Analysis of type I signal peptidase affinity and specificity for preprotein substrates.
Type I signal peptidases (SPases) are membrane-bound endopeptidases responsible for the catalytic cleavage of signal peptides from secretory proteins. Here, we analysed the interaction between a bacterial type I SPase and preprotein substrates using surface plasmon resonance. The use of a home-made biosensor surface based on a mixed self-assembled monolayer of thiols on gold allowed qualitative and kinetic analysis. In vitro binding of purified preproteins to a covalently immobilised bacterial SPase was found to be rather efficient (apparent K(D)=10(-7)-10(-8)M). The signal peptide was shown to be a prerequisite for SPase binding and the nature of the mature part of the preprotein significantly affected SPase binding affinity. The developed biosensor containing immobilised SPase is of great importance for analysis of specificity at substrate binding level and for drug screening. In fact, this is the first report of a membrane protein that was covalently attached to a biosensor surface and that retained binding capacity.
Geukens
Nick
N
Laboratory of Bacteriology, Rega Institute, K.U.Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
Frederix
Filip
F
Reekmans
Gunter
G
Lammertyn
Elke
E
Van Mellaert
Lieve
L
Dehaen
Wim
W
Maes
Guido
G
Anné
Jozef
J
D015374:Biosensing Techniques; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry; D008565:Membrane Proteins; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D013302:Streptomyces / Q000378:metabolism; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D013696:Temperature; D013997:Time Factors
D008081:Liposomes; D008565:Membrane Proteins; D009841:Oligonucleotides; D011498:Protein Precursors; D012697:Serine Endopeptidases; C026515:type I signal peptidase