9 research outputs found
Schematic representation of functionality of YsaN and its regulation by YsaL.
<p>YsaN exists in solution as mixture of monomer and higher order oligomer (dodecamer) and act as Mg <sup>2+</sup> dependent ATPase. The oligomeric form of YsaN is highly active compared to monomeric form. Computational studies predicted YsaL as a putative ATPase regulator. YsaL exists as dimer in solution and form stable heterotrimeric complex with monomeric YsaN. This results in significantly loss of ATPase activity of YsaN, probably due to loss of oligomeric state. The complex is unstable at high salt concentration (>500 mM NaCl). Furthermore, N-terminal (1β20) residues of YsaN are involved in YsaL-YsaN interaction, as revealed from interaction studies of deletion mutants.</p
Strains, plasmid and constructs used in the work.
*<p>ATCC-American Type Culture Collection.</p
Co-Purification, stoichiometry and functional analysis of YsaL-YsaN complex.
<p>(<b>A</b>) SDS-PAGE analysis of YsaL-YsaN complex after co-purification in Ni-NTA affinity chromatography: M-Marker, UI- Uninduced, I- induced, E1 and E2- Ni-NTA eluates of untagged YsaL-YsaN-His complex. (<b>B</b>) Size exclusion chromatography (SEC) profile of untagged YsaL-YsaN-His complex in pre-calibrated Superdex 200 hi-load16/60 column (GE Healthcare). Molecular weight standards are indicated with triangles: Thyroglobulin (T) β669 kDa, Ferritin (F) β440 kDa, Aldolase (A) β158.4 kDa, Ovalbumin (O) β48 kDa, Carbonic anhydrase (C) β29 kDa and Ribonuclease A (R) β14 kDa. SDS PAGE analysis of Gel filtration profile: M β marker, P1 (Peak1) - YsaN-His dodecamer P2 (Peak2) - untagged (UT) YsaL-YsaN-His heterotrimer and P3 (Peak3) - YsaN-His monomer <b>(inset)</b>. (<b>C</b>) Molecular mass estimation from size exclusion profile of untagged YsaL-YsaN-His complex using the known molecular weight standards. Peak1 corresponds to YsaN-His (Dodecamer) - βΌ603 kDa, Peak2 corresponds to heterotrimeric assembly of untagged YsaL-YsaN-His complex (2βΆ1) - βΌ102 kDa and Peak3 corresponds to YsaN-His (monomer) - βΌ49.5 kDa. (<b>D</b>) Chemical crosslinking profile of untagged (UT) YsaL-YsaN-His complex in SDS-PAGE using Sulfo - EGS (EGSS) - Ctrl-Control, C1 and C2-crosslinking of untagged YsaL-YsaN-His using 1.0 mM EGSS incubated for 5 min and 10 min respectively, M-Marker. (<b>E</b>) Relative ATPase activity (%) with increasing molar ratio of refolded YsaL-His/Ye3555 and YsaN-His. Maximum inhibition of ATPase activity occurs in the ratio 2βΆ1 shown by arrow. Relative ATPase activity of untagged YsaL-YsaN-His complex (NL<sub>C</sub>) and YsaN-His incubated with YsaL-His refolded (N +L<sub>HR</sub>) in the molar ratio 2βΆ1. YsaN-His (N) was used as a control <b>(inset)</b>.</p
Binding propensity of YsaL with YsaN wild type and different deletion mutants using Surface Plasmon resonance.
<p>Interactions of untagged (UT) YsaL with (<b>A</b>) YsaN-His (<b>B</b>) YsaN<sub>flr</sub>-His (<b>C</b>) YsaN Ξ<sub> (1β5)</sub> -His (<b>D</b>) YsaN Ξ<sub> (411β430)</sub> -His and (<b>E</b>) YsaN Ξ <sub>(426β430)</sub>-His. Untagged YsaL was used as an analyte in the concentration 5 nM, 10 nM, 20 nM and 50 nM.</p
Purification, stoichiometric characterization and secondary structure analysis of YsaL/Ye3555.
<p>(<b>A</b>) SDS PAGE analysis of refolded YsaL-His after Ni-NTA affinity chromatography: M-Marker, UI- Uninduced, I- induced, E1 and E2- Ni-NTA eluates. (<b>B</b>) Chemical crosslinking profile of refolded YsaL-His in SDS-PAGE using 1.5% Gluteraldehyde - M- Marker Ctrl- Control, C- crosslinking of YsaL-His using 1.5% Gluteraldehyde incubated for 5 min. (<b>C</b>) Size exclusion chromatography (SEC) profile of refolded YsaL-His in pre-calibrated Superdex 200 hi-load16/60 column (GE Healthcare). Molecular weight standards are indicated with triangles: Thyroglobulin (T) β669 kDa, Ferritin (F) β440 kDa, Aldolase (A) β158.4 kDa, Ovalbumin (O) β48 kDa, Carbonic anhydrase (C) β29 kDa and Ribonuclease A (R) β14 kDa. SDS PAGE analysis of Gel filtration profile: M β marker, P1 (peak1) - YsaL-His <b>(inset)</b>. (<b>D</b>) Molecular mass estimation from size exclusion profile of refolded YsaL-His using the above known standards. Refolded YsaL-His was dimeric (βΌ48 kDa). (<b>E</b>) Far UV-CD (Circular dichroism) spectra of refolded YsaL-His (HT) and refolded untagged (UT) YsaL.</p
Identification of critical residues of YsaN for stable YsaL-YsaN complex formation.
<p>(<b>A</b>) Schematic diagram of deletion mutant constructs of YsaN. (<b>B</b>) Far-UV CD spectra of his tagged deletion mutants of YsaN [YsaN Ξ<sub>(1β5)</sub>, YsaN Ξ<sub>(1β20)</sub>, YsaN Ξ<sub>(426β430)</sub>, YsaN Ξ<sub>(411β430)</sub> and YsaN<sub>(21β410)</sub>] compared with full length YsaN (YsaN-His) and refolded YsaN full length (YsaN<sub>flr</sub>-His). (<b>C</b>) Relative ATPase activity (%) of deletion mutants of YsaN with YsaN<sub>flr</sub>-His as a control. (<b>D</b>) Relative ATPase activity (%) of YsaN-His with YsaN<sub>flr</sub>-His. (<b>E</b>) SDS-PAGE profile of Ni-NTA pull-down assay of deletion mutants of YsaN (His tagged) with untagged YsaL; YsaN <sub>Ξ(1β20)</sub> and YsaN<sub> (21β410)</sub> does not bind to untagged (UT) YsaL. M-denotes molecular weight marker.</p
Pfam analysis of BLASTp hits with YscL as a query.
*<p>Belonged to same clan HrpE/YscL/FliH and V-type ATPase subunit E (CL 0255).</p
Oligomerization analysis and enzymatic characterization of YsaN ATPase.
<p>(<b>A</b>) SDS PAGE analysis of YsaN-His after purification by Ni-NTA affinity chromatography: M- Marker, UI- Uninduced, I- induced, E1 and E2- Ni-NTA eluates. (<b>B</b>) Chemical crosslinking profile of YsaN-His in SDS-PAGE using Sulfo - EGS (EGSS) - Ctrl-Control, C1 and C2- crosslinking of YsaN-His using 0.5 mM EGSS incubated for 2 min and 5 min respectively, M-Marker. (<b>C</b>) Size exclusion chromatography (SEC) profile of YsaN-His in pre-calibrated Superdex 200 hi-load16/60 column (GE Healthcare). Molecular weight standards are indicated with triangles: Thyroglobulin (T) β669 kDa, Ferritin (F) β440 kDa, Aldolase (A) β158.4 kDa, Ovalbumin (O) β48 kDa, Carbonic anhydrase (C) β29 kDa and Ribonuclease A (R) β14 kDa. SDS PAGE analysis of Gel filtration profile: P1 (Peak1) - YsaN-His Dodecamer and P2 (Peak2) - YsaN-His monomer, M - marker <b>(inset)</b>. (<b>D</b>) Molecular mass estimation from size exclusion profile of YsaN-His using the above known standards. Peak1 from SEC profile corresponds to βΌ603 kDa (Dodecamer) and peak 2 corresponds to βΌ49.5 kDa (Monomer). (<b>E</b>) Measurement of hydrodynamic diameter of YsaN-His using Dynamic Light Scattering. Hydrodynamic radii (R<sub>H</sub>) of YsaN corresponded to monomer and dodecamer. (<b>F</b>) ATPase activity of Ni-NTA eluates of YsaN-His measured in terms of Phosphate (Pi) released per minute per mg of protein. Hill fit equation provided the kinetic parameters - V<sub>max</sub>, K<sub>0.5</sub> and hill coefficient (n). (<b>G</b>) Relative ATPase activity of YsaN Dodecamer and YsaN Monomer separated after SEC. YsaN mixture (monomer and oligomer) was used as a control.</p
Primers used in the work.
<p>Letters in bold indicate restriction sites for NdeI (sense primer) and XhoI (Antisense primer).</p