Physical nature of signal peptide binding to DmsD.
Here we describe the biophysical characterization of the interaction of the redox enzyme maturation protein DmsD with the signal peptide of its target protein, DmsA. Isothermal titration calorimetry (ITC), size exclusion chromatography (SEC), and an in vitro Far-Western assay is used to show that DmsD binds the twin-arginine signal peptide from DmsA in the micromolar range and in a 1:1 molar ratio. The SEC also shows that there is no oligomerization upon binding. Urea and guanidium hydrochloride denaturation profiles demonstrate the stability of DmsD and give insights on how electrostatic and hydrophobic interactions are important within this binding process. Furthermore, by use of N- and C-terminal fusions of DmsA signal peptide to GST, we observe that N-terminal display of the peptide is important for binding DmsD. In addition, all the folding forms of DmsD were found to bind the DmsA signal peptide as observed with the Far-Western assay.
Winstone
Tara L
TL
Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada T2N 1N4.
Workentine
Matthew L
ML
Sarfo
Kwabena J
KJ
Binding
Andrew J
AJ
Haslam
Bronwyn D
BD
Turner
Raymond J
RJ
D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D011485:Protein Binding / Q000187:drug effects; D011489:Protein Denaturation; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D002352:Carrier Proteins; C434268:DmsD protein, E coli; D029968:Escherichia coli Proteins; D047908:Intracellular Signaling Peptides and Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D014508:Urea; D005982:Glutathione Transferase; D019791:Guanidine