Filter binding assay for the geldanamycin-heat shock protein 90 interaction.
A filter binding assay to measure affinity of [3H-allyl]17-allylamino geldanamycin ([3H]AAG) for the ATP binding site of the N-terminal domain of human Hsp90alpha (hHsp90alpha9-236) was developed. Diethylaminoethyl cellulose or glass fiber filters impregnated with polyethyleneimine were used to capture the [3H]AAG-Hsp90 complex, and conditions which washed >98% of free [3H]AAG from the filters were developed. The complex formed at a rapid rate (k(on)=2.5 x 10(7)Lmol(-1) x s(-1)) and dissociated with a half-life of 2.3 min (k(off)=5 x 10(-3) x s(-1)). hHsp90alpha9-236 bound to [3H]AAG with a K(d) value of 0.4+/-0.1 microM. [3H]AAG had similar affinities for full-length hHsp90alpha and for hHsp90alpha9-236 variants containing biotinylated N-terminal biotinylation signal sequences and N- or C-terminal His(6) tags. Geldanamycin, ADP, ATP, and radicicol-all known to bind to the ATP domain of Hsp90-competed with [3H]AAG for binding to hHsp90alpha9-236, showing K(d) values in good agreement with reported values.
Department of Pharmacological Sciences, Kosan Biosciences, Inc, 3832 Bay Center Place, Hayward, CA 94545, USA. carreras@kosan.com
Christopher W
CW
Carreras
Andreas
A
Schirmer
Ziyang
Z
Zhong
Daniel V
DV
Santi
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D016227:Benzoquinones; D001665:Binding Sites; D001667:Binding, Competitive; D019921:Biotinylation; D005374:Filtration / Q000295:instrumentation; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D047029:Lactams, Macrocyclic; D011485:Protein Binding; D011809:Quinones / Q000737:chemistry / Q000378:metabolism*; D011869:Radioligand Assay / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013302:Streptomyces / Q000737:chemistry / Q000235:genetics; D014316:Tritium
D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D011809:Quinones; D011993:Recombinant Fusion Proteins; D014316:Tritium; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C001277:geldanamycin