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Time Domain EPR: Membrane-binding Proteins
Using R1 from EPR as a probe of the Structure-function and the Dynamics-function relation in
biologyGraduate Students:
Tamara Okonogi
Robert Nielsen
Faculty:
Michael Gelb
Kate Pratt
Post Docs:
Andy Ball
Ying Lin
Stephane Canaan
Kepeng CheSupported by NSF and NIH
Time Domain EPR: Outline
• Time Domain: Saturation Recovery and Pulsed Electron Double Resonance Methods– Comparison with CW methods– Spectrometer, experiment, data
• Theory of relaxation Rates
• Application to the Spin Relaxant Method
• Using site directed mutagenesis– Orienting a Membrane-binding Protein– Determine an Oxygen gradient in a membrane
CW Power Saturation
The method to obtain spin-lattice relaxation rates using CW methods. Plot the Peak to Peak height as a function of microwave power (or really amplitude).
Details of CW Power Saturation
Peak-to-Peak height
1
21
2
Y
1P
hC
h
1 3
2 2
2 1 2P = R R
A product of spin-spin and spin-lattice relaxation rates.
CW
Cr
O
O O
O
O
O
CC
O
O
CC
O
O
CC
O
O
3
Field (Gauss)
+Crox -Crox2 2 2R R R CROX
15N
O
C
O
NH2
TD ESR Spectrometer
AB
C
D
G
90
0
90
0 0
90
observe osc.
pump osc.
E
Amplifier/Digitizer
F
Sig.2Sig.1
Balancecontrol
Phasecontrol
0
90 RFIF
RFIF
0
90 LO
LO
H
Pulsed Bridge
Free Induction Decay
Z'
X'
Y'
Z'
X'
Y'
Measures spin-spin dephasing
Pulsed Saturation Recovery
Z'
X'
Y'
Z'
X'
Y'
Measures relaxation to equilibrium
Pulsed Electron-Electron Double Resonance
pSR; the effect of a relaxant
+Crox -Crox1 1 1R R R CROX
Collision with Oxygen
14N
OH
O
Redfield Theory (or BWRT)
Relaxation rate theory began with Bloch and Wangness, and was amplified by Alfred Redfield to be a complete theory for the effects of dynamics and stochastic processes on spins in condensed matter. Relaxation theory is often called BWRT (Bloch Wangness and Redfield Theory).
BWRT Predicts Spin-Lattice (R1) and Spin-Spin (R2) relaxation rates.
Relaxation Rates are related to Relaxation times:
1 21 2
1 1 and R RT T
What Redfield Theory (BWRT) Uses
• There is a system Hamiltonian • BWRT requires a bilinear operator, which couples
the spin system (S) to the lattice (or bath), F – The Hamiltonian is:
• F is the fluctuating variable that causes the spins to have a fluctuating environment
• The fluctuation of the lattice, coupled to the spin system, then causes the spins to relax or dissipate the absorbed (microwave or r.f.) energy, non-radiatively.
H S F
sH
A problem: R2 diverges
The coupling Hamiltonian (e.n.d.) is:
The orientation variable, , is a stochastic function of time.
The correlation function (at high temperature) is:
This shows the statistical origin of the rotational correlation time. Exponential decay of the correlation function with time is typical of such functions.
212 where 1 3cosz zH S I F F a
2 214
11 3cos 1 3cos 0
5
t
ct e
Nielsen, R. D. and Robinson, B. H. "A Novel Relaxation Equation of Motion". J. Physical Chemistry 2004; 108: 1589-1600.
CW Spectra
CTPO
14N
O
C
O
NH2
R2 from BWRT, diverging R2 rates from
Kubo Theory
R2 rates from modified BWRT
log c
Rel
axat
ion
Rat
es (
MH
z)D.A.Haas, C. Mailer, and B.H. Robinson, Biophysical J. (1993) 64, 594
R1 does not diverge
• R1 for the electron and R1 for the nitrogen nucleus in a nitroxide spin label as a function of rotational correlation times can be computed from BWRT.
• If R2 diverges for correlation times longer than a few nanoseconds how can we rely on the theory to give us R1 values out to milliseconds and beyond?
• The Problem: Why does the theory fail for R2
rates but not for R1 rates?
• It is important to understand why R1 works and to understand why R2 fails.
Nitroxide Nitrogen Spin Lattice Relaxation Rates
Electron spin-lattice relaxation rates:
With O2
and Without O2
Correlation Time (sec)
Rel
axat
ion
Rat
es (
sec-1
)
B.H. Robinson, D. A. Haas and C. Mailer (1994). Science 263(5146): 490-3.
Mechanisms of R1
• Sum the rates from statistically independent processes– Spin-Rotation, rate goes as – Electron-Nuclear Dipolar Coupling
• Electron rate peaks at the spectrometer frequency
• Nuclear rate Peaks at coupling
– Oxygen relaxation (used later)– Empirical “Spin-Diffusion” process
• Just a catch-all effect, goes at
• Partially due to spins local to the nitroxide
1c
2a
1
8c
2
0x x t
tRS S e
2 2
0x x t
R t R tS S a e a e
The ideal form of the solution is:
The actual form of the solution is a bit more complicated:
2
222
1 1 11 1 2
2 2 2o c oc c c
R f f
The two rates are:
The slower rate dominates.
Time (sec)
Solution (Signal)Black: two rate solution
Blue: rate Green: BWRT rate
0
0.1
2 1
10
c f
4 10 5 10 secf
2eal R
Dominant Rate in all limits
2
2c ofR
In the fast motion limit: In the slow motion limit:
2
12 cR
BWRT gives only the fast motion limit, which predicts that the rate goes to infinity as the correlation time goes to infinity. The new theory avoids this and correctly predicts coherent oscillations of (at frequency ) as the interaction becomes coherent, in the no motion limit.
2
2 21 2
co
c o
R ff
The rates in the two limits may be “combined” into a rate that does cover both motional regimes (for both R1 and R2):
2
1 2 21
c o
c s c o
fR
f
New Term in both R2 and R1 rates
ofxS
Spin Labeled-Fatty Acids in DOPC
• Different spectrometer frequencies (from 2 to 35 GHz) with the best possible single effective correlation time.
• A poor fit. The frequency dependence of simple isotropic rotational motion is incomplete.
Spin-Lattice Relaxation rates for varied Doxyl-Steric Acids in DOPC.
Data from Jin and Hyde
SL at 5 position
SL at 12 position
SL at 16 position
Same Data Different Model
• Improving the model to include anisotropic dynamics.
• For simplicity the anisotropy ratio was kept constant.
• Improved agreement indicates the need to improve the model, and the frequency dependence of the relaxation rates can rule-out some incorrect models.
Relaxation rates from 60 different experimentsCorrelation among all
the data and the model. Model has 1 adjustable parameter (the mean rotational correlation time) for each sample at all 5 different frequencies and two different isotopic forms.
Bee venom phospholipase
Oriented on a membrane surface by
Site Directed Mutagenesis
EPR spin relaxant method
Lin, Y., Nielsen, R., Murray, D.,
Hubbell, W. L., Mailer, C., Robinson, B. H. and Gelb, M. H. Science 1998; 279 (5358): 1925-9
Membrane Binding Proteins
Labeling a protein (PLA2) with a Spin Probe
Use site directed mutagenesis techniques to prepare proteins with a single
properly placed cytsteine. General Reaction for adding
relaxants
H3C S S CH2
N OO
OS CH2
N O
PLA2 C SHPLA2 C S
+
..
The protein should contain only one cysteine for labeling.
Protein labeled at only one site at a time per experiment.
Relaxant Method: Nitroxide Spectra depend on concentration of relaxants
Spin-Lattice: T1-1 or R1
Spin-Spin: T2-1 or R2
1 1
2 2
o
o
R R Rlxnt
R R Rlxnt
Rates are increased by the same amount due to additional relaxing agents (relaxants).
2 1 2 1 2
2 1 2
02 2 2 1 2
o o
o o o
o o
P R R R Rlxnt R Rlxnt
R R R Rlxnt
P P P R R Rlxnt
Human (HGIIA) Secretory Phospholipase sPLA2
A highly charged (+20 residues) lipase, 14kDa protein
And a highly charged (-70 mV) membrane
All exposure data was determined by SR and pELDOR directly measuring spin-lattice relaxation rates.
CW Spectra of hGIIA on Micelles
hGIIACTPO14N
O
C
O
NH2
CW Spectrum of site N70C with CROX
Probing the hGIIA protein surface potential using CW and TD EPR
8 [ ]
[ ] [ ] o
B
B
r NaClo k Tcrox oz ek T
CROX CROX e
rates from pSR and pELDOR for CTPO
solvent accessibility
Spin Lattice Relaxation Rates for sl-sPLA2
Power Saturation Curves site S120C
O2 Relaxant: hGIIA on LUV
TD
CW
Compare O2 Relaxant Effects from TD-SR and CW
Summary of Vesicle data
• Large protein surface charge determined by CW and TD data
• Complete protection from Crox for all EPR data
• Oxygen effect reduced relative to solution
• Light scattering occurs
Aggregation model
~50 enzymes (36 Angstrom diameter)
LUV of DOPM (100 nm diameter)
TD data, Vesicles vs. Mixed Micelles
Vesicle
(DTPM)
Mixed
Micelles
hGIIA-sPLA2 on mixed micelles
Ni2+
OC NH
O
CH2
OC NH
O
CH2
Cr
O
O O
O
O
O
CC
O
O
CC
O
O
CC
O
O
3
NiEDDA
Crox
sPLA2 on MembraneView from membrane
Yellow: Hydrophobic Residues
Blue: Charged (pos) residues
Orientation perpendicular to that predicted by M. Jain.
Anchored by hydrophobic residues. Charges not essential
sPLA2 Conclusions
• sPLA2 causes the vesicles to aggregate.
Explains much other data and misconceptions about the kinetics and processive nature of sPLA2 action.
• sPLA2 was oriented on micelles (instead) using spin-lattice relaxation rates alone.
Orientation different from that of another model.
• Hydrophobic residues are the main points of contact.
• Charges provide a general, non-specific
attraction.•Substrate binding site identified by orientation on the mixed micelles
The WALP Proteins
WALP proteins are single alpha helical membrane-spanning proteins.
The sequence is 23 residues long:
HCO-NH-G-WW-L-(AL)8-WW-A-CO-NH2
Leucine and Alanine are both hydrophobic. In a membrane this forms a single turn alpha helix.
The membrane, di-oleic (DO) PC, is about 28-30 Ang thick. The two outer Tryptophans (W) are about 30 Angs apart. The membrane will stretch (or shrink) to accommodate the protein.
Demmers et al: J. Biol. Chem., 276, 34501-34508, 2001
WALP23The sequence is 23 residues long: HCO-NH-G-WW-L-(AL)8-WW-A-CO-NH2
Subcyznski et al. Biochemistry, 2003, 42, 3939
WALP23-sl CW spectraCW EPR spectra of spin labeled WALP23 at various
positions.
Oxygen Transport Parameter
The Oxygen transport parameter is the change in the spin-lattice relaxation rate due to oxygen collision-relaxation
,
where
1 2 1 2 1 2 2 21 1
1 1R O R O R O O OT T
1 2 2 R O O
Depends on transport
properties (e.g. Diffusion) of Oxygen in the local environment of the spin label
Typical WALP/DOPC Saturation Recovery EPR
CW
With Oxygen
Without Oxygen
Walp23 in DOPM: Oxygen Transport Parameter
From SR
11
1e
e
RT
2eREstimated from the CW line width
Estimated from the CW line width2eR11
1e
e
RT
From SR
Walp23 in DOPC: Oxygen Transport Parameter
Ratio Parameter*
1 2
1
lnR O
R Ni
*Altenbach, C. et al. PNAS (1994) 91 (5), 1667-71.
Conclusions
• The gradient of the oxygen transport parameter, measured on WALP 23, is ideal as a ruler for determination of spin label position in membranes.
• The spin-lattice and spin-spin relaxation rates show dependence on local mobility of the spin label in the bi-layer.
• The oxygen transport parameter cannot be separated into its two
components: the oxygen concentration and transport-dependent coefficient.
• The ratio parameter, designed to cancel out transport effects, provides a profile of relative relaxant concentration.
• Ratio parameter can be used to position nitroxide in the membrane.