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Location of the TEMPO Moiety of TEMPO-PC in Lipid Bilayers

2017; Elsevier BV; Volume: 113; Issue: 4 Linguagem: Inglês

10.1016/j.bpj.2017.04.057

1542-0086

Alex M. Schrader, Songi Han,

Advanced NMR Techniques and Applications

In a recent article in Biophysical Journal entitled “Effects of Dimethyl Sulfoxide on Surface Water near Phospholipid Bilayers” by Lee, Pincus, and Hyeon (1Lee Y. Pincus P.A. Hyeon C. Effects of Dimethyl Sulfoxide on Surface Water near Phospholipid Bilayers.Biophys. J. 2016; 111: 2481-2491Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), the authors conducted molecular dynamics (MD) simulations of fluid-phase phosphatidylcholine (PC) membranes in the presence of DMSO/water mixtures. They plotted trends for the spatial variation of water diffusivity along the membrane normal as a function of DMSO concentration in the solvent (Figure 1 a, upper panel) and found that the computed diffusivity trends with DMSO concentration at the height of the choline group (about 2 Å above the phosphate group, |z|=22 Å in their computational study) disagreed with the experimental trends for surface water diffusivity near unilamellar PC vesicle surfaces (Figure 1 b). The authors further show that when the surface water diffusivity is normalized by the bulk water diffusivity, there is qualitative agreement between the simulated and experimental results (even when simulated values are taken any point between the choline level and 8 Å outward, |z|=22-30 Å), but state that agreement is “coincidental.” At the center of the dispute is the experimental location of the surface water measurements as probed by Overhauser dynamic nuclear polarization (ODNP) (2Cheng C.-Y. Song J. Han S. et al.DMSO induces dehydration near lipid membrane surfaces.Biophys. J. 2015; 109: 330-339Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). ODNP relies on a small fraction of lipids functionalized with a nitroxide radical spin label, here TEMPO tethered on top of the PC moiety of the PC head group, termed TEMPO-PC. We, the authors of (2Cheng C.-Y. Song J. Han S. et al.DMSO induces dehydration near lipid membrane surfaces.Biophys. J. 2015; 109: 330-339Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), relied on the established knowledge of the wider electron paramagnetic resonance (EPR) community that TEMPO of TEMPO-PC is on average located at about 5 Å above the phosphate level, with literature support for this statement provided below. It should also be noted that ODNP averages local contributions of water within about 5–10 Å of the TEMPO labels. In other words, the ODNP measurements with TEMPO-PC are capturing the surface water diffusivity within 0 – 10 Å distance above the phosphate level (which would correspond to (|z|=20-30 Å). The authors of (1Lee Y. Pincus P.A. Hyeon C. Effects of Dimethyl Sulfoxide on Surface Water near Phospholipid Bilayers.Biophys. J. 2016; 111: 2481-2491Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) rely on their computed values to be quantitative to within a few Å, and consequently conclude that the shift of the computed versus experimental trends by about 5 Å must be due to the erroneously assigned location of TEMPO-PC for the experimental interpretation. Hyeon and coworkers rationalized the discrepancy by simulating TEMPO-PC in a lipid membrane, and claiming that the TEMPO moiety primarily resides 8–10 Å beneath the phosphate group (|z|=12-14 Å). The simulated values for water diffusivity around the buried TEMPO group (as measured by water residence time) agreed better with the experimental ODNP results than the simulated results (without TEMPO) taken at the choline level or higher (|z|=22-30 Å, Figure 1 a, upper panel). They consequently concluded that the prior ODNP measurements in (2Cheng C.-Y. Song J. Han S. et al.DMSO induces dehydration near lipid membrane surfaces.Biophys. J. 2015; 109: 330-339Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) did not measure properties of the water solvent at the liposome surface, but instead below the head group. Notably, TEMPO-PC is a commonly used spin label for lipid membrane studies in the literature, such that the conclusion by Hyeon and coworkers would impact all past studies relying on TEMPO-PC as a probe. In fact, there are many experimental EPR and ODNP observations in the literature that directly and convincingly counter the claim that the TEMPO of TEMPO-PC is mostly buried within the bilayer to such a depth. Below, we summarize select published experimental results from the literature that show that the TEMPO moiety largely probes the solvent phase, and that TEMPO indeed is partitioned at the bilayer surface at about 5 Å above the phosphate group, as reported in our study published in Biophysical Journal (2Cheng C.-Y. Song J. Han S. et al.DMSO induces dehydration near lipid membrane surfaces.Biophys. J. 2015; 109: 330-339Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). If the TEMPO moiety was buried, as suggested by Hyeon and coworkers, one would expect sharp changes in TEMPO mobility and local water diffusivity as the system temperature crosses the melting point of the lipid hydrocarbon chain, Tm—a phenomenon that is not observed when TEMPO is attached to the end of the PC head group, as experimentally verified by both EPR (3Kausik R. Han S. Dynamics and state of lipid bilayer-internal water unraveled with solution state 1H dynamic nuclear polarization.Phys. Chem. Chem. Phys. 2011; 13: 7732-7746Crossref PubMed Scopus (44) Google Scholar) and ODNP (4Kausik R. Han S. Ultrasensitive detection of interfacial water diffusion on lipid vesicle surfaces at molecular length scales.J. Am. Chem. Soc. 2009; 131: 18254-18256Crossref PubMed Scopus (42) Google Scholar) measurements as a function of temperature. Specifically, EPR measurements of the rotational diffusion rate of the TEMPO moiety were conducted on DPPC vesicles containing TEMPO-PC (3Kausik R. Han S. Dynamics and state of lipid bilayer-internal water unraveled with solution state 1H dynamic nuclear polarization.Phys. Chem. Chem. Phys. 2011; 13: 7732-7746Crossref PubMed Scopus (44) Google Scholar), and no such jump or sharp transition was observed when crossing the phase transition temperature (from the ripple phase, Pβ’, to the fluid phase, Lα), Tm of 313 K, of the DPPC lipid bilayer, as shown in Fig. 2 (filled square symbols). In contrast, when local viscosity/lipid packing in the interior of the bilayer was probed using a stearic acid labeled with DOXYL (a moiety of essentially the same volume as TEMPO and with comparable magnetic parameters) at the 16th carbon position (16DSA), the expected step transition in spin label mobility (∼40% increase) was observed as the temperature crossed Tm (empty square symbols). It should also be noted that EPR measurements on spin-labeled unilamellar vesicles in water by Marsh, Watts, and Knowles (5Marsh D. Watts A. Knowles P.F. Cooperativity of the phase transition in single- and multibilayer lipid vesicles.Biochim. Biophys. Acta. 1977; 465: 500-514Crossref PubMed Scopus (136) Google Scholar) displayed a steep change in the outer hyperfine splitting, A||, when crossing Tm for both 5DSA and 5PC, indicating that 1) the lipid or surfactant head group (COOH versus PC) does not qualitatively affect this transition, and 2) steep transitions in EPR parameters are observed even when probing at the fifth carbon position. Further contradicting the claim that TEMPO is buried, ODNP measurements of local water diffusivity of unilamellar DPPC vesicle surfaces using TEMPO-PC showed no jump in diffusivity as the temperature crossed Tm (4Kausik R. Han S. Ultrasensitive detection of interfacial water diffusion on lipid vesicle surfaces at molecular length scales.J. Am. Chem. Soc. 2009; 131: 18254-18256Crossref PubMed Scopus (42) Google Scholar). Hyeon and coworkers also referenced the analysis of fluorescence quenching measurements by Kyrychenko and Ladokhin (6Kyrychenko A. Ladokhin A.S. Refining membrane penetration by a combination of steady-state and time-resolved depth-dependent fluorescence quenching.Anal. Biochem. 2014; 446: 19-21Crossref PubMed Scopus (12) Google Scholar), where measurements of fluorescence intensity were made on suspensions of fluid-phase PC unilamellar vesicles that contained 1 mol% of lipids with a fluorophore, NBD, on the head group, and up to 30 mol% of spin-labeled PC lipids in the membrane. The spin-labeled lipids quench the fluorescence of NBD, and increasingly so as the spin label nears the NBD moiety. The results indicate that the order of spin label depth into the bilayer is as follows: 5PC<TEMPO-PC≈7PC<12PC. However, given the high error of the measurement (Fig. 1 C in (6Kyrychenko A. Ladokhin A.S. Refining membrane penetration by a combination of steady-state and time-resolved depth-dependent fluorescence quenching.Anal. Biochem. 2014; 446: 19-21Crossref PubMed Scopus (12) Google Scholar)), the authors had to use spin-labeled PC lipids up to 30 mol% to achieve a decent correlation of spin label position versus fluorescence intensity. As described above, when examining bilayers containing <2 mol% of 5PC, 5DSA, or 16DSA (3Kausik R. Han S. Dynamics and state of lipid bilayer-internal water unraveled with solution state 1H dynamic nuclear polarization.Phys. Chem. Chem. Phys. 2011; 13: 7732-7746Crossref PubMed Scopus (44) Google Scholar, 5Marsh D. Watts A. Knowles P.F. Cooperativity of the phase transition in single- and multibilayer lipid vesicles.Biochim. Biophys. Acta. 1977; 465: 500-514Crossref PubMed Scopus (136) Google Scholar), EPR spectra show sharp changes when crossing Tm, and (3Kausik R. Han S. Dynamics and state of lipid bilayer-internal water unraveled with solution state 1H dynamic nuclear polarization.Phys. Chem. Chem. Phys. 2011; 13: 7732-7746Crossref PubMed Scopus (44) Google Scholar) shows no such change for TEMPO-PC. Thus, it may be that the large fraction of spin-labeled lipids used in the fluorescence study skew their results, but currently this is simply a hypothesis. Furthermore, an average 8–10 Å depth of TEMPO below the phosphate group of the lipid would suggest that ODNP measurements using TEMPO-PC would give similar results to the measurements using spin labels that explicitly probe shallow depths of the bilayer (for example, the labelled DSA variants at the fifth carbon position, 5DSA). This is in contrast to the experimental ODNP observations. Specifically, ODNP-derived local water diffusion at different depths within the bilayer (using TEMPO-PC and DSA labeled with DOXYL at different carbon positions of the acyl chain) displays a monotonic gradient as a function of the nominal distance from the phosphate level of the fluid-phase membranes of POPC:POPS (7:3) (7Cheng C.-Y. Varkey J. Han S. et al.Hydration dynamics as an intrinsic ruler for refining protein structure at lipid membrane interfaces.Proc. Natl. Acad. Sci. USA. 2013; 110: 16838-16843Crossref PubMed Scopus (65) Google Scholar), as reproduced in Fig. 3. The reported values shown in the figure for the retardation factor, ρt, are approximately inversely proportional to the surface water diffusivity. The results showed a smooth and gradual decrease in water diffusion with depth, probing at –5, 8.1, 10.5, 14.0, and 16.0 Å above (negative value) or below (positive value) the phosphate level, as measured by the spin probes TEMPO-PC, 5DSA, 7DSA, 10DSA, and 12DSA, respectively. There are several literature studies of the EPR properties of TEMPO-PC in lipid bilayer membranes that clearly show the nitrogen hyperfine coupling constant (2Az) for TEMPO-PC to reflect its location in a solvent-exposed local volume, on average, unlike when the nitroxide is located at the fifth or seventh position of the acyl chain of 5PC or 7PC. The published 2Az values by Subczynski et al. (8Subczynski W.K. Raguz M. Widomska J. Studying lipid organization in biological membranes using liposomes and EPR spin labeling.Methods Mol. Biol. 2010; 606: 247-269Crossref PubMed Scopus (44) Google Scholar) on pure POPC bilayers show a clear trend for the location of TEMPO-PC to be more hydrophilic compared to that of 5 and 7PC (at T = –165°C, Fig. 8 B in (8Subczynski W.K. Raguz M. Widomska J. Studying lipid organization in biological membranes using liposomes and EPR spin labeling.Methods Mol. Biol. 2010; 606: 247-269Crossref PubMed Scopus (44) Google Scholar)). Hyeon and coworkers note this, but selectively single out the observation by the same authors of an increased oxygen transport parameter (Fig. 9 of (8Subczynski W.K. Raguz M. Widomska J. Studying lipid organization in biological membranes using liposomes and EPR spin labeling.Methods Mol. Biol. 2010; 606: 247-269Crossref PubMed Scopus (44) Google Scholar)) at the nitroxide location that is comparable to that of 5 or 7PC, suggesting a more lipophilic environment for TEMPO-PC than expected. Although the oxygen transport parameter is generally regarded as a decent gauge of membrane fluidity, one must keep in mind that it is directly proportional to local oxygen concentration (9Subczynski W.K. Hyde J.S. Kusumi A. Oxygen permeability of phosphatidylcholine--cholesterol membranes.Proc. Natl. Acad. Sci. USA. 1989; 86: 4474-4478Crossref PubMed Scopus (246) Google Scholar), and not just translational diffusivity. Thus, it is likely not as good a metric of the local “viscosity” as the mobility of spin labels at select spots within the bilayer. Regardless, the oxygen transport results of (8Subczynski W.K. Raguz M. Widomska J. Studying lipid organization in biological membranes using liposomes and EPR spin labeling.Methods Mol. Biol. 2010; 606: 247-269Crossref PubMed Scopus (44) Google Scholar) can be explained by a partial partitioning or occasional dipping of TEMPO-PC below the phosphate head group or the hydrophobic character of the TEMPO moiety itself. What Hyeon and coworkers miss is that Subczynski et al. prepared multi-lamellar liposomes, as opposed to the unilamellar liposomes that were employed in both the simulation (1Lee Y. Pincus P.A. Hyeon C. Effects of Dimethyl Sulfoxide on Surface Water near Phospholipid Bilayers.Biophys. J. 2016; 111: 2481-2491Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) as well as experiments (2Cheng C.-Y. Song J. Han S. et al.DMSO induces dehydration near lipid membrane surfaces.Biophys. J. 2015; 109: 330-339Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). If TEMPO has a somewhat hydrophobic character, its confinement within the interstitial regions of multi-lamellar liposomes could potentially enhance its partitioning into lipophilic environments because of any repulsive steric-hydration forces between TEMPO and the apposing bilayer. To be clear, the dispute is not whether TEMPO (of TEMPO-PC) spends a fraction of the time below the phosphate head group—this is possible for highly dynamic structures such as lipid bilayers. The dispute is whether TEMPO-PC is on average located 5 Å above or 8–10 Å below the lipid phosphate head group. While the simulation results are intriguing, one must keep in mind that the published EPR and ODNP studies provide convincing evidence that the TEMPO moiety of TEMPO-PC largely resides in the solvent. The simulations of water diffusivity (in pure water—no DMSO) as a function of the nitroxide position along the membrane normal in Figure 1 a do bear a qualitative resemblance to the ODNP measurements in Fig. 3 at varying depths within the membrane, but quantitative differences (especially when adding a co-solvent) are expected, possibly given any shortcomings of the model used in the simulations. Certainly, a single computer simulation study relying on one specific set of force fields for water, DMSO, and the lipid membrane is not sufficient to take a broad and collective swipe at all experimental studies that rely on TEMPO-PC as an effective tool for probing average surface properties of the solvent near lipid membrane surfaces.