Journal: The Journal of Biological Chemistry

Article Title: The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species

doi: 10.1074/jbc.RA117.001145

Figure Lengend Snippet: Clotrimazole spectral binding assay. A, stepwise clotrimazole addition induced a type-II spectral binding perturbation in rat CYP24A1 at ligand concentrations between 0.05 μm (blue trace) and 3.0 μm (black trace). B, the difference in absorbance between A432 and A411 is plotted against ligand concentration. The data are fitted to a sigmoidal binding curve. Error bars, S.D.

Article Snippet: At induction, growth medium was augmented with clotrimazole (Gold Biotechnology) to a final concentration of 100 μ m . Clotrimazole was reintroduced during lysis and maintained at 20 μ m in all purification buffers.

Techniques: Binding Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species

doi: 10.1074/jbc.RA117.001145

Figure Lengend Snippet: Peak broadening of the [15N]Adx HSQC spectra upon the addition of CYP24A1. A, overlay of [15N]Adx alone (gray) and with increasing amounts of human CYP24A1 (clotrimazole) corresponding to the red and blue spectra, respectively. All spectra are shown at an equivalent contour level. B, peak broadening of the [15N]Adx backbone resonances quantified as ratios of free [15N]Adx/[15N]Adx in the presence of CYP24A1 from human (top), rat (middle), and opossum (bottom). The red markers represent residues broadened beyond one S.D. from the mean. Error bars, S.D.

Article Snippet: At induction, growth medium was augmented with clotrimazole (Gold Biotechnology) to a final concentration of 100 μ m . Clotrimazole was reintroduced during lysis and maintained at 20 μ m in all purification buffers.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species

doi: 10.1074/jbc.RA117.001145

Figure Lengend Snippet: Differential line broadening of [15N]Adx side chain amides. A, distribution of Gln and Asn residues of Adx in relation to the conserved acidic residues (Protein Data Bank code 1CJE) (3). B, line-broadening pattern of [15N]Adx side chain resonances upon titration with unlabeled CYP24A1 corresponding to clotrimazole-bound human, rat, and opossum P450 isoforms. Red arrows indicate broadening resulting in ratios that are greater than one root mean square S.D. from the mean. Error bars, S.D.

Article Snippet: At induction, growth medium was augmented with clotrimazole (Gold Biotechnology) to a final concentration of 100 μ m . Clotrimazole was reintroduced during lysis and maintained at 20 μ m in all purification buffers.

Techniques: Titration

Journal: The Journal of Biological Chemistry

Article Title: The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species

doi: 10.1074/jbc.RA117.001145

Figure Lengend Snippet: Effect of [15N]Adx charge-neutralizing mutations on the CYP24A–Adx complex formation. Amino acid intensity ratios representing broadening of the backbone [15N]Adx resonances for wildtype and all charge neutralizing mutations were examined. Data are shown for each mutant in a 1:0.25 molar ratio with human (A), rat (B), and opossum (C) CYP24A1 bound to clotrimazole. An increase in the intensity ratio reflects loss of line broadening for each redox complex, consistent with disruption of the CYP24A1–Adx interaction. Error bars, S.D.

Article Snippet: At induction, growth medium was augmented with clotrimazole (Gold Biotechnology) to a final concentration of 100 μ m . Clotrimazole was reintroduced during lysis and maintained at 20 μ m in all purification buffers.

Techniques: Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species

doi: 10.1074/jbc.RA117.001145

Figure Lengend Snippet: 15N HSQC spectra of the E65Q side chain resonances upon binding to human, rat, and opossum CYP24A1 (clotrimazole). 2D resonances corresponding to the Asn36 and Asn37 resonances maintain observable intensity when bound to human and rat CYP42A1 (clotrimazole) (top two panels). However, upon complex formation with opossum CYP24A1 (clotrimazole) (bottom), the Asn36 side chain undergoes significantly enhanced broadening (dashed lines indicate the expected location of these resonances) concurrent with an increase in intensity at the Asn75 side-chain resonances. All spectra are displayed at a normalized contour level.

Article Snippet: At induction, growth medium was augmented with clotrimazole (Gold Biotechnology) to a final concentration of 100 μ m . Clotrimazole was reintroduced during lysis and maintained at 20 μ m in all purification buffers.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The cytochrome P450 24A1 interaction with adrenodoxin relies on multiple recognition sites that vary among species

doi: 10.1074/jbc.RA117.001145

Figure Lengend Snippet: Modulation of the opossum CYP24A1 complex with the E65Q mutation of [15N]Adx. Redistribution of peak broadening was observed for the complex between opossum CYP24A1 (clotrimazole) and [15N]Adx harboring the E65Q substitution on helix 2. A, intensity ratios for the [15N]Adx E65Q side chains reflect a non-uniform pattern of broadening in which the Asn36 side chain has become significantly more broadened along with that of Asn13 (red asterisks) combined with a loss of line broadening at the Asn75 side chain. In the mutant 15N HSQC, only one of the Gln61 side chains (located one helical turn from the mutated site) is detectable. B, the non-uniform line broadening was also observed along the backbone resonances of [15N]Adx E65Q, in which N- and C-terminal β strands underwent enhanced broadening compared with wildtype [15N]Adx, particularly at residues His10 and Thr20, whereas the helix-3 resonances were concurrently less broadened. C, mapping of the more affected sites, consisting of the His10 and Thr20 backbone amides along with the Asn36 and Asn13 side chain amides, forms a nearly contiguous surface on E65Q Adx. Error bars, S.D.

Article Snippet: At induction, growth medium was augmented with clotrimazole (Gold Biotechnology) to a final concentration of 100 μ m . Clotrimazole was reintroduced during lysis and maintained at 20 μ m in all purification buffers.

Techniques: Mutagenesis