1. Volume 105, Issue 3, Pages 667-678 (August 2013)
Investigation of Homeodomain Membrane Translocation Properties: Insights from the Structure Determination of Engrailed-2 Homeodomain in Aqueous and Membrane- Mimetic Environments 
Ludovic Carlier, Stéphane Balayssac, François-Xavier Cantrelle, Lucie Khemtémourian, Gérard Chassaing, Alain Joliot, Olivier Lequin 
Biophysical Journal 
Volume 105, Issue 3, Pages (August 2013)
DOI: /j.bpj
Copyright © 2013 Biophysical Society Terms and Conditions

2. Figure 1
Comparison of chicken En2HD and Drosophila homeodomain structures. (A) Sequence alignment of chicken and human Engrailed 2, and Drosophila melanogaster Engrailed homeodomains. (B) NMR structure of chicken En2HD in aqueous solution (PDB:3ZOB). The backbone of the 20 lowest energy conformers is indicated. Structures were superimposed over residues 8–56. Disordered residues 1–3 and 57–60 at the extremities are not shown. (C) Superimposition of chicken En2HD solution NMR structure and Drosophila Engrailed homeodomain crystal structure (PDB:1ENH), represented as ribbon diagrams. Structures were superimposed on backbone atoms of residues 8–56.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2013 Biophysical Society Terms and Conditions

3. Figure 2
NMR parameters of En2HD in aqueous solution (left) and in DPC micelles (right). (A) Hα chemical shift deviations from random coil values. The location of helical secondary structure elements is shown at the bottom. (B) Summary of sequential and medium-range HN/HN and Hα/HN NOEs.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2013 Biophysical Society Terms and Conditions

4. Figure 3
(A) CD spectra and (B) intrinsic tryptophan fluorescence spectra of En2HD in phosphate buffer, SDS micelles, DPC micelles, DMPC LUVs, DMPG LUVs, and DMPC/DMPG (1:1) LUVs. Samples contained 25 μM protein, and detergent and lipid concentrations, were 20 mM and 1.25 mM, respectively.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2013 Biophysical Society Terms and Conditions

5. Figure 4
Two-dimensional 1H-15N HSQC spectra of En2HD in aqueous solution (A), in DPC micelles (B), in zwitterionic bicelles (C), and in anionic bicelles (D). Assignments of back-bone and side-chain Trp resonances are indicated in aqueous solution. All the spectra were recorded at 500 MHz and 30°C except the DPC spectrum, which was acquired at 40°C. DPC concentration was 80 mM. Zwitterionic bicelles contained 50 mM DHPC and 25 mM DMPC and anionic bicelles contained 50 mM DHPC, 18.75 mM DMPC, and 6.25 mM DMPG.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2013 Biophysical Society Terms and Conditions

6. Figure 5
Paramagnetic relaxation enhancements induced by site-directed spin labeling in aqueous solution (A) and in DPC micelles (B). The paramagnetic tag was attached to a Cys residue (indicated by an asterisked (∗) label) in position –3 (upper), in position 39 (middle), or in position 61 (lower panel). The paramagnetic relaxation enhancements are shown as intensity ratios between the paramagnetic (I) and the diamagnetic state (I0). (Solid circles) Backbone amide groups; (shaded squares) side-chain groups.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2013 Biophysical Society Terms and Conditions

7. Figure 6
Use of paramagnetic reagents to probe the accessibility of En2HD residues in DPC micelles. (A) Paramagnetic relaxation enhancements of gadodiamide probe measured as amide proton relaxivities (differences in R1 longitudinal rates per mM concentration of gadodiamide). (B) Paramagnetic relaxation enhancements of 5-doxylstearic acid probe measured as residual intensities of HSQC cross-peaks. (Solid circles) Backbone amide groups; (shaded squares) side-chain groups. Hydrophobic side chains are indicated with asterisked (∗) labels.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2013 Biophysical Society Terms and Conditions

8. Figure 7
Model of DPC-bound En2HD structure. (A) Structural model based on chemical shifts, NOEs, and site-directed spin-labeled PREs data. The model was calculated with DYANA using 166 NOE-based distance constraints, 43 ϕ-, ψ-helical angular restraints, and two PRE-derived long-range distance constraints between the amide groups of Thr-60, Ala-7, and Phe-8, respectively (20 Å). (Dotted circle) The dimension of a spherical DPC micelle containing 60 monomers. (B) Positioning of En2HD three helices with respect to DPC micelle, inferred from paramagnetic effects induced by gadodiamide and 5-DSA probes.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2013 Biophysical Society Terms and Conditions