17 research outputs found
Computer simulations mimic ITC titration.
<p><b>A.</b> Concentrations of the five species as a function of progress of the titration experiment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036102#pone-0036102-g004" target="_blank">Figure 4A</a> using the K values that were derived from the SEC data. <b>B.</b> ITC experimental data (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036102#pone-0036102-g004" target="_blank">Figure 4A</a>) together with the best fit to the data (see Supplement for details).</p
ITC reveals interaction energetics.
<p>A. A solution of 666 ”M Cu-Atox1 (in the syringe) is titrated into a solution of 52 ”M apoWD4 (in the reaction chamber) at 3°C. B. A solution of 666 ”M apo-Atox1 (in the syringe) is titrated into a solution of 62 ”M apoWD4 (in the reaction chamber) at 3°C. The top plots are the raw data of heats versus time and the bottom plots are integrated heats as a function of molar ratio of Atox1/WD4. Noise estimation based on the data in B predicts uncertainties for individual ITC points of 0.03 kcal/mol.</p
SEC analysis of concentrations of species.
<p>Concentrations of the five species in <i>scheme 1</i> determined from SEC measurements as described in the text using different initial concentrations (1â¶1:1 of Atox1:Cu:WD4) as indicated. Also, the % of the total copper found in hetero-complex is reported. The equilibrium concentrations established are used to derive K<sub>1</sub> and K<sub>2</sub> and from this the copper exchange factor K<sub>1</sub>*K<sub>2</sub> is calculated. Two experiments with 150 ”M starting concentrations are reported. For 75 ”M, also the opposite reaction, mixing Cu-WD4 with apo-Atox1 was performed.</p
Thermodynamic parameters for steps 1 and 2, and overall reaction.
<p>The parameters are based on <i>scheme 1</i> and equilibrium constants from SEC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036102#pone-0036102-t001" target="_blank">Table 1</a>) and ITC data, as described in the text.</p
Illustration of Scheme 1.
<p>Upon mixing Cu-loaded Atox1 (purple) and apo-WD (green), the proteins interact and form a hetero-complex, Atox1-Cu-WD4 (shown with Cu coordinating one Cys in Atox1 and both Cys in WD4; however, there are other possible Cu coordinations in the hetero-complex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036102#pone.0036102-RodriguezGranillo1" target="_blank">[17]</a>), and also products, apo-Atox1 and Cu-WD4, according to the equilibrium constants K<sub>1</sub> and K<sub>2</sub>.</p
SEC probed at 2 wavelengths is used to separate equilibrium species in Scheme 1.
<p><b>A.</b> SEC analysis of a mixture of the two apo proteins (300 ”M each) at 280 and 254 nm. <b>B.</b> SEC analysis of holo Atox1 (300 ”M) at 280 and 254 nm. <b>C.</b> SEC analysis at 280 nm of a mixture of 300 ”M Cu-Atox1 and 300 ”M apo-WD4. For comparison, the 280 nm elution trace for the mixture of the two apo-proteins is shown. <b>D.</b> SEC analysis at 280 nm and 254 nm of a mixture of 300 ”M Cu-Atox1 and 300 ”M apo-WD4. <b>E.</b> De-convolution of the underlying peaks in the elution trace of the Cu-Atox1+apo-WD4 mixture.</p
CD reveals ternary complex.
<p>A. Near-UV CD of apo and holo-forms of Atox1 and WD4. B. Near-UV CD of a 1-to-1 mixture of Cu-Atox1 and apo-WD. For comparison the theoretical CD signal derived for no reaction (i.e., sum of Cu-Atox1 and apo-WD4 signals) and for 100% reaction (i.e., signals for apo-Atox1 and holo-WD4) are also shown. The dotted line is the calculated CD signal for 100% pure heterocomplex at the same concentration (see text).</p
Structural Topology and Activation of an Initial Adenylate KinaseâSubstrate Complex
Enzymatic activity is ultimately defined by the structure,
chemistry,
and dynamics of the Michaelis complex. A large number of experimentally
determined structures between enzymes and substrates, substrate analogues,
or inhibitors exist. However, transient, short-lived encounter and
equilibrium structures also play fundamental roles during enzymatic
reaction cycles. Such structures are inherently difficult to study
with conventional experimental techniques. The enzyme adenylate kinase
undergoes major conformational rearrangements in response to binding
of its substrates, ATP and AMP. ATP is sandwiched between two binding
surfaces in the closed and active enzyme conformation. Thus, adenylate
kinase harbors two spatially distant surfaces in the substrate free
open conformation, of which one is responsible for the initial interaction
with ATP. Here, we have performed primarily nuclear magnetic resonance
experiments on <i>Escherichia coli</i> adenylate kinase
(AK<sub>eco</sub>) variants that allowed identification of the site
responsible for the initial ATP interaction. This allowed a characterization
of the structural topology of an initial equilibrium complex between
AK<sub>eco</sub> and ATP. On the basis of the results, we suggest
that the ATP binding mechanism for AK<sub>eco</sub> is a mixture between
âinduced fitâ and âconformational selectionâ
models. It is shown that ATP is activated in the initial enzyme-bound
complex because it displays an appreciable rate of nonproductive ATP
hydrolysis. In summary, our results provide novel structural and functional
insights into adenylate kinase catalysis
Comparative densitometric analysis of pH and calcium-dependent Yops secretion in <i>Y. pseudotuberculosis</i>.
<p>To compare and quantify the Yop secretion efficiency in wild-type <i>Y. pseudotuberculosis</i> under different conditions, Coomassie stained Yop secretion profiles were subjected to densitometric analysis with Multi Gauge software (Fuji Film). The protein YopJ (boxed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049349#pone-0049349-g004" target="_blank">Figure 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049349#pone-0049349-g005" target="_blank">Figure 5E</a>) was selected for quantitative analysis. Growth kinetics in media with different pHâs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049349#pone.0049349.s004" target="_blank">Figure S4B</a>) showed attenuation of bacterial growth directly linked to the observed Yop secretion efficiency.</p>a<p>secretion efficiency was set to 100%;</p>b<p>secretion efficiency at pH 7.5 was set to 100%.</p
<i>In vivo</i> dissociation and secretion of YscU<sub>CC</sub> in different <i>Yersinia</i> strains.
<p>Calcium dependent regulation of Yop and YscU<sub>CC</sub> secretion in wild-type <i>Y. pseudotuberculosis</i>, in a Î<i>ysc</i>C mutant and Î<i>ysc</i>N mutant strain without and with <i>in trans</i> complementation of YscU<sub>CC</sub>. Bacteria transformed with empty pBADmycHis B (pBAD), or pBAD with one additional <i>yscU<sub>CC</sub></i> copy (pBAD(YscU<sub>CC</sub>)), were grown for 2 h at 26°C and 3 h at 37°C in calcium depleted (â) or calcium supplemented (+) medium. The expression of <i>yscU<sub>CC</sub></i> was induced by addition of arabinose. Yop secretion is coupled to the secretion of YscU<sub>CC</sub> in all analysed <i>Yersinia</i> strains and required a functional T3SS. Secreted Yops visualized on Coomassie stained PAGE gels; YscU<sub>CC</sub> visualized on immunoblots with anti-YscU<sub>CC</sub> peptide antibodies. âpelletâ indicates intracellular proteins; âsupernatantâ denotes secreted proteins. The YopJ protein (black box) was subjected to densitometric analysis for quantification of secretion levels (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049349#pone-0049349-t002" target="_blank">Table 2</a>).</p