Spin probe

Abstract: The hydrophobicity profiles across phosphatidylcholine (PC)-cholesterol bilayer membranes were estimated in both frozen liposome suspensions and fluid-phase membranes as a function of alkyl chain length, unsaturation, and cholesterol mole fraction. A series of stearic acid spin labels, with the probe attached to various positions along the alkyl chain, cholesterol-type spin labels (cholestane and androstane spin labels), and Tempo-PC were used to examine depth-dependent changes in local hydrophobicity, which is determined by the extent of water penetration into the membrane. Local hydrophobicity was monitored primarily by observing the z component of the hyperfine interaction tensor (A,) of the nitroxide spin probe in a frozen suspension of the membrane at -150 "C and was further confirmed in the fluid phase by observing the rate of collision of Fe(CN)63- with the spin probe in the membrane using saturation recovery ESR. Saturated-PC membranes show low hydrophobicity (high polarity) across the membrane, comparable to 2-propanol and 1-octanol, even at the membrane center where hydrophobicity is highest. Longer alkyl chains only make the central hydrophobic regions wider without increasing the level of hydrophobicity. Introduction of a double bond at C9-C10 decreases the level of water penetration at all locations in the membrane, and this effect is considerably greater than the cis configuration than with the trans configuration. Incorporation of cholesterol (30 mol %) dramatically changes the profiles; it decreases hydrophobicity (increases water penetration) from the polar headgroup region to a depth of approximately C7 and C9 for saturated- and unsaturated-PC membranes, respectively, which is about where the bulky rigid steroid ring structure of cholesterol reaches in the membrane. Membrane hydrophobicity sharply increases at these positions from the level of methanol to the level of pure hexane, and hydrophobicity is constant in the inner region of the membrane. Thus, formation of effective hydrophobic barriers to permeation of small polar molecules requires alkyl chain unsaturation and/or cholesterol. The thickness of this rectangular hydrophobic barrier is less than 50% of the thickness of the hydrocarbon regions. Results obtained in dioleoyl-PC- cholesterol membranes in the fluid phase are similar to those obtained in frozen membranes. These results correlate well with permeability data for water and amino acids in the literature.

Abstract: The ordering of the hydrocarbon chains and the rates of lipid motion are two independent parameters characterizing the structure and the dynamics, respectively, of a bilayer membrane. In this work, deuterium magnetic resonance has been used to elucidate the influence of a single cis double bond on the hydrocarbon chain ordering of a phospholipid bilayer. 1-Palmitoyl-2-oleoyl-3-sn-phosphatidylcholine was specifically deuterated at various segments of the palmitic acyl chain and at the 9, 10 position of the oleic acyl chain, and the segmental order parameters were deduced from the quadrupole splittings of the unsonicated bilayer phases. The shape of the order profile of the palmitic acyl chain is similar to that observed for the corresponding fully saturated membrane, but the magnitude of the order parameters is distinctly smaller in the unsaturated system. This demonstrates that the presence of a double bond in a membrane causes a more disordered conformation of the hydrocarbon chains. Considering the relative flexibility within the palmitic acyl chain, the deuterium resonance data indicate a local stiffening of those segments which are located in the vicinity of the double bond. The membrane fluidity was investigated using a nitroxide-labeled stearic acid spin probe. The smaller electron paramagnetic resonance line width in bilayers of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine demonstrates an increased fluidity compared to bilayers of 1,2-dipalmitoyl-3-sn-phosphatidylcholine.

Abstract: 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...

Abstract: The development of ESR methods that measure long-range distance distributions has advanced biophysical research. However, the spin labels commonly employed are highly flexible, which leads to ambiguity in relating ESR measurements to protein-backbone structure. Herein we present the double-histidine (dHis) Cu2+-binding motif as a rigid spin probe for double electron–electron resonance (DEER) distance measurements. The spin label is assembled in situ from natural amino acid residues and a metal salt, requires no postexpression synthetic modification, and provides distance distributions that are dramatically narrower than those found with the commonly used protein spin label. Simple molecular modeling based on an X-ray crystal structure of an unlabeled protein led to a predicted most probable distance within 0.5 A of the experimental value. Cu2+ DEER with the dHis motif shows great promise for the resolution of precise, unambiguous distance constraints that relate directly to protein-backbone structure and flexibility.