Journal: Biochimica et biophysica acta. General subjects
Article Title: Mutational analysis of TlyA from Brachyspira hampsonii reveals two key residues conserved in pathogenic bacteria responsible for oligomerization and hemolytic activity.
doi: 10.1016/j.bbagen.2021.130045
Figure Lengend Snippet: Fig. 1. TlyA from Brachyspira hampsonii possesses hemolytic activity and rRNA methyltransferase activity. A, homology model of TlyA from Brachyspira hampsonii. Homology modelling was carried out using the phyre2 server [34] utilizing a putative hemolysin from Streptococcus thermophilus (PDB ID 3HP7) as a model. Amino acid residues comprising the N-terminal S4 domain are coloured red, amino acid residues comprising the Rossman-like methyltransferase fold are coloured blue, and amino acids mutated in this study (S9, C27, H40, C80, and C93) are labelled and coloured cyan. B, purified Brachyspira hampsonii TlyA-His protein visualized by Coomassie blue staining (CB) and western blotting utilizing an anti-His-tag antibody (WB). Sizes of molecular weight markers (M) are indicated. C, far-UV circular dichroism spectrum of TlyA-His protein. D, hemolytic activity of TlyA-His. Purified TlyA-His protein was incubated with an equal volume of a 2% (v/v) pig erythrocyte suspension for 18–24 h at room temperature, after which point hemolysis was quantified by spectrophotometric measurement of hemoglobin release into the supernatant. Percent hemolysis was calculated by comparison to values obtained from a pig erythrocyte suspension in PBS (0% hemolysis) and a suspension lysed in distilled water (100%) hemolysis. Hemolytic activity was inhibited by addition of 10 mM β-mercaptoethanol to reaction mixtures (grey bars). Data are presented as mean + SEM (n = 10 per group). TlyA protein constructs were expressed in E. coli and purified from the soluble fraction by Ni2+ IMAC chromatography. E, estimation of TlyA-His pore size. TlyA-His was incubated with a 2% pig erythrocyte suspension and various osmoprotectants (mannitol/PEG 300/400/600/1000/3000/6000). Osmoprotectants between 1.88 and 2.88 nm in diameter (PEG 1000/3000) blocked hemolysis to a significant degree, while a 5.00 nm diameter osmoprotectant (PEG 6000) completely blocked hemolysis (One Way-Anova, Holm-Sidak Post-hoc, *** indicates significant difference from TlyA-His with no osmoprotectants at p < 0.001, n = 10 per group, data are presented as mean ± SEM). F, rRNA methyltransferase activity of TlyA-His. TlyA-His could incorporate a 3H labelled methyl group into E. coli rRNA (Student’s t-test, *** indicates significance at p < 0.001, n = 4 per group, data are presented as mean + SEM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Article Snippet: Insoluble cell debris was pelleted by centrifugation, and the soluble fraction was loaded onto an equilibrated XK 26/20 column (GE Healthcare) packed with Ni2+ charged Profinity Ni2+ IMAC resin (Bio-Rad).
Techniques: Activity Assay, Purification, Staining, Western Blot, Molecular Weight, Circular Dichroism, Incubation, Suspension, Comparison, Construct, Chromatography, Pore Size
Bio-Rad is a verified supplier 
![Fig. 1. TlyA from Brachyspira hampsonii possesses hemolytic activity and rRNA methyltransferase activity. A, homology model of TlyA from Brachyspira hampsonii. Homology modelling was carried out using the phyre2 server [34] utilizing a putative hemolysin from Streptococcus thermophilus (PDB ID 3HP7) as a model. Amino acid residues comprising the N-terminal S4 domain are coloured red, amino acid residues comprising the Rossman-like methyltransferase fold are coloured blue, and amino acids mutated in this study (S9, C27, H40, C80, and C93) are labelled and coloured cyan. B, purified Brachyspira hampsonii TlyA-His protein visualized by Coomassie blue staining (CB) and western blotting utilizing an anti-His-tag antibody (WB). Sizes of molecular weight markers (M) are indicated. C, far-UV circular dichroism spectrum of TlyA-His protein. D, hemolytic activity of TlyA-His. Purified TlyA-His protein was incubated with an equal volume of a 2% (v/v) pig erythrocyte suspension for 18–24 h at room temperature, after which point hemolysis was quantified by spectrophotometric measurement of hemoglobin release into the supernatant. Percent hemolysis was calculated by comparison to values obtained from a pig erythrocyte suspension in PBS (0% hemolysis) and a suspension lysed in distilled water (100%) hemolysis. Hemolytic activity was inhibited by addition of 10 mM β-mercaptoethanol to reaction mixtures (grey bars). Data are presented as mean + SEM (n = 10 per group). TlyA protein constructs were expressed in E. coli and purified from the soluble fraction by <t>Ni2+</t> <t>IMAC</t> chromatography. E, estimation of TlyA-His pore size. TlyA-His was incubated with a 2% pig erythrocyte suspension and various osmoprotectants (mannitol/PEG 300/400/600/1000/3000/6000). Osmoprotectants between 1.88 and 2.88 nm in diameter (PEG 1000/3000) blocked hemolysis to a significant degree, while a 5.00 nm diameter osmoprotectant (PEG 6000) completely blocked hemolysis (One Way-Anova, Holm-Sidak Post-hoc, *** indicates significant difference from TlyA-His with no osmoprotectants at p < 0.001, n = 10 per group, data are presented as mean ± SEM). F, rRNA methyltransferase activity of TlyA-His. TlyA-His could incorporate a 3H labelled methyl group into E. coli rRNA (Student’s t-test, *** indicates significance at p < 0.001, n = 4 per group, data are presented as mean + SEM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_5264/pm34715264/pm34715264__page4_image1.jpg)