Bacillus subtilis alpha-amylase: interactions of a partially folded conformer with small unilamellar vesicles.
We studied the interactions between conformers of exocellular alpha-amylase and small unilamellar vesicles (SUV) composed of the major membrane lipids of Bacillus subtilis under physiological conditions of pH, temperature and ionic strength. Using fluorescence spectroscopy, surface plasmon resonance (SPR) and phase separation, we show that the native alpha-amylase has no affinity for the SUV, whereas a partially folded form, displaying structural properties in common with the competent state for secretion, binds to the vesicles (KA approximately 10(5) M(-1)). This association prevented its subsequent folding. The complex was destabilized in the presence of PrsA, a major peripheric lipoprotein of B. subtilis which displays a strong affinity for SUV (KA approximately 1.5x10(8) M(-1)). Vesicles coated with PrsA lost their ability to bind the partially folded conformer. The approach in vitro, in which our aim was to mimic the last stage of alpha-amylase translocation, indicates that PrsA possibly helps, in vivo, the secreted protein to acquire its native conformation by modulating the interaction between the latter and the lipid polar heads on the trans side of the cytoplasmic membrane.
Colomer-Pallas
Anne
A
Institut Jacques Monod, Laboratoire Génétique et Membranes, Centre National de la Recherche Scientifique-Universités Paris 6 et Paris 7, Tour 43-2, place Jussieu, 75251 Paris cedex 05, France.
Petit-Glatron
Marie-Françoise
MF
Chambert
Régis
R
D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins; D003377:Countercurrent Distribution; D008051:Lipid Bilayers / Q000737:chemistry*; D008074:Lipoproteins / Q000494:pharmacology / Q000502:physiology; D008565:Membrane Proteins / Q000494:pharmacology / Q000502:physiology; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D008051:Lipid Bilayers; D008074:Lipoproteins; D008565:Membrane Proteins; D010743:Phospholipids; C082160:prsA protein, bacteria; D000516:alpha-Amylases