Study of Structure and Function of Recombinant Pea Root Plastid Porin by Biophysical Methods

Abstract

Pea root plastid porin (Fischer et al. (1994) J. Biol. Chem. 269, 25754-25760), which belongs to the family of mitochondrial (eukaryotic) porins, was expressed in Escherichia coli in high amounts using the pQE expression system. The recombinant protein was reconstituted into lipid bilayer membranes, and its characteristic properties were compared to those of the native porin isolated from pea root plastids. No significant difference was found between the native and the recombinant form when the protein was preincubated in detergent and sterol. The recombinant porin seems to be a valuable model system for the study of eukaryotic porins by spectroscopic methods, in which high amounts of protein are needed. CD spectroscopy was performed to determine the secondary structure of the porin under different conditions. It was found to have a high degree of beta-sheet structure in the nonionic detergent Genapol X-80 and in lipid vesicles. The more polar detergent sodium dodecyl sulfate (SDS) induced a large amount of alpha-helix structure in the protein. Addition of sterol to the porin in Genapol buffer did not influence its secondary structure to any measurable extent, whereas it had a strong influence on channel forming activity in black lipid bilayers. First refolding experiments performed in decreasing urea concentrations are discussed together with the results of the other measurements with regard to protein folding and channel formation.

Characterization of the Structure, Function, and Conformational Stability of PorB Class 3 Protein from Neisseria meningitidis

“…Therefore, the SDSinduced conformational changes of class 3 protein are characterized by the generation of a partially unfolded state as demonstrated by the SDS-dependent generation of a molecular species exhibiting faster electrophoretic migration than that observed for the native trimeric state. These results suggest that such species might populate the in vivo folding pathways of porins, particularly in the case of class 3 protein, and supports the notion that porins may undergo folding/unfolding through monomeric intermediates as suggested before for other membrane proteins including porins (54). In fact, the effects of SDS may be envisioned as the initial step on the folding pathway of class 3 protein in which a partially unfolded intermediate inserts into the membrane and eventually assumes its final ␤-sheet oligomeric conformation.…”

The N-terminus of VDAC: Structure, mutational analysis, and a potential role in regulating barrel shape

“…Both modeling and artificial bilayer experiments revealed that the S193E variant opens and closes less frequently than does the wild-type [43] although the open state conductances are similar (3.7 and 3.9 nS, respectively, [44]). In this case, the N-terminal methionine is not present in the native form [45], but presumably remains in the recombinant one.…”

“…This archaeal lipid forms very densely packed and stable planar bilayers, suitable for these measurements [81]. Low concentrations of sterol may added to the lipid mixture used to create the bilayer [80] or added to the detergent solubilized porin [44] to encourage insertion of the protein into the bilayer. While sterol is a component of native membranes, its interactions with DPhPC may be subtly different, and influence the lipid environment of the VDAC.…”

VDAC, a multi-functional mitochondrial protein regulating cell life and death

“…For example, adapting unfolding and refolding protocols that consider the presence of lipids that are naturally present in the functional unit (e.g. cholesterol (De Pinto et al, 1989b) or ergosterol for Neurospora crassa (Freitag et al, 1982) VDAC) or of non-ionic detergents in the protein solution (Engelhardt et al, 2007;Koppel et al, 1998;Popp et al, 1997;Ujwal et al, 2008) can be considered. Indeed, because of the artificial process of refolding in the presence of detergents, the proposed VDAC1 structure has been questioned (Colombini, 2009).…”