Download Biophysical Society Poster, 2004

Introduction
The equilibrium concentration of oxygen in membranes can be 3 to
4 times that in water. This concentration enhancement has been
used to assess the depth of penetration of protein residues into
membranes combined with site direct spin labeling and EPR-based
collisional relaxation measurements.
These studies have been very useful in characterizing the nature of
protein-membrane interactions and in determining the orientation of
a protein with respect to the membrane. However, the profile and
absolute concentration of oxygen is not well characterized and, thus
far, any method to position an amino acid residue using oxygen
alone has only been qualitative. Therefore, we have prepared a
series of polypeptides consisting of around 23 amino acids that form
a single alpha-helix that spans the membrane. The polypeptide, a
variation on the WALP-23 [of Demmers, et al., JBC, 276, 3450134508], consists of Leu-Ala repeats that form an alpha helix within
the membrane and terminate in a tryptophan and uncharged groups
at either end. The terminal groups aid in registering the polypeptide
with respect to the membrane.
A series of polypeptides was made in which the position of a single
cysteine was systematically varied from one end of WALP23 to the
other. A spin label was covalently attached to the cysteine. We
recorded the spin lattice relaxation rate for the spin-label on the
polypeptide as a function of residue position with and without
oxygen using pulsed saturation recovery. We determined the oxygen
transport parameter, which is the product of the relaxivity and the
local concentration of oxygen, as a function of position on the alpha
helix. The profile is symmetric about the middle of the membrane,
and the effect is about three fold larger in the middle of the
membrane than in bulk water. The effect of the spin relaxant
Ni(EDDA) at 50mM was also measured.
The WALP23 Polypeptide
•
WALP23 is a single alpha helical
membrane-spanning polypeptide.
• The sequence is 23 residues long:
HCO-NH-G-WW-L-(AL)8-WW-A-CO-NH2
•
•
•
L and A are both hydrophobic. In a
membrane this forms a single turn alpha
helix.
The membrane using di-oleic (DO) 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
Oxygen Transport Parameter
The Oxygen transport parameter is the
change in the spin-lattice relaxation rate
due to oxygen collisional relaxation
R O

1 

2 
 O




2 
,

Depends on transport
properties (e.g. Diffusion)
of Oxygen in the local
environment of the spin
label
where
R1 O2   R1 O2   R1 O2   1  O2   1  O2 


T1 
T1 






WALP in DOPC LUVs
DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)
Label Position
WALP
in
DOPM
LUVs
DOPM (1,2-dioleoyl-sn-glycero-3-phosphomethanol)
Label Position
Typical WALP/DOPC Saturation
Recovery EPR
With Oxygen
CW
Without Oxygen
Walp23 in DOPC: Oxygen Transport Parameter
R1e 
1
T1e
From SR
R2e Estimated from the CW line width
Walp23 in DOPM: Oxygen Transport Parameter
Ratio Parameter*

 R1
  ln 
 R1


O2  

Ni
  
*Altenbach, C. et al. PNAS (1994) V 91 n5 pgs. 1667-71
Results
• The Oxygen transport parameter achieves a maximum in the center
of the bi-layer for both DOPC and DOPM membranes (filled
squares)
• The oxygen transport parameter approaches a common solution
value at each end of the polypeptide, indicating the ends are
exposed to solvent
• The spin-lattice (filled diamonds) and spin-spin (open squares)
relaxation rates have a maximum and a minimum ,respectively, in
the center of the bi-layer; and reflect local mobility of the spin label.
The bi-layer is more fluid in the middle and near the ends.
• Ni(EDDA) data (filled circles, DOPC) shows partial solubility of the
Ni relaxant in the bi-layer. A gradient of the Ni(EDDA) transport
parameter is observed, and diminishes toward the center of the bilayer.
• The ratio of oxygen and nickel transport parameters introduced by
Hubbell and co-workers is measured here by time domain EPR for
first time
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 because WALP is a uniform
helical polypeptide that has known registration in various
membrane bi-layers
The spin-lattice and spin-spin relaxation rates show
dependence on local mobility of the spin label in the bilayer. Therefore, the oxygen transport parameter cannot
be separated into its two components: the oxygen
concentration and transport-dependent coefficient.
The ratio parameter, which is designed to cancel out
transport effects, and can be a measure of relaxant
concentration seems not to scale simply with nitroxide
positions in the membrane.