Study of structure and function of recombinant pea root plastid porin by biophysical methods.
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.
Popp
B
B
Lehrstuhl fur Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universitat Wurzburg, Germany.
Gebauer
S
S
Fischer
K
K
Flügge
U I
UI
Benz
R
R
D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D018532:Pisum sativum / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D018087:Plastids / Q000737:chemistry; D018272:Porins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014508:Urea
D007473:Ion Channels; D008081:Liposomes; D010940:Plant Proteins; D018272:Porins; D011994:Recombinant Proteins; C089963:pspor1 protein, Pisum sativum; D014508:Urea