1. Volume 114, Issue 3, Pages 592-601 (February 2018)
ESR Resolves the C Terminus Structure of the Ligand-free Human Glutathione S- Transferase A1-1 
Matthew J. Lawless, John R. Pettersson, Gordon S. Rule, Frederick Lanni, Sunil Saxena 
Biophysical Journal 
Volume 114, Issue 3, Pages (February 2018)
DOI: /j.bpj
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2. Figure 1
Views of the hGSTA1-1 dimer bound to GSH. (A) Looking down the twofold symmetry axis of the hGSTA1-1 dimer (PDB: 1K3L). (B and C) Views generated by 90° rotations around the x and y axes, respectively. In this image, residues 212 and 214 were converted to Cys residues and residues 211 and 215 were converted to His residues for illustrative purposes. The first domain is colored light gray, the second domain is darker gray, and the C-terminal helix is colored black and labeled. The bound ligand, GS-hex, is highlighted with a surface. The attachment points for R1 on either 212 or 214 are shown as dark gray spheres. The Cu2+ that interacts with the side chains of H211 and H215 is represented by a lighter gray sphere between the two His side chains.
Biophysical Journal  , DOI: ( /j.bpj )
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3. Figure 2
Labeling strategies for hGSTA1-1. (A) Reaction scheme of MTSSL with cysteine thiol to produce the R1 side chain. (B) Schematic of the Cu2+-binding dHis motif.
Biophysical Journal  , DOI: ( /j.bpj )
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4. Figure 3
DEER data for R1-labeled hGSTA1-1 mutants. Background-corrected time-domain DEER data (left) and the resultant distance distributions (right) for both unliganded hGSTA1-1 mutants S212R1 and E214R1 (top and bottom, respectively). The fit modeled by DEERAnalysis 2013 is shown as a dashed line. The bimodal distance distributions are strong evidence that the unliganded hGSTA1-1 exists in equilibrium between two conformations. The distance distribution predicted by MTSSLWizard based on the liganded GST structure (PDB: 1K3L) is shown as a dotted line.
Biophysical Journal  , DOI: ( /j.bpj )
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5. Figure 4
Structural model of the unliganded state of hGSTA1-1. Comparison of the known conformation of liganded hGSTA1-1 (light gray; PDB: 1K3L) with the MMM-generated model of the second conformation of unliganded hGSTA1-1 (dark gray). DEER data indicate that unliganded hGSTA1-1 exists in conformational equilibrium between these two helical conformations.
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6. Figure 5
MTSSL simulations of R1 DEER data. Simulated R1-R1 distance distributions of both S212 (left) and E214 (right) mutants of hGSTA1-1 produced by both MTSSLWizard (dashed lines) and MMM (shaded area) overlaid upon actual experimental data (solid lines). Insets show the orientation of R1 distribution in both the ligand-free model (dark gray) and the ligand-bound crystal structure (gray).
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7. Figure 6
dHis motif DEER data for 211H/215H hGSTA1-1. (A) The C-terminal α-helix of hGSTA1-1 mutant K211H/E215H containing the Cu2+-IDA binding dHis site. (B) The background-subtracted time-domain DEER data (solid line) and corresponding fit (dashed line). (C) The distance distribution showing two distinct distances. Expected distances shown by gray lines.
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8. Figure 7
DEER distribution population shift due to GS-hex. DEER distance distributions in the presence (dotted lines) and absence (solid lines) of GS-hex for hGSTA1-1 S212R1, E214R1, and K211H/E215H. Upon addition of GS-hex, the population of the conformation with the larger distance occurs for all three sites. This larger distance coincides with expected distances of the liganded conformation based on x-ray crystallography (PDB: 1K3L).
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9. Figure 8
CW-ESR comparison of liganded and unliganded R1-labeled hGSTA1-1. Nitroxide CW spectra of S212R1 (top) and E214R1 (bottom) both unliganded (solid lines) and in the presence of GS-hex (dotted lines) at 294 K. Spectral changes are highlighted by the black arrows. Spectra were simulated using the MOMD model, and differences upon ligand addition could be accounted for by changing only the relative population between components. This change in population is shown by the bar graph (black lines represent errors in fits).
Biophysical Journal  , DOI: ( /j.bpj )
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