PpiA and PpiB have hydrophobic patches on the surface and are present in oligomeric form in solution.

<p><b>A.</b> Green color shows the hydrophobic patches on the surface of protein structures of PpiA and PpiB, blue, red and limon yellow colors show negative, positive and polar residues, respectively. <b>B.</b> Elution profile of Gel Filtration Chromatography: Green color indicates elution profile of rPpiA and blue indicates elution profile of rPpiB.</p

Purified rPpiases of <i>M</i>.<i>tb</i> are enzymatically active.

<p><b>A.</b> Isomerization activity of rPpiA and rPpiB at a concentration of 50nM was measured in a coupled assay using the chromogenic peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and α chymotrypsin compared with the spontaneous background rate of cis-trans isomerization in the absence of the recombinant enzymes. <b>B.</b> Molecular weight of purified histidine-tagged rPpiA and GST tagged rPpiB as checked on 10% SDS polyacrylamide gel was around 25kDa and 69kDa, respectively.</p

rPpiases protected <i>Nde</i>1 is refractile to thermal denaturation.

<p>Enzymatic activity of thermal denatured <i>Nde</i>1 was assayed in presence and absence of rPpiA, rPpiB and control protein BSA. Lane M, 1-kb molecular size marker; lane 1, uncut pET22b; lane 2, pET22b digested with native <i>Nde</i>1; lane 3, with heat denatured <i>Nde</i>1; lane4, with rPpiA treated heat denatured <i>Nde</i>1; lane 5, with rPpiB treated heat denatured <i>Nde</i>1; lane 6, with BSA treated heat denatured <i>Nde</i>1.</p

ANS Florescence spectra reveal surface hydrophobicity in <i>M</i>.<i>tb</i> rPpiases.

<p>Concentration of ANS, rPpiA and rPpiB used were 20μM, 0.1 mg/ml and 0.1mg/ml, respectively. Blue shift in the position of peak and increase in the intensity of peak was observed upon addition of rPpiA and rPpiB. The ANS emission was scanned in the range of 400 to 600 nm.</p

<i>Mycobacterium tuberculosis</i> Peptidyl-Prolyl Isomerases Also Exhibit Chaperone like Activity <i>In-Vitro</i> and <i>In-Vivo</i>

<div><p>Peptidyl-prolyl cis-trans isomerases (Ppiases), also known as cyclophilins, are ubiquitously expressed enzymes that assist in protein folding by isomerization of peptide bonds preceding prolyl residues. <i>Mycobacterium tuberculosis</i> (<i>M</i>.<i>tb</i>) is known to possess two Ppiases, PpiA and PpiB. However, our understanding about the biological significance of mycobacterial Ppiases with respect to their pleiotropic roles in responding to stress conditions inside the macrophages is restricted. This study describes chaperone-like activity of mycobacterial Ppiases. We show that recombinant rPpiA and rPpiB can bind to non-native proteins <i>in vitro</i> and can prevent their aggregation. Purified rPpiA and rPpiB exist in oligomeric form as evident from gel filtration chromatography.<i>E</i>. <i>coli</i> cells overexpressing PpiA and PpiB of <i>M</i>.<i>tb</i> could survive thermal stress as compared to plasmid vector control. HEK293T cells transiently expressing <i>M</i>.<i>tb</i> PpiA and PpiB proteins show increased survival as compared to control cells in response to oxidative stress and hypoxic conditions generated after treatment with H₂O₂ and CoCl₂ thereby pointing to their likely role in adaption under host generated oxidative stress and conditions of hypoxia. The chaperone-like function of these <i>M</i>.<i>tuberculosis</i> cyclophilins may possibly function as a stress responder and consequently contribute to virulence.</p></div

<i>M</i>.<i>tb</i> rPpiases suppress aggregation of MalZ.

<p>The aggregation pattern was monitored by light scattering at O.D. 500 nm with excitation and emission slit width 5 and 2.5 nm, respectively. GroEL was used as a positive control. Lysozyme was used as a negative control. <b>A.</b> Increasing molar ratio of rPpiA (10, 20, 40) was used. <b>B</b>. Increasing molar ratio of rPpiB (5,10, 20) was used.</p

rPpiases can rescue <i>E</i>.<i>coli</i> from thermal shock: <i>E</i>.<i>coli</i> expression strain was transformed with Ec_VCy, pGEX6p-1 only, Ec_ppiA and Ec_ppiB.

<p>After heat treatment at 50^°C different dilutions of 100ul culture was plated at one hour interval. The quantification of bacterial growth was carried out by counting the bacterial colony forming unit (cfu).<i>E</i>. <i>coli</i> transformed with <i>ppiA</i> and <i>ppiB</i> exhibited approximately 10 fold more survival compared with vector control.</p

Electron microscopic observation and analysis of HpDnaBWt, DelN2, DelN3 and DelC1

<p><b>Copyright information:</b></p><p>Taken from "The domain structure of DnaB helicase: the N-terminal domain can be dispensable for helicase activity whereas the extreme C-terminal region is essential for its function"</p><p></p><p>Nucleic Acids Research 2007;35(9):2861-2874.</p><p>Published online 11 Apr 2007</p><p>PMCID:PMC1888833.</p><p>© 2007 The Author(s)</p> Above four proteins were processed for electron microscopy as described in the materials and methods and individual sample was scanned under a Morgagni 268 transmission electron microscope at 80 kV voltage. More than hundred images were captured in each case and the raw images were processed using IMAGIC software. The left panel in each pair shows the unprocessed image and the right panel shows the processed image. HpDnaBWt was found in both C6 and C3 conformations whereas DelN2 was found only in C3 conformations. Bars in the panels are equivalent to 10 nm

() Far-UV circular dichroism spectra of wild-type and different mutant variants of the helicase

<p><b>Copyright information:</b></p><p>Taken from "The domain structure of DnaB helicase: the N-terminal domain can be dispensable for helicase activity whereas the extreme C-terminal region is essential for its function"</p><p></p><p>Nucleic Acids Research 2007;35(9):2861-2874.</p><p>Published online 11 Apr 2007</p><p>PMCID:PMC1888833.</p><p>© 2007 The Author(s)</p> CD spectra were measured for each protein in a 2 mm path length quartz cell and data were collected at 1.0 nm wavelength resolution. The arrowheads indicate the CD spectra of the respective protein. () Intrinsic fluorescence spectra of wild-type and different deletion mutants. The fluorescence emission spectra of wild-type and different mutant forms of HpDnaB (as indicated) were recorded from 300 to 400 nm at 25°C. The excitation wavelength was 278 nm and data were collected at 0.5 nm wavelengths resolution. The arrowheads indicate the CD spectra of the respective protein

ATPase activity of the wild-type and different deletion mutant proteins

<p><b>Copyright information:</b></p><p>Taken from "The domain structure of DnaB helicase: the N-terminal domain can be dispensable for helicase activity whereas the extreme C-terminal region is essential for its function"</p><p></p><p>Nucleic Acids Research 2007;35(9):2861-2874.</p><p>Published online 11 Apr 2007</p><p>PMCID:PMC1888833.</p><p>© 2007 The Author(s)</p> ATPase assay using various HpDnaB forms. Enzyme-linked ATPase assays were performed using protocol as described in the materials and methods either in the absence or presence of DNA. The rates of the reactions against different substrate (ATP) concentrations were plotted for HpDnaBWt, HpDnaBDelN1, HpDnaBDelN2 and HpDnaBDelN3 as shown in panels A, C, E and G, respectively. Lineweaver-Burk plots of the same enzymes were also plotted either in the absence or presence of DNA in each case (panels B, D, F and H, respectively). Maximum velocity of the reaction (Vmax) and turn-over number (Kcat) were calculated in each case (as shown in )