPosition 78 : change of the EPR signal due to increased dipolar interaction between the two spin labels

32


Low Temperature CW EPR (1-2 nm)

Double Electron Electron Resonance (DEER) (2-8 nm)

Simulation of EPR distance distributions

...or how to translate the EPR data into a model of your biomolecule

º molecular dynamics (MD) simulations

º the rotamer library approach (RLA)

33


Dipolar InteractionsDipolar Interactions

w



broadening ofcw EPR spectrabroadening ofcw EPR spectra

34

Steinhoff et al. (1997) Biophys. J. 73, 3287-3298.

Low Temperature CW EPR:

35



36



37

Pulse Electron Double Resonance (PELDOR)or Double Electron-Electron Resonance (DEER)



38

PELDOR/DEERPELDOR/DEER

G domain dimerization of the tRNA-modifying enzyme MnmE monitored with PELDOR.(Meyer et al., PLoS Biol. 7, e1000212 (2009))

39


Proteins

Spin labeling of cysteines Spin labeling by peptide synthesis Spin labeling using “click chemistry” Spin labeling using the nonsense suppressor methodology

ProteinsProteins

Nucleic Acids

Usage of spin labeled nucleotide analogs in oligonucleotide synthesis Postsynthetic modification of nucleotide analogs with reactive groups Postsynthetic modification at the sugar/phosphate backbone

Nucleic AcidsNucleic Acids

40

Spin Labeling of Cysteines

1. Methanethiosulfonate spin labels

MTSSL

MTSSL comprises a flexible linker minimizing disturbances of the

native fold of the protein !Size: comparable to that of a

tryptophane side chain.

MTSSL structure

MTSSL: (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate

Formation of adisulfide bond:

→ sensitivity towardsreducing conditions !

MTS-4-oxyl spin label

Besides MTSSL, a variety of other different nitroxide radical compoundsare commercially available, which

- have a longer or shorter linker- are sterically more demanding- are pH sensitive- ....

41

Spin Labeling of Cysteines

2. Maleimide spin labelsFormation of aC-S bond:

→ no sensitivity towardsreducing conditions !

...but... sterically more demanding

(disturbance of protein structure!) can react also with primary amines

(N-term., Lysine, Arginine)

3. (Iodo)acetamide spin labels

Formation of aC-S bond:

→ no sensitivity towardsreducing conditions !

→ long, flexible linker

...but... can react also with secondary amines

(esp. Histidine)

42

Spin Labeling by (solid phase) peptide synthesis (SPPS)

Using the method of (solid phase)peptide synthesis, polypeptideswith arbitrary amino acids (incl.spin labeled amino acids !) can besynthesized.

Spin label building blocks for thestandard Boc- or Fmoc-basedpeptide synthesis have been syn-thesized or are commerciallyavailable.

The most popular spin label building block is the paramagnetic α-amino acid TOAC(4-amino-1-oxyl-2,2,6,6,-tetramethyl-piperidine-4-carboxylic acid).

TOAC exhibits only one degree of freedom: the ring conformation.

direct information about the orientation of secondarystructure elements !

Drawback of the method: Peptide synthesis is still limited to < 200 amino acids !Drawback of the method: Peptide synthesis is still limited to < 200 amino acids !

43

Spin Labeling by peptide synthesis - Expressed Protein Ligation (EPL)

Semisynthesis of proteins from recombinant and synthetic fragmentsSemisynthesis of proteins from recombinant and synthetic fragments

two polypeptide fragments:

peptide 1 (recombinant)peptide 2 (synthetic,

carrying the spin label)

formation of a native peptide bondbetween the two fragments by chemical ligation.

44

Spin Labeling using „click chemistry“

Highly selective formation of a carbon-heteroatom bond under mild conditions (→ stability of the nitroxide radical)and with high yield.

1,3-dipolar cycloaddition of organic azides with alkynes in the presence of Cu(I) (copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition,CuAAC)

45Spin Labelling

Spin Labeling of nucleic acids

labeling of the nucleobase

labeling of the “backbone” sugar

46Spin Labelling

1. Usage of spin labeled nucleotide analogs in oligonucleotide synthesis

Chemically synthesized nucleotide analogs are used in standardoligonuleotide synthesis.Example: Spin-labeled thymidine analog, where a methyl group

is replaced by an acetylene-tethered nitroxide →

2. Postsynthetic modification of nucleotide analogs with reactive groups

Usage of nucleotide analogs chemically modified with reactive groups suitable forpost-synthetic modification with a spin labeling reagent.Example: 4-thiouridine (for RNA) derivatives, which can be modified with

thiol-specific reagent like MTSSL.

Spin Labeling of nucleic acids

47

Postsynthetic modification at the sugar/phosphate backbone

Alternatively, the spin label can be attached to the sugar moiety of specific nucleotides

Example: Postsynthetic derivatization of a 2’-amino group introduced into oligonucleotides with a isocyanatederivative of a TEMPO-like moiety.

Ward et al., ChemBioChem 8, 1957-1964 (2007)

48


1. Spin labeling in solution

Incubate the purified protein for 1-2 h with a reducing agent (DTT, DTE) to reduce oxidized thiol groups.

Remove the reducing agent completely (!) by an appropriate method (washing in centrifugal concentrators, gel filtration, affinity chromatography (for proteins carrying an affinity tag), desalting columns, dialysis). Use a protective atmosphere (N2, Ar) to prevent re-oxidation.

Adjust the protein concentration be < 500 µM.

Incubate the protein with a 2-5fold excess (subject to optimization) of the spin label reagent.(Spin label compounds like MTSSL are usually quite hydrophobic. Stock solutions of 100 mM spin labelin e.g. DMSO have been proven to be convenient for most cases.)

The duration and the temperature at which incubation is performed depends on the actualsystem. Incubations over night at 4°C or 2-12h at RT usually lead to good labeling efficienciesfor well-accessible labeling sites. (Also this point is subject to optimization)

Remove unbound/unreacted spin label. The same procedure as for the removal of the reducingagent could/should be applied.

Check the labeling efficiency !

Check the functionality of the labeled protein protein/biomolecule !!!

49


2. Spin labeling on affinity columns

Bind the protein to the affinity column. (Spin labelling could be the last step of your protein purification, just before you would elute it from the column)

Incubate the purified protein for 1-2 h with a reducing agent (DTT, DTE) to reduce oxidized thiol groups.

Wash out the reducing agent completely (!).

Incubate the protein with a 2-5fold excess (subject to optimization) of the spin label reagent.(Spin label compounds like MTSSL are usually quite hydrophobic. Stock solutions of 100 mM spin labelin e.g. DMSO have been proven to be convenient for most cases.)

The duration and the temperature at which incubation is performed depends on the actualsystem. Incubations over night at 4°C or 2-12h at RT usually lead to good labelling efficienciesfor well-accessible labelling sites. (Also this point is subject to optimization)

Wash out unbound/unreacted spin label.

Elute the protein from the column.

Check the labeling efficiency !

Check the functionality of the labeled protein protein/biomolecule !!!

Date post:	04-Apr-2018
Category:	Documents
Upload:	vuongthu
View:	222 times
Download:	0 times

Site-directed Spin Labeling (SDSL) and Electron ... · Electron Paramagnetic Resonance (EPR)...

Documents