Membrane binding spin labeling

Fig. 2. Phase diagram describing lateral phase separations in the plane of bilayer membranes for binary mixtures of dielaidoylphosphatidylcholine (DEPC) and dipalmitoyl-phosphatidylcholine (DPPC). The two-phase region (F+S) represents an equilibrium between a homogeneous fluid solution F (La phase) and a solid solution phase S presumably having monoclinic symmetry (P(J. phase) in multilayers. This phase diagram is discussed in Refs. 19, 18, 4. The phase diagram was derived from studies of spin-label binding to the membranes.

We assume that the affinities of these specific IgG molecules for spin-labeled lipid haptens such as (V), (IX), or (X) are of the same order of magnitude. Figure 10 illustrates the effect of antibody binding on the paramagnetic resonance spectra of (X) incorporated into lipid membrane vesicles. 

Xin WJ, Zhao BL, Zhang JH. 1984. Studies on the property of sulfhydryl binding site on the lung normal and cancer cell membrane of Chinese hamster with maleimide spin labels. Sci Sin [B] 27 1008-1014. 

The fluorescence probe l-anilinonaphthalene-8-sulfonate was used to examine the binding of spin-labeled local anesthetics to membranes of human blood cells and rabbit sarcoplasmic reticulum. Two distinct fluorescence lifetimes are exhibited by this probe the shorter life time represents the probe associated with pure lipid, the... 

Hammerstedt, R.H., Keith, A.D., Snipes, W., Amann, R.P., Arruda, D., and Griel, L.C. (1978). Use of spin labels to evaluate effects of cold shock and osmolality on sperm. Biol. Reprod. 75 686-696. Hardy, D.M. and Garbers, D.L. (1995). A sperm membrane protein that binds in a species- specific manner to the egg extracellular matrix is homologous to von Willebrand factor. J. Biol. Chem. 

Spin labels are stable, paramagnetic molecules that, by their structme, easily attach themselves to various biological macromolecular systems such as proteins or cell membranes. Examples of spin labels that can be covalently bonded to specific sites of biological systems include nitroxide derivatives of A-ethylmaleimide, which bind specifically to -SH groups, and nitroxide derivatives of iodoacetamide, which bind specifically to methionine, lysine, and arginine residues of amino acids. Nonco-valently bonded spin-labels that can be incorporated into biological systems include nitroxide derivatives of stearic acid, of phospholipids, and of cholesterol. 

To characterize the structural changes induced by membrane binding, the EPR spectra of 47 singly spin labeled ASYN derivatives were recorded in solution and upon binding to small unilamellar vesicles [42, 128]. 

Fig. 11 Spin-label EPR revealed that ASYN membrane binding is triggered by its N-terminus. Schematic representation of ASYN at the membrane-water interface. Positions of spin labels used in this study are depicted as red circles, and the number of the labeled residue is given. Representing electrostatic interactions, the cationic residue K80 is shown as a white circle. Adapted from [126]...

Drescher M, Godschalk F, Veldhuis G, van Rooijen BD, Subramaniam V, Huber M (2008) Spin-label EPR on alpha-synuclein reveals differences in the membrane binding affinity of the two antiparallel helices. Chembiochem 9 2411-2416... 

Relaxation-related work on proteins and polypeptides makes typically use of H, and NMR. A couple of papers have dealt with measurements on in fluorine-labelled aminoacids incorporated into peptide stuctures. Shi and co-workers introduced, at some specific sites, an unnatural fluorine-containing aminoacid and a nitroxide spin-label into a multidomain protein known to exist in different conformations. Measurements of F PRE allowed to determine the conformation under different conditions. Suzuki et used another fluorine-containing amino acid, inserted into different parts of a membrane-active peptide, as a local dynamics probe. They measured F transverse relaxation to examine changes in the mobility in different regions of the peptide upon binding to a lipid bilayer. 

This parallels the kinetic study conclusions(52) that the two sites are initially kinetically identical with respect to toxin binding, and yet differ in reactivity toward the affinity alkylating agent, 4-(N-maleimido)-benzyltrimethylammonium iodide. The 2 1 stoichiometry and structural similarity of the toxin combining sites of AChR have been recently confirmed also by EPR studies of interaction of nonselectively spin labeled Naja naja siamensis long-type a-neuro toxin with Torpedo californica AChR solubilized protein and AChR-rich membranes(53). 

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