In vitro reconstitution and biophysical characterization of OEP16, an outer envelope pore protein of pea chloroplasts.
More than 30% of all proteins in the living cell are membrane proteins; most of them occur in the native membranes only in very low amounts, which hinders their functional and structural investigation. Here we describe the in vitro reconstitution of overexpressed Outer Envelope Protein 16 (OEP16) from pea chloroplasts, a cation-selective channel, which has been purified from E. coli inclusion bodies. Reconstitution in detergent micelles was monitored by CD and fluorescence spectroscopy. Electron microscopy showed a homogeneous size distribution of the reconstituted protein, and differential scanning calorimetry gave an estimate of the enthalpy of protein folding. First protein crystals were obtained that have to be further refined for X-ray structural analysis. The described methods of membrane protein reconstitution and biophysical analysis might prove helpful in the study of other membrane proteins.
Linke
D
D
Max Volmer Institut für Biophysikalische Chemie und Biochemie, Technische Universität Berlin, Strasse des 17, Juni 135, 10623 Berlin, Germany.
Frank
J
J
Holzwarth
J F
JF
Soll
J
J
Boettcher
C
C
Fromme
P
P
D002152:Calorimetry, Differential Scanning; D060365:Chloroplast Proteins; D002736:Chloroplasts / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D002942:Circular Dichroism; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D009994:Osmolar Concentration; D018532:Pisum sativum; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry
D060365:Chloroplast Proteins; D007473:Ion Channels; D010940:Plant Proteins; D011994:Recombinant Proteins; D014364:Tryptophan