Transmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative diseases that are associated with conformational conversion of the normally monomeric and α-helical prion protein, PrPC, to the β-sheet-rich PrPSc. This latter conformer is believed to constitute the main component of the infectious TSE agent. In contrast to high-resolution data for the PrPC monomer, structures of the pathogenic PrPSc or synthetic PrPSc-like aggregates remain elusive. Here we have used site-directed spin labeling and EPR spectroscopy to probe the molecular architecture of the recombinant PrP amyloid, a misfolded form recently reported to induce transmissible disease in mice overexpressing an N-terminally truncated form of PrPC. Our data show that, in contrast to earlier, largely theoretical models, the con formational conversion of PrPC involves major refolding of the C-terminal α-helical region. The core of the amyloid maps to C-terminal residues from ≈160–220, and these residues form single-molecule layers that stack on top of one another with parallel, in-register alignment of β-strands. This structural insight has important implications for understanding the molecular basis of prion propagation, as well as hereditary prion diseases, most of which are associated with point mutations in the region found to undergo a refolding to β-structure.

https://www.pnas.org/content/pnas/104/48/18946.full.pdf

Molecular architecture of human prion protein amyloid: A parallel, in-register β-structure

A spin-probe method is described that can detect changes in the relative aggregation numbers in SDS micelles with a precision of about one molecule. The method is based on the fact that the 14N hyperfine coupling constant is sensitive to the average fraction of the volume occupied by water in the region of the nitroxide moiety that is located on average near the micelle surface. Defining A0(NA) to be the 14N hyperfine coupling constant at an aggregation number NA, we find A0(NA) = A0(0) + (∂A0/∂NA)NA, where NA is controlled by varying either the SDS or the NaCl concentrations. For the spin probe 5-doxylstearic acid ester(5DSE), by combination of the results of experiments in which the SDS and/or the NaCl concentrations were varied, linear least-squares fits gave A0(0) = (15.498 ± 0.009) G and ∂A0/∂NA = − 3.99 ± 0.02 mG/molecule (constant). A0(NA) depends only on the aggregation number despite the fact that a given value of NA may be prepared by choosing different combinations of NaCl and SDS concentration...

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Precision Relative Aggregation Number Determinations of SDS Micelles Using a Spin Probe. A Model of Micelle Surface Hydration

Molecular oxygen is paramagnetic and gives strong EPR signals in the gas phase. At sufficiently low pressure the number of observed lines is large indeed (see Figure 1 obtained at 180 micron pressure). Tinkham and Strandberg (1955a, 1955b) developed the theory for the spectrum of molecular oxygen and were able to assign 120 lines observed at X-band. As the pressure increases, the linewidths increase greatly. Even at atmospheric pressure, intense EPR signals can be detected from molecular oxygen (Figure 2). However, to the authors’ best knowledge no EPR spectra have been reported from oxygen dissolved in fluids near room temperature. Apparently lines are so broadened as to be undetectable. Thus there seems to be no possibility for the directdetection of oxygen in biological systems using magnetic resonance techniques. However, an indirect method does exist and is the subject of this chapter. Bimolecular collisions of oxygen with free radicals (and we consider particularly spin labels) alter the resonance characteristics of the radical. As will become apparent, it is a remarkable fact that effects can be detected at dissolved oxygen concentrations as low as 10−7M in a measurement that requires only a few seconds. This method has been called “spin-label oximetry.” The National Biomedical ESR Center has been active in the development of the field. A rigorous foundation has been laid down, and it is believed that the method can be applied with confidence to a wide range of biological systems.

Spin-Label Oximetry

Linewidth analysis of spin labels in liquids. I. Theory and data analysis

Molecular organization and dynamics in protein-rich membranes have been studied by investigating transport (diffusion-concentration product) of molecular oxygen at various locations in reconstituted membranes of bacteriorhodopsin (BR) and L-alpha-dimyristoylphosphatidylcholine. Oxygen transport was evaluated by monitoring the bimolecular collision of molecular oxygen with four types of nitroxide lipid spin labels placed at various locations in the membrane. The collision rate was estimated from the spin-lattice relaxation times (T1's) measured at various oxygen partial pressures by analyzing the short-pulse saturation recovery ESR signals. CD spectra and decay of polarized flash-induced photodichroism of bacteriorhodopsin indicated that BR molecules are monomers in reconstituted membranes with a lipid/BR molar ratio of 80 (80-rec) and are 25% monomers and 75% trimers plus oligomers of trimers when the lipid/BR ratio is 40 (40-rec). In the 80-rec, the lipid environment is homogeneous on a microsecond scale (T1), probably because the exchange rate of lipids between the bulk and the boundary regions is greater than the T1 relaxation rate (approximately 10(6) s-1). The oxygen collision rate in the hydrophobic region of the 80-rec membrane is smaller by a factor of 1.6 than in that of the lipid membrane without BR, and the effect of BR in decreasing the collision rate is independent of the "depth" in the hydrophobic region. In the 40-rec, two collision rates were observed, one of which is close to those for purple membrane (or the gel-phase membrane), while the other is about the same as was measured in the 80-rec.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular organization and dynamics in bacteriorhodopsin-rich reconstituted membranes: discrimination of lipid environments by the oxygen transport parameter using a pulse ESR spin-labeling technique.

1. Introduction.- 2. Theory of Spin Exchange.- 2.1 Exchange Interaction Between Two Paramagnetic Particles.- Dependence of Interaction Energy on Spin.- Spin-Hamiltonian of Exchange Interaction.- The Nature of Exchange Interaction. The Two-Electron System.- Interaction Between Many-Electron Systems. Delocalization and Spin Polarization.- Anisotropy of Exchange Interaction.- Semi empirical Estimates of Exchange Integral.- 2.2 Qualitative Description of Spin Exchange Process.- Spin Exchange.- Spin Exchange in a Two-Electron System.- Spin Density Matrix.- Spin Motion Due to Exchange Interaction.- 2.3 Formal Kinetics of Spin Exchange in Magnetically Dilute Solutions.- Binary Collision Approximation.- Rate of Bimolecular Spin Exchange Process.- Kinetics of Spin Exchange for Particles with Spins S = 1/2.- Kinetic Equations for Free Radicals Showing Hyperfine Structure (HFS) in Their ESR Spectra.- 2.4 Spectroscopic Manifestations of Spin Exchange.- Spectral Diffusion Due to Spin Exchange.- Shape of ESR Spectra.- Slow Exchange.- Fast Exchange.- Other Methods of Measuring the Rate of Spin Exchange.- Effect of Saturation.- Electron-Electron Double Resonance (ELDOR).- Electron Spin Echo.- 2.5 Theory of Sudden Collisions.- 2.5.1 Model of Sudden Collisions.- Basic Assumptions.- Equations for the Estimation of xc.- 2.5.2 Kinetic Equations.- Steady-State Equation for the Operator of Collision Efficiency.- 2.5.3 Spin Exchange Between Radicals.- Model System.- Spin Exchange Between Radicals in the Presence of Hyperfine Interaction.- 2.5.4 Spin Exchange Between Radicals with Anisotropic Interaction.- Rotation of Partners at the Moment of Their Contact.- Orientation Relaxation of Particles in Intervals Between Reencounters.- 2.5.5 Spin Exchange of Radicals with Other Paramagnetic Particles.- Spin Exchange Between Radicals and Spins SB with Long Times of Paramagnetic Relaxation.- Shape of ESR Spectra.- The Influence of the Relaxation of Spin SB on the Rate of Exchange.- Anisotropy of Spin Exchange.- 2.5.6 Spin Exchange in Biradicals.- The Model.- Kinetic Equations.- The ESR Spectrum of Nitroxide Biradicals.- 2.6 The Model of Diffusive Passage.- The Model.- An Equation for the Operator of Collision Efficiency.- The Kinetic Equations.- The Spin Exchange Between Two Kinds of Particles with Spins SA = SB = 1/2.- Comparison of Results Obtained from Models of Sudden Collisions and Those of Diffusive Passage.- 3. Experimental Measurement of Spin Exchange Rate.- 3.1 Experimental Measurement of Spin Exchange Rate Constants from ESR Spectra.- 3.1.1 Exchange Between Identical Particles with S = 1/2.- Doublet Spectrum with a Zero Initial Width.- Complex Spectra.- Determination of Rate Constants from Exchange Broadening of Complex Spectra.- Determination of Rate Constants from Exchange Shift of the Lines of Complex Spectra.- Determination of Rate Constants from Exchange Narrowing of Complex Spectra.- Peculiarities of Exchange Narrowing in Concentrated Solutions.- Comparison of Spin Exchange Rate Constants Measured by Different Techniques.- 3.1.2 Exchange Between Different Particles.- 3.2 Other Methods of Measuring the Spin Exchange Rate Constants.- 3.2.1 Continuous Wave Saturation Method.- 3.2.2 Electron-Electron Double Resonance.- 3.2.3 Electron Spin Echo.- 3.3 Separation of Exchange and Dipole-Dipole Contributions to Concentration Broadening of ESR Lines.- Theoretical Estimation of Dipole Broadening.- Experimental Estimation of Dipole-Dipole Contribution at Low Viscosity.- Separation of the Cases of Exchange and Dipole-Dipole Broadening According to Viscosity and Temperature Dependences.- 3.4 Separation of the Cases of Weak and Strong Exchange.- 4. Spin Exchange in Chemistry and Biology.- 4.1 Study of Diffusion Collisions in Solutions.- Hydrodynamic Model of Diffusion Collisions.- Potentialities of Spin Exchange.- 4.1.1 Collisions in Single-Component Solvents.- Spin Exchange Between Radicals.- Spin Exchange Between Free Radicals and Paramagnetic Complexes.- 4.1.2 Collisions in Complex Systems.- Binary Solvents.- Liquids Near Critical Points.- Polymeric and Heterogeneous Systems.- Collisions of Macroradicals.- 4.1.3 Study of Intramolecular Collisions of Paramagnetic Fragments.- 4.2 Estimation of the Energy of Exchange Interaction During Collisions.- 4.2.1 Free Radicals.- 4.2.2 Spin Exchange with Metal Complexes.- 4.2.3 Discussion of Results.- 4.3 Collisions of Charged Particles.- 4.3.1 Experimental Results.- Spin Exchange in Solutions of Anion Radical (S03)2N02-.- Spin Exchange Involving Charged Nitroxide Radicals.- Spin Exchange Between Anion Radicals in Organic Solvents.- 4.3.2 Comparison of Experiments on Spin Exchange with the Diffusion Theory of Collisions of Charged Particles in Liquid.- 4.3.3 Study of Collisions Between Aquo Complexes of Transition Metals.- Role of Counterions in Collisions of Aquo Complexes of Vanadyl.- An Example of the Process with Weak Influence of Electrostatic Repulsion.- 4.4 Biological Applications of Spin Exchange.- Paramagnetics Used as Spin Probes.- 4.4.1 Measurement of Binary Collision Rate of Molecules in Biological Systems.- 4.4.2 Study of Location of Paramagnetic Centers in Biopolymers.- Quantitative Interpretation of the Values of Kg for Spin Exchange Processes Involving Paramagnetic Centers of Biopolymers.- 4.4.3 Development of Individual ESR Spectra of Spin Labels via Spin Exchange.- 4.4.4 Spin Exchange Titration.- Kinetics of Reactions Involving Oxygen Molecules.- Coordination of Paramagnetic Ions by Macromolecules.- 4.4.5 Other Applications.- 4.5 Conclusion.- List of Frequently Used Notations and Abbreviations.- References.

I︠u︡. N. Molin

Papers

Spin exchange : principles and applications in chemistry and biology