Fig 1.
Urea induced denaturation of Ace and Ace complexed with CaCC inhibitors (1:1 molar ratio) at 25°C.
The protein concentration used was 5 μM in 0.1 M potassium phosphate buffer pH 8.0. The unfolding was measured from the ratio of fluorescence intensities (F340/F350) after exciting the protein at 295 nm at different urea concentrations.
Table 1.
Urea induced unfolding of Ace and Ace complexed with various CaCC inhibitors†.
Fig 2.
Far-UV CD of Ace and Ace complexed with CaCC inhibitors.
Spectra were measured in 0.1 M potassium phosphate buffer (pH 8.0) with protein-ligand molar ratio of 1:1 at 25°C.
Table 2.
Secondary structural content of native Ace and Ace-CaCC inhibitor complexes†.
Fig 3.
Temperature induced unfolding, measured using CD spectra, of Ace and Ace complexed with CaCC inhibitors.
Fig 4.
(A) Quenching of tryptophan fluorescence of Ace with CaCC inhibitors. (B) log [(Fo –Fc)/Fc] versus log [ligand] plot for the fluorescence quenching of Ace with CaCC inhibitors.
Table 3.
Parameters of CaCC inhibitors binding to Ace obtained from tryptophan fluorescence quenching.
Fig 5.
Effects of CaCC inhibitors on basal and Ace-stimulated short circuit current (Isc) in mouse tissue by Ussing chamber experiment, as described in the Materials and Methods.
Basal (unstimulated) and Ace stimulated Isc was measured in the absence (control) and presence of (A) CaCCinh-A01 and (B) tannic acid. Two different concentrations of small molecules, low (5 μM) and high (100 μM), were used in each case and pre-incubated luminally before being treated with Ace toxin to the luminal side of the tissue. Results represent mean ± SEM of 6 tissue pairs; NS indicates the difference is not significant.
Fig 6.
Effect of CaCC inhibitors on Ace stimulated intestinal fluid accumulation.
Representative mouse ileal loops 6 h after luminal injection with Ace in the presence or absence of (A) DGA and CaCCinh-A01 and (B) tannic acid in 1:1 molar ratio (left panel). The right panel provides the bar graph showing the quantified averaged fluid accumulation in the loop experiment (n = 5–10). Values are mean ± SEM.
Fig 7.
Structures of (A) Ace alone, and (B) complexed with DGA and (C) CaCCinh-A01; in each case the left panel indicates the start and the right panel the end of simulation time period.
The A subunit of the Ace protein is shown in green cartoon and the B subunit is in blue: tryptophan residues 10, 17 and 38 are shown in red sticks. DGA is represented as cyan sticks (B), and CaCCinh-A01 as magenta sticks (C).
Table 4.
Binding energetics of DGA and CaCCinh-A01 with Ace from MD simulation†.
Fig 8.
The backbone (A) root mean square deviations (RMSD) and (B) root mean square fluctuation (RMSF) of uncomplexed Ace along with the protein bound to DGA and CaCCinh-A01.
Both the subunits are considered in (A); in (B) residue numbers 1–96 correspond to chain A, and 97–192 to chain B.