Probing Structural Dynamics of Membrane Proteins Using Electron Paramagnetic Resonance Spectroscopic Techniques

Round 1

Reviewer 1 Report

The work is good and interesting. In biological systems, magnetic complexes like those associated with metals and free radicals play a very important role. They are very small and dynamic and static magnetic measurements can be studied using EPR and SQUID methods.

Author Response

Please see attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

This well written manuscript reviews the literature on the use of  Electron Paramagnetic Resonance Spectroscopic Techniques including site directed spin labeling (SDSL) in the investigation of membrane protein structures and their conformational dynamics.        The authors point out that membrane proteins represent targets of more than 50% of modern medical drugs and therefore are of special interest.  The authors point out that membrane proteins are imbedded in the lipid membrane bilayer and difficult to investigate as they can not be studied  in a purely aqueous environment as is done for most other protein structures.     The authors focus on the use of EPR techniques but do point out the other commonly use methods for investigating these systems such as NMR, FRET, CryoEM and point out the advantages of EPR techniques.  The authors have many publications in the area and are very well qualified to review this area of work.     There are only a few comments that need attention.

1. Figure 2 does not appear to show the appropriate chemical structures after binding of MTSL.

The labeling process releases a sulfonic acid group from the MTSL and forms and disulfide bond to the protein.

   RSO₂S-nitroxide + protein-SH → protein-S-S-nitroxide + RSO₂H

  It appears the link is not shown properly as the sulfonic acid remains.

2. Lines 283-285 The authors need to point out how the membrane depth parameter was determined in ref 102.
3. The use of CW dipolar broadening EPR  and DEER for short range and long range spin spin interactions is well described in several studies.  The authors should discuss the possible effect of the spin label on protein structure as a confounding issue.

Author Response

Please see the cover letter.

Author Response File: Author Response.pdf

Reviewer 3 Report

I recommend publishing the article after some revising is done (see attached pdf file)

Kind regards

Comments for author File: Comments.pdf

Author Response

please see the cover letter.

Author Response File: Author Response.pdf