Comparative analysis of the methods used for finding surface energy to investigate protein interaction behavior on chromatographic supports.

Hydrophobic interaction chromatography, an important and effective purification strategy, is generally used for the purification of variety of biomolecules. A basic understanding of the protein interaction behavior is required to effectively separate these biomolecules. A colloidal type extended Derjaguin, Landau, Verwey, and Overbeek calculations were utilized to study the interactions behavior of model proteins to commercially available hydrophobic chromatographic materials that is, Toyopearl Phenyl 650C and Toyopearl Butyl 650C. Physicochemical properties of selected model proteins were achieved by contact angle and zeta potential measurements. The contact angle of chromatographic materials used was achieved through sessile drop method on disrupted beads and capillary penetration method (CPM) on intact beads. The surface properties were further used to calculate the interactions of the proteins to chromatographic supports. The calculated secondary energy minimum of the proteins with the chromatographic materials (from the contact angle values determined through both methods can be correlated with the retention volumes from the real chromatography. The secondary energy minimum values are higher for each protein to the chromatographic materials calculated from the inputs derived through sessile drop method compared to CPM. For instance, immunoglobulin G has secondary energy minimum value of 0.17 kT compared to 0.11 kT, obtained through sessile drop method and CPM, respectively. Average relative values of the energy minimum calculated for all proteins are as 1.51 kT and 1.29 kT for Toyopearl Butyl 650C and Toyopearl Phenyl 650C, respectively, as a conversion factor for estimation of secondary energy minimum for both methods.

Quantitative analysis of the chloroplast molecular chaperone ClpC/Hsp93 in Arabidopsis reveals new insights into its localization, interaction with the Clp proteolytic core, and functional importance.

The molecular chaperone ClpC/Hsp93 is essential for chloroplast function in vascular plants. ClpC has long been held to act both independently and as the regulatory partner for the ATP-dependent Clp protease, and yet this and many other important characteristics remain unclear. In this study, we reveal that of the two near-identical ClpC paralogs (ClpC1 and ClpC2) in Arabidopsis chloroplasts, along with the closely related ClpD, it is ClpC1 that is the most abundant throughout leaf maturation. An unexpectedly large proportion of both chloroplast ClpC proteins (30% of total ClpC content) associates to envelope membranes in addition to their stromal localization. The Clp proteolytic core is also bound to envelope membranes, the amount of which is sufficient to bind to all the similarly localized ClpC. The role of such an envelope membrane Clp protease remains unclear although it appears uninvolved in preprotein processing or Tic subunit protein turnover. Within the stroma, the amount of oligomeric ClpC protein is less than that of the Clp proteolytic core, suggesting most if not all stromal ClpC functions as part of the Clp protease; a proposal supported by the near abolition of Clp degradation activity in the clpC1 knock-out mutant. Overall, ClpC appears to function primarily within the Clp protease, as the principle stromal protease responsible for maintaining homeostasis, and also on the envelope membrane where it possibly confers a novel protein quality control mechanism for chloroplast preprotein import.

The chloroplast protein CPSAR1, dually localized in the stroma and the inner envelope membrane, is involved in thylakoid biogenesis.

Thylakoid biogenesis is a crucial step for plant development involving the combined action of many cellular actors. CPSAR1 is shown here to be required for the normal organization of mature thylakoid stacks, and ultimately for embryo development. CPSAR1 is a chloroplast protein that has a dual localization in the stroma and the inner envelope membrane, according to microscopy studies and subfractionation analysis. CPSAR1 is close to the Obg nucleotide binding protein subfamily and displays GTPase activity, as demonstrated by in vitro assays. Disruption of the CPSAR1 gene via T-DNA insertion results in the arrest of embryo development. In addition, transmission electron microscopy analysis indicates that mutant embryos are unable to develop thylakoid membranes, and remain white. Unstacked membrane structures resembling single lamellae accumulate in the stroma, and do not assemble into mature thylakoid stacks. CPSAR1 RNA interference induces partially developed thylakoids leading to pale-green embryos. Altogether, the presented data demonstrate that CPSAR1 is a protein essential for the formation of normal thylakoid membranes, and suggest a possible involvement in the initiation of vesicles from the inner envelope membrane for the transfer of lipids to the thylakoids.