Fig 1: Secondary structure of linker histone H1 variants H1d, H1t, and HILS1. a Schematic representation of Hils1 gene on rat chromosome 10q31 located within the intron 9 of the α-sarcoglycan gene (Sgca). HILS1 protein consists of 169 amino acid and has three predicted domains similar to other linker histones that include N-terminal domain (NTD: 1–42 amino acid residues), globular domain (GD: 43-115 amino acid residues), and C-terminal domain (CTD: 116-169 amino acid residues). b Over expression of His-tagged HILS1 protein after induction with 0.1 mM IPTG. Lane 1 and lane 2 represents total cell extract from un-induced and 0.1 mM IPTG induced E. coli culture for 12 h at 18 °C, respectively. Protein expression was confirmed by western blotting analysis with anti-His (H1029, Sigma, WB: 1:1000) antibodies. c Coomassie-stained image of a 12% SDS-polyacrylamide gel depicting fractions collected at different steps of purification of recombinant HILS1 (rHILS1) by Ni-NTA affinity column chromatography. E. coli Rosetta(DE3)pLysS cells harboring the plasmid pHILS1 were induced with 0.1 mM IPTG for 12 h and purified as described in methods. Lane 1 represents total cell extract of E. coli Rosetta(DE3)pLysS cells expressing HILS1, lane 2 represents proteins not bound to Ni-NTA column, lanes 3, 4 and 5 represent proteins eluted with elution buffer containing 30, 50 and 80 mM imidazole, respectively. HILS1 protein migrates at ~ 25 kDa (lane 5). d Western blot analysis of purified HILS1 protein showing a single band at ~ 25 kDa with anti-HILS1 antibodies. e Coomassie-stained image of a 12% SDS-polyacrylamide gel showing purified recombinant His-tagged rat H1d, H1t, and HILS1 proteins. Rat H1d and H1t proteins were purified using Ni-NTA agarose column followed by binding with heparin agarose and HILS1 protein was purified by one-step Ni-NTA affinity chromatography as described in methods. f Sequence alignment of rat linker histones H1d, H1t, and HILS1, aligned by Clustal Omega software. Globular domain has been highlighted in red. S/TPXK motif in the CTD has been highlighted in orange. Rat H1d possesses four S/TPXK motifs, rat H1t has two, whereas rat HILS1 lacks S/TPXK motif in the CTD. g–i, Circular dichroism spectra of rat recombinant linker histones H1d, H1t, and HILS1, respectively. A 200 µg/ml solution of purified histone H1 subtypes was used for recording the CD spectrum in a Jasco J-810 spectropolarimeter. The spectra were recorded in 10 mM sodium phosphate buffer, pH 7.5; 10 mM sodium phosphate buffer, pH 7.5 containing 1 M NaCl; and 10 mM sodium phosphate buffer, pH 7.5 containing 60% trifluoroethanol. j Summary of α-helicity induced in rat H1d, H1t, and HILS1 in different buffers