https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2872%2980300-4

Dynamics of lipids in membranes: Heterogeneity and the role of cholesterol.

Biological systems provide the physical chemist with an abundance of interesting, challenging and significant problems. One example is the problem of the molecular basis of co-operative or allosteric interactions between distant ligand or substrate binding sites in hemoglobin and in enzymes. This problem has been discussed recently in This Journal by Eigen (1968) and by Wyman (1968). Another particularly challenging problem is the molecular organization of biological membranes. Such problems tend to be particularly resistant to solution by the straight-forward application of most spectroscopic techniques, in large part because of the enormous chemical and spectroscopic complexity of biological macromolecules. This spectroscopic complexity has stimulated the use of various ‘probes’ that can be introduced into selected sites in complex systems to provide spectroscopic signals that are comparatively free from interference. The use of heavy metal atoms (‘isomorphous replacement’) in X-ray studies of protein crystals (Green, Ingram & Perutz, 1954), and fluorescent dyes in the study of proteins in solutions (Weber, 1953; Steiner & Edelhoch, 1962) are early examples. Spin labels represent a new member of the family of spectroscopic structural probes. A spin label is a synthetic paramagnetic organic free radical, usually having a molecular structure and/or chemical reactivity that results in its attachment or incorporation at some particular target site in a biological macromolecule, or assemblage of macromolecules (Ohnishi & McConnell, 1965; Stone et al. 1965). This type of probe is being used in our laboratory to study allosteric interactions in proteins, and molecular dynamics and organization in membranes.

/pdf/physics-and-chemistry-of-spin-labels-1c3ektehba.pdf

Physics and chemistry of spin labels.

The first publication on the use of ESR techniques in connection with biological systems appeared only nine years after the first demonstration of paramagnetic resonant absorption.† In that first report, a variety of biological systems, such as leaves, seeds, and tissue preparations, were shown to contain free radicals. A definite correlation was found between the concentration of the radicals and the metabolic activity of the material. This work appears to confirm earlier ideas that free radicals are involved as intermediates in metabolic processes. However, the question as to which free radicals are involved in a given metabolic process proved to be a much more difficult question to answer.

Biological applications of electron spin resonance

In general biological membranes possess no intrinsic paramagnetism and hence in the unlabeled state do not give rise to an electron spin resonance (ESR) spectrum. The introduction of a stable free radical (“spin label”) thus enables one to use ESR spectroscopy to study specific environments within the membrane. The spin label which is invariably used is the nitroxide radical, which has a three-line nitrogen hyperfine structure whose splitting varies with the orientation of the magnetic field relative to the nitroxide axes. It is this spectral anisotropy which has made spin label ESR such a powerful tool in the study of the molecular motions which are the characteristic feature of the highly dynamic structure of biological membranes. The nitroxide hyperfine splittings are partially averaged by the anisotropic motion, which gives a measure of the motional amplitude, and the linewidths are differentially broadened by an extent which depends on the rate of molecular motion. Other important features of the spin label spectra are the broadening by intermolecular label-label interactions and the ability to detect compartmentation of the label by quantitating spectral lineheights after selectively removing accessible spin-label signals by chemical reducing agents. Label-label interactions arise from two sources: the Heisenberg exchange interaction which is essentially a contact interaction and is therefore capable of measuring translational diffusion; and the dipole-dipole interaction which depends on the distance apart of the labels and is therefore capable of measuring intermolecular separations. Quantitation after treatment with reducing agents is chiefly concerned with the measurement of transport properties: of spin-label substrates or reducing agents, or the translocation of labelled lipid molecules.

Electron Spin Resonance: Spin Labels

Nitroxide free radicals: spin labels for probing biomolecular structure

are remarkably stable and inert,1 and show sharp, well-resolved, and simple paramagnetic resonance spectra2 that are sensitive to molecular motion,3 and, to a lesser extent, sensitive to polarity of the molecular environment.4 As shown below, such nitroxide radicals containing an isocyanate group can be bound to proteins and synthetic polypeptides in a manner similar to that used previously to attach fluorescent dyes.5 Here we report a preliminary study of the paramagnetic resoiiance of a nitroxide radical bonded to bovine serum albumin and to poly-L-lysine. It is evident from the present study that there are many other potential applicatioiis of this method to biological systems. Materials and Methods.-Preparation of 2, 2, 6, 5-tetramethyl-3-isocyanatopyrrolidine-1-oxyl (II): 2, 2, 5, 5-Tetramethyl-3-aminopyrrolidone-1-oxyl (III) was synthesized from triacetonamine by the method of Rosantzev and Krivitzkaya.6 NCO NH2

http://www.pnas.org/content/54/4/1010.full.pdf

P L Nordio

Papers

Spin-labeled biomolecules.