PMC

SAXS analysis for cortactin repeats (cortactinCRH). (A) Intensity profiles for small angle scattering of two concentrations of cortactinCRH. 1.1 mg/mL (blue) and 0.4 mg/mL (red) samples. (B) Linearity of the Guinier plots. Manual selection of the Guinier region is shown. (C) Dimensionless Kratky and (D) Perod-Debye plots indicate the profile of an intrinsically disordered protein.

Hydrogen-deuterium exchange mass spectrometry for cortactinCR. Percentage of deuterium uptake is indicated for HDX incubation periods ranging from 30 s to 240 min. Minimal changes in deuterium uptake are observed over the time course suggesting a minimal hydrophobic core for cortactinCR, and that the protein is largely unprotected and in an unfolded state. Alternating orange and black sequences indicate cortactin repeats.

Cortactin repeats and helical domain do not homo-oligomerize. (A) Schematic diagram of the defined domains of cortactin. Abbreviations: NTA, amino-terminal acidic region; H, helical domain; SH3, Src-homology 3. Cortactin repeats 1 through 6 are indicated. The cortactin regions included in constructs cortactinCR and cortactinCRH are indicated. (B) Amino acid sequence of the 6½ cortactin repeats shown. (C) SEC-MALS for cortactinCRH (green) and cortactinCR (blue). Predicted molecular masses for monomeric proteins including N-terminal vector derived residues are 36.5 kDa and 27.4 kDa for cortactinCRH and cortactinCR. SEC-MALS observed experimental molecular masses are 43.9 (±1.2%) kDa and 29.9 (±1.5%) kDa for cortactinCRH and cortactinCR, respectively.

Structure of the cortactin repeats. (A) Normalized pair distribution function P(r) for cortactinCRH calculated with GNOM. 1.1 mg/mL (blue) and 0.4 mg/mL (red) samples. (B) Ab initio models of CortactinCRH show extensive conformational diversity (blue). The averaged model (orange) is elongated. Models were calculated by use of DAMMIF. (C) Calculated R _g values for the cortactinCRH sequence. Relative frequency of calculated R _g values from analysis of 100,000 molecular models of cortactinCRH as an unfolded protein based on its sequence by use of the program Flexible-Meccano. Frequency of calculated R _g values (green) is compared to the observed R _g for cortactinCRH. The experimental R _g of cortactinCRH falls at the distribution peak of the calculated range.

Circular Dichroism for the cortactin repeat domain. (A) Far UV CD spectrum for cortactinCR at 4 °C (solid line) shows a negative peak at 202 nm, but no features that could be interpreted as α-helical or β-sheet. A red shift of ~2 nm occurs on increase in temperature from 4 °C to 90 °C (dashed line). (B) Temperature dependence of molar ellipticity from 4 °C to 90 °C monitored at 202 nm does not show a melting point typical of folded proteins. (C) CD spectra for a well folded α-helical control protein, CCM3, at 4 °C and 90 °C. Secondary structure is lost at 90 °C. (D) Melting point analysis for CCM3 shows that this control protein melts between 60 °C and 70 °C. (E) Analysis of [θ]₂₂₂ vs [θ]₂₀₀ for cortactinCR. Plotting [θ]₂₂₂ vs [θ]₂₀₀ suggests that cortactinCR is falls into the coil-like unfolded protein class and not the pre-molten globule class. Analysis based on.