Results: 5

1.
Figure 2

Figure 2. Saxiphilin c-lobe characterization using MALDI-TOF MS. From: Dynamics of Saxitoxin Binding to Saxiphilin C-lobe Reveals Conformational Change.

Expected c-lobe sequence. Underlined residues confirmed by Edman degradation; italicized residues are the signal tag (a). MALDI-TOF mass spectrum of intact (purified) c-lobe (MH+) obtained using 2,5-dihydroxybenzoic acid (DHB) matrix (b). MALDI-TOF peptide mass fingerprints obtained using DHB and α-cyano-4-hydroxycinnamic acid (HCCA) (c and d, respectively), with assignment of peaks corresponding to expected tryptic fragments.

Penny Lewis, et al. Toxicon. ;51(2):208-217.
2.
Figure 1

Figure 1. Secreted saxiphilin c-lobe, present in stainable quantities in the media, is purified to homogeneity. From: Dynamics of Saxitoxin Binding to Saxiphilin C-lobe Reveals Conformational Change.

Saxiphilin c-lobe that has been secreted into the media is present in stainable quantities after 6 days of expression (a, Coomassie, indicated by arrow). High Five™ insect cells were transformed with plasmid lacking (a, −C-lobe) or containing (a, +C-lobe) the coding sequence for His6-tagged SAX c-lobe. Transformed cells were grown for 6 days. An equal volume of media from each population was examined by electrophoresis using 12.5% SDS-PAGE and stained (Coomassie) or blotted and probed with antibody against saxiphilin c-lobe or c-terminal His6. (b) c-lobe purified by nickel affinity chromatography as described in Materials and Methods was analyzed by electrophoresis using 12.5% SDS-PAGE and either stained (b, Coomassie) or blotted and probed with antibody to c-lobe (b, Anti-SAX).

Penny Lewis, et al. Toxicon. ;51(2):208-217.
3.
Figure 4

Figure 4. From: Dynamics of Saxitoxin Binding to Saxiphilin C-lobe Reveals Conformational Change.

(a) van’t Hoff analysis of saxitoxin:c-lobe interaction. Thermodynamic values were determined from a plot of equilibrium association constants calculated from kinetic data for each temperature using the van’t Hoff equation: ln KD = −(ΔH/R)(1/T) + (ΔS/R) in which the term ΔH/R corresponds to the slope of the van’t Hoff plot. Values of ΔG were calculated from dissociation constants at each temperature using the equation: ΔG = −RT ln KD in which R and T have their usual meanings. The value of ΔS at 273 K was then calculated from ΔH and ΔG using the relationship ΔG = ΔH − T ΔS. Error bars based on one standard deviation lie within the markers on the plot. (b) Plot of ΔG of saxitoxin:c-lobe dissociation vs temperature. Error bars are set at ±0.1 kcal/mol. The midpoint temperature (402 K), where KD = 1, was determined from the least squares fit of the data.

Penny Lewis, et al. Toxicon. ;51(2):208-217.
4.
Figure 3

Figure 3. SPR analysis of saxitoxin:c-lobe interaction shows saxitoxin concentration-dependent response at multiple temperatures. From: Dynamics of Saxitoxin Binding to Saxiphilin C-lobe Reveals Conformational Change.

Sensorgrams collected at 11, 17, 25, 31, and 37 °C. The concentrations of toxin were 250, 50, and 10 nM, and are illustrated in that order from top to bottom at each temperature. Each toxin sample was flowed over biotinylated c-lobe captured on an immobilized-streptavidin chip. 20 mM sodium phosphate, 150 mM NaCl, pH 7 (buffer A) containing saxitoxin was flowed over the surface beginning at time 0 s and was switched to buffer A lacking toxin only at 60 s. N = 5 for each data set. Smooth curves (which are sometimes obscured by experimental traces) represent the best fit of the binding responses to a 1:1 interaction model.

Penny Lewis, et al. Toxicon. ;51(2):208-217.
5.
Figure 5

Figure 5. Fluorescence intensity of ANS increases and shifts wavelength in the presence of c-lobe and c-lobe with saxitoxin. From: Dynamics of Saxitoxin Binding to Saxiphilin C-lobe Reveals Conformational Change.

(a) Fluorescence intensity of 160μM ANS (dotted line) increases from 335 to 772 (130%), with concomitant shift of λmax from 513 nm to 494 nm, in the presence of 10 μM c-lobe (dashed line). When 10 μM STX is also present (solid line) a further increase in fluorescence is observed (Fmax = 952). Control experiments under identical conditions but without c-lobe showed no change in ANS fluorescence; also, no fluorescence was observed from either c-lobe or saxitoxin. Data are an average of seven scans. λex = 390 nm; Buffer = 20 mM Na2HPO4, 150 mM NaCl, pH 7. (b) ANS associated with c-lobe in the saxitoxin binding site would be displaced by the natural ligand, saxitoxin, resulting in an ANS fluorescence spectrum like that of ANS in the absence of c-lobe. This effect is not observed. (c) ANS associated with c-lobe in a hydrophobic patch exterior to the STX binding site remains associated with c-lobe when saxitoxin is introduced, consistent with data shown in Figure 5a.

Penny Lewis, et al. Toxicon. ;51(2):208-217.

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk