Bunits. Residues present in the crystal structures but not present in our expression constructs were deleted for consistency. For EOM analysis, the 14 amino acid linker (residues 171?84; Figure 1A) was modeled with RANCH [37]. Data were ?cut off at qmax = 0.23 A21. Theoretical scattering curves for the two subunits were obtained with CRYSOL [39]. Superpositions of models were performed in SUPCOMB with no symmetry (P1) applied; superpositions did not exclude fits rotated by two-fold rotation [40]. Porod-Debye analysis was performed, with scatter??ing data in the range of qmin = 0.1222 A21 to qmax = 0.156 A21 plotted as q46I(q) vs q4 used for linear fitting [33].(ILK-pKD) [11?3] (Figure 1A). ILK expression constructs containing the pseudokinase domain are largely insoluble when expressed alone in E. coli; however, co-expression with a-parvinCH2 results in a soluble protein complex [[9] and data not shown]. Therefore, we co-expressed full-length ILK as a GSTfusion protein and a-parvin-CH2 as a His-tagged protein in E. coli by co-transformation of compatible expression vectors under dual selection, which rescues ILK protein from insolubility (data not shown). Codon-optimized synthetic cDNA MNS site encoding ILK showed improvement of total yield over native cDNA, and was used in subsequent purifications (Figure 1B). His-tagged PINCH1-LIM1, which is soluble in E. coli, was expressed alone, and cells mixed with the ILK/a-parvin-CH2 expressing cells prior to co-sonication. The IPPmin complex was affinity purified on glutathioneagarose beads, eluted, and the GST- and (His)- tags simultaneously removed by TEV proteolysis (Figure 1C). IPPmin complex was then purified by anion exchange chromatography using a shallow gradient, which allowed for differential 370-86-5 elution of GST and IPPmin. Remaining GST protein was then removed by incubation with glutathione-agarose beads and size-exclusion chromatography to achieve purity of crystallization quality, and immunoblot analysis confirms the presence of ILK in the 22948146 complex (Figure 1D). Notably, the IPPmin complex remains intact during all steps of purification. The migration of ILK, a-parvin-CH2 and PINCH-1LIM1 on SDS-PAGE is consistent with their expected molecular weights (51.4 kDa, 14.7 kDa, and 7.5 kDa, respectively). Purified IPPmin protein resolved on a calibrated gel-filtration column results in a single peak with an elution volume corresponding to a molecular weight of approximately 80 kDa, in close agreement with the expected molecular weight of 73.6 kDa (Figure 1E). The purified IPPmin complex migrates as a single species on native gel electrophoresis and its migration is unchanged upon addition of excess purified PINCH1-LIM1 and/or a-parvin-CH2 proteins, confirming that the purified IPPmin complex is stable and monodisperse in solution (Figure 1F).SAXS analysis of IPPmin complexTo study the overall conformation of the IPP in solution, we collected SAXS data on a concentration series of IPPmin complex protein (Figure 2A). First, we analyzed the SAXS intensity data to confirm that IPPmin protein is monodisperse and monomeric in solution. We find that Guinier plots are linear in the Guinier region (q*Rg,1.3) (Figure 2B) and that Guinier approximation for the radius of gyration (Rg) values are consistent for all of the ??IPPmin concentrations measured, ranging from 33.9 A to 35.3 A (Table 1), confirming a lack of aggregation in the sample. Automatic generation of Guinier plots using AutoRg [29] results in similar.Bunits. Residues present in the crystal structures but not present in our expression constructs were deleted for consistency. For EOM analysis, the 14 amino acid linker (residues 171?84; Figure 1A) was modeled with RANCH [37]. Data were ?cut off at qmax = 0.23 A21. Theoretical scattering curves for the two subunits were obtained with CRYSOL [39]. Superpositions of models were performed in SUPCOMB with no symmetry (P1) applied; superpositions did not exclude fits rotated by two-fold rotation [40]. Porod-Debye analysis was performed, with scatter??ing data in the range of qmin = 0.1222 A21 to qmax = 0.156 A21 plotted as q46I(q) vs q4 used for linear fitting [33].(ILK-pKD) [11?3] (Figure 1A). ILK expression constructs containing the pseudokinase domain are largely insoluble when expressed alone in E. coli; however, co-expression with a-parvinCH2 results in a soluble protein complex [[9] and data not shown]. Therefore, we co-expressed full-length ILK as a GSTfusion protein and a-parvin-CH2 as a His-tagged protein in E. coli by co-transformation of compatible expression vectors under dual selection, which rescues ILK protein from insolubility (data not shown). Codon-optimized synthetic cDNA encoding ILK showed improvement of total yield over native cDNA, and was used in subsequent purifications (Figure 1B). His-tagged PINCH1-LIM1, which is soluble in E. coli, was expressed alone, and cells mixed with the ILK/a-parvin-CH2 expressing cells prior to co-sonication. The IPPmin complex was affinity purified on glutathioneagarose beads, eluted, and the GST- and (His)- tags simultaneously removed by TEV proteolysis (Figure 1C). IPPmin complex was then purified by anion exchange chromatography using a shallow gradient, which allowed for differential elution of GST and IPPmin. Remaining GST protein was then removed by incubation with glutathione-agarose beads and size-exclusion chromatography to achieve purity of crystallization quality, and immunoblot analysis confirms the presence of ILK in the 22948146 complex (Figure 1D). Notably, the IPPmin complex remains intact during all steps of purification. The migration of ILK, a-parvin-CH2 and PINCH-1LIM1 on SDS-PAGE is consistent with their expected molecular weights (51.4 kDa, 14.7 kDa, and 7.5 kDa, respectively). Purified IPPmin protein resolved on a calibrated gel-filtration column results in a single peak with an elution volume corresponding to a molecular weight of approximately 80 kDa, in close agreement with the expected molecular weight of 73.6 kDa (Figure 1E). The purified IPPmin complex migrates as a single species on native gel electrophoresis and its migration is unchanged upon addition of excess purified PINCH1-LIM1 and/or a-parvin-CH2 proteins, confirming that the purified IPPmin complex is stable and monodisperse in solution (Figure 1F).SAXS analysis of IPPmin complexTo study the overall conformation of the IPP in solution, we collected SAXS data on a concentration series of IPPmin complex protein (Figure 2A). First, we analyzed the SAXS intensity data to confirm that IPPmin protein is monodisperse and monomeric in solution. We find that Guinier plots are linear in the Guinier region (q*Rg,1.3) (Figure 2B) and that Guinier approximation for the radius of gyration (Rg) values are consistent for all of the ??IPPmin concentrations measured, ranging from 33.9 A to 35.3 A (Table 1), confirming a lack of aggregation in the sample. Automatic generation of Guinier plots using AutoRg [29] results in similar.
