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Reply to Comment on "Distinct Populations in Spin-Label EPR Spectra from Nitroxides" Derek Marsh J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b11960 • Publication Date (Web): 15 Feb 2019 Downloaded from http://pubs.acs.org on February 17, 2019
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The Journal of Physical Chemistry
Reply to Comment by Meirovitch et al. on “Distinct Populations in Spin-Label EPR Spectra from Nitroxides.”
Derek Marsh, Max-Planck-Institut für biophysikalische Chemie, Göttingen, Germany
The reader can be excused for not recognizing, in the Comment by Meirovitch et al., the paper that I wrote. Much of the emphasis is on topics not touched upon by me, and attributes to me statements that I did not make; and never would make. In view of this misrepresentation, it is useful first to describe what my paper actually is about. I identify a very clear and serious error in the paper entitled “Analysis of Protein-Lipid Interactions Based on Model Simulations of ESR.” by Meirovitch et al. (referred to as MNF). The conclusion they reached was that experimental spectra from lipid-protein systems that were interpreted as consisting of two distinct components (one from lipids interacting directly with the protein and the other from more remote environments) could be simulated with a single-component model. However, the only simulated line shapes in MNF that can be interpreted at all credibly as consisting of two independent components are those of Fig. 8, which I show to be incorrect. MNF state specifically in their Abstract that these spectra account for experimental two-component line shapes, but the correct simulations do not resemble two components at all. Thus their claim that two-component line shapes can be reproduced by single-component simulations is not justified by the MNF paper. Further, the claim of MNF as regards their title subject, namely “Protein-Lipid Interactions”, is therefore as I state: misleading and unhelpful. I do not address other simulations given in MNF because they cannot be misinterpreted as two-component line shapes. (Except, I point out inconsistency in the field scan of Fig.1, which is analogous to that in Fig. 8.) Although buried in irrelevancies (as far as my paper is concerned), the authors do now admit the error in MNF (finally after 34 years, as they pointedly remark). Their explanation turns out to be very salutary. In the Comment, they instruct the reader about the importance of checking for convergence. But, to the best of my knowledge, MNF is the only case in the literature where this basic error is made. (Surely, it is the only case where such a mistake has been inflated to a sweeping criticism of the competent interpretation of experimental work by others.) I now turn explicitly to the many misrepresentations of my paper that are made by the authors of the Comment: 1) The authors begin their Comment by stating that I disregard eight of the sets of simulated spectra in MNF. On the contrary, I point out specifically that none of these spectra can be misconstrued as representing two-component spectra. 2) They then imply that I conclude these latter sets of simulated spectra to be wrong. This simply is not true. Contrary to a corresponding misstatement in their opening paragraph, I do not comment on whether these simulations are correct. This is because they cannot be mistaken for twocomponent spectra, and therefore are not germane to the central issue. 3) Building on misrepresentation 2), the authors go on to imply that I conclude the whole of the MNF article is misleading and unhelpful. What I actually state is: that MNF “makes a misleading and unhelpful contribution to the otherwise valuable application of spin-label EPR for studying lipid-
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protein interactions.” No more and no less. As I summarized at the outset, the reasons for saying this are given in my paper. 4) Next, the authors imply that I claim the software used by MNF is flawed, although the version that I use derives from it. My paper demonstrates that the results in Fig. 8 of MNF are seriously flawed; but nowhere do I suggest that the software said to produce them is flawed. Quite the opposite, I give specific reference (ref. 25) in Methods to the congruence of the two sets of software. 5) Immediately following, the authors then state that I use the VAR model in the paper commented upon “mainly to interpret ESR spectra from dispersions of lipid bilayers.” This is a complete misrepresentation. I use the VAR model because this is the model said to be used for Fig. 8 of MNF, which I show in my paper to be incorrect. 6) When the authors eventually get to lipid-protein interactions (i.e., the title subject of MNF), they say that I ignore a demonstration in Fig. 11B(ii) of MNF that a single-component model reproduces experimental two-component line shapes. I do not ignore this; it is simply that this claim by MNF is unwarranted. Comparing Fig. 11B(ii) with the original experimental spectrum that corresponds to Fig. 11B(i) (from Cornell et al., 1978 FEBS Lett. 90,29-35; not even cited in MNF) not with the small and inadequately reproduced version in MNF one sees immediately that the simulation is simply not an adequate representation. Nor does it even remotely approach Fig. 11A(iii) at 37oC, which is the experimental spectrum reproduced in Fig. 11 of MNF that is most clearly two-component. 7) The authors also say that I ignore that single-component simulations can reproduce EPR spectra from pure lipids. Nobody doubts this, but it is utterly irrelevant to the central question of whether a single-component model can reproduce two-component line shapes. The fluid pure lipid samples are single-component, as are the simulations. 8) They then go on to say that, in the paper commented on, I simulate spectra from pure lipids with the VAR model. No, I merely simulate VAR spectra with the parameters given for this model in Fig. 8 of MNF (see point 5). I also use earlier numerical results from VAR simulations (ref. 21), simply to illustrate that fast-motion VAR spectra given in Fig. 8 of MNF are impossible for their stated model, whereas those that I present are entirely reasonable. Note that hyperfine splittings in fast-motion spectra are determined by the order parameter S zz of the nitroxide z-axis, which is a purely geometrical construct (Saupe, 1964, Z. Naturforsch. A19, 161171). The Commentators confuse the issue by concentrating on the physical ordering of the molecule that is spin-labeled, which is only part of S zz . We get the total contribution from the addition theorem for spherical harmonics (see, e.g., Marsh et al., 2007, Biophys. J. 92, 473-481; Marsh et al., 2007, Biophys. J. 92, 4003-4011). This has long been recognized in spin-label EPR (Seelig 1976, in Biological Magnetic Resonance, Vol. 1, 373-409, New York: Academic Press; Griffith and Jost 1976 in Biological Magnetic Resonance, Vol. 1, 453-523, New York: Academic Press), and analogously in 2H-NMR where the quantity measured is, for instance, SCD – the order parameter of the C-D bond. Now I turn to consider the spectral simulations, presented towards the end of the Comment: The authors compare spectral simulations using EasySpin with those from the standard Cornell suite of programmes. This is unnecessary: nobody doubts that EasySpin faithfully reproduces stochasticLiouville simulations. Indeed, I state specifically that parameters from MNF are transferrable directly to EasySpin and give the appropriate reference (my ref. 25; omitted from the present authors’ Comment). 2 ACS Paragon Plus Environment
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The Journal of Physical Chemistry
In Fig. 1A, the authors repeat some of the simulations in Fig. 1 of MNF; these are not simulated in my paper. They obtain reasonable agreement with MNF, but none of their line shapes contain any feature that can be interpreted as a second component. Therefore, Fig. 1A is irrelevant to the central issue of protein-lipid interactions and to my paper, which the authors supposedly are commenting on. In Fig. 1B, the authors repeat the simulations from Fig. 8B of MNF. They obtain line shapes that diverge strongly from those of MNF. None of them appear two-component. In fact, they recapitulate almost exactly the line shapes from Fig. 1B of my paper. The authors conspicuously refrain from making this comment in their Comment on my paper. Exactly in this context, it also would be closer to the facts for them to comment that I, and not they, corrected or “repaired” the errors in MNF. In conclusion, the authors of the Comment fail to refute my demonstration that MNF does not provide evidence that a single-component model can produce a line shape with the appearance of a two-component spectrum. After introducing my correction, no simulated line shapes in MNF contain any feature that can be interpreted as a second component. Nor do the authors of the Comment produce any other simulation from a single-component model that contains such a feature. They are, however, able to reproduce important simulation results from my paper. The final “22-year” sentence of the introductory paragraph in the Comment may cause some perplexity. I take it to mean that the authors still lack a direct and meaningful comparison between one-component simulations and two-component experimental spectra from lipid-protein systems. Certainly, the issue is not moot as they seem to claim. The introductory paragraphs of my paper list direct, unambiguous experimental demonstrations (Refs. 3-19) of two-component spectra; this is ignored totally by the authors of the Comment. From the standpoint of reality, an experimental demonstration is always preferable to simulation, even when the simulation is not fatally flawed.
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