Surface plasmon resonance characterization of calspermin-calmodulin binding kinetics.
We cloned, expressed, and purified a chimeric fusion between a soluble green fluorescent protein (smGFP) and the calmodulin binding protein calspermin. We have shown that the fusion protein, labeled smGN, has a K(i) in the calmodulin-dependent cyclic nucleotide phosphodiesterase activity assay of 1.97 nM, i.e., 3800 times smaller than that of the commonly used calmodulin inhibitor W7. Association and dissociation rate constants (k(a) and k(d)) and the dissociation equilibrium constant (K(D)) of smGN for calmodulin were determined using surface plasmon resonance (SPR). The k(a)=1.24 x 10(6)M(-1)s(-1), the k(d)=5.49 x 10(-3)s(-1), and the K(D)=4.42 x 10(-9)M. We also found that the GFP moiety was important for successfully binding calspermin to the surface of the CM5 flow cell at a sufficiently high concentration for SPR, and that this procedure may be used for SPR analysis of other acidic polypeptides, whose pI< or =4. To determine whether smGN might also bind to other calmodulin-like proteins in a heterologous system, we purified proteins from a plant total cell extract or a plant total protein extract by affinity chromatography against smGN. The purified proteins were identified as calmodulins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry, indicating a high level of specificity. We conclude that the high affinity and specific binding between smGN and calmodulin make it an easily localized recombinant alternative to chemical calmodulin inhibitors.
Murphy
Andrew J
AJ
School of Biosciences, The University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter, Devon EX4 4QD, UK.
Kemp
Fred
F
Love
John
J
D000818:Animals; D029681:Arabidopsis Proteins / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*
D029681:Arabidopsis Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D011993:Recombinant Fusion Proteins; C051666:calspermin; D049452:Green Fluorescent Proteins