Journal: International Journal of Molecular Sciences

Article Title: The ALS-Related σ1R E102Q Mutant Eludes Ligand Control and Exhibits Anomalous Response to Calcium

doi: 10.3390/ijms21197339

Figure Lengend Snippet: Secondary structure, charge distribution map, and 3D model of sigma receptor type 1 (σ1R). ( A ) Protein sequence and secondary structure of σ1R, with helices indicated by green waves and β-sheets by yellow arrows. The amino acid change in human σ1R associated with the juvenile form of amyotrophic lateral sclerosis (ALS) is indicated in red. ( B ) Charge distribution map of wild-type (WT) σ1R and σ1R E102Q (the images were created using NovaFold v17, DNASTAR, Inc., Madison, WI, USA). Positive charges are indicated by blue and negative charges are indicated by red. The E102Q mutant protein lacks a negatively charged cluster located in the linker region between β2 and β3 (residues 98–106, ASLSEYVLL). ( C ) 3D structure of σ1R showing the WT and mutated amino acid as a colored tube. E102 is indicated by green and Q by pink. The structural model of σ1R and its secondary structure shown here were generated with NovaFold v. 17 (DNASTAR).

Article Snippet: Human σ1R is composed of 223 amino acids that form seven helices and ten β-sheets, with the rest of the protein being linear sequences ( A; NovaFold v17, DNASTAR).

Techniques: Sequencing, Mutagenesis, Generated

Journal: International Journal of Molecular Sciences

Article Title: The ALS-Related σ1R E102Q Mutant Eludes Ligand Control and Exhibits Anomalous Response to Calcium

doi: 10.3390/ijms21197339

Figure Lengend Snippet: Effect of σ1R ligands on the interactions of WT σ1R and the E102Q mutant with the NR1-C1 subunit and BiP. Recombinant C0-C1-C2 region of NR1 and BiP were covalently attached to NHS-activated Sepharose® and incubated with WT σ1R or mutant σ1R (100 nM). Unbound σ1R was removed by three cycles of washing/resuspension. The protein–σ1R complexes were incubated for 30 min with rotation at room temperature (RT) in the presence of increasing concentrations of σ1R ligands in a final volume of 300 μL (50 mM Tris-HCl, pH 7.5, and 0.2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)), and 2.5 mM CaCl 2 was used throughout the procedure. Finally, σ1R that remained attached to the NR1 subunit or BiP was resolved by SDS-PAGE and evaluated by immunoblotting. The assays were performed two times, and the samples were analyzed in duplicate for each ligand concentration. Representative blots are shown. Inset, WT σ1R and the E102Q mutant showing position 102 (solid arrows) and the loss of a negative charge in the mutant surrounding the Q residue (dashed arrows) (Novafold v. 17; DNASTAR Inc., Madison, WI, USA).

Article Snippet: Human σ1R is composed of 223 amino acids that form seven helices and ten β-sheets, with the rest of the protein being linear sequences ( A; NovaFold v17, DNASTAR).

Techniques: Mutagenesis, Recombinant, Incubation, SDS Page, Western Blot, Concentration Assay