Flow cytometry for real-time measurement of guanine nucleotide binding and exchange by Ras-like GTPases.
Ras-like small GTPases cycle between GTP-bound active and GDP-bound inactive conformational states to regulate diverse cellular processes. Despite their importance, detailed kinetic or comparative studies of family members are rarely undertaken due to the lack of real-time assays measuring nucleotide binding or exchange. Here we report a bead-based flow cytometric assay that quantitatively measures the nucleotide binding properties of glutathione-S-transferase (GST) chimeras for prototypical Ras family members Rab7 and Rho. Measurements are possible in the presence or absence of Mg(2+), with magnesium cations principally increasing affinity and slowing nucleotide dissociation rates 8- to 10-fold. GST-Rab7 exhibited a 3-fold higher affinity for guanosine diphosphate (GDP) relative to guanosine triphosphate (GTP) that is consistent with a 3-fold slower dissociation rate of GDP. Strikingly, GST-Rab7 had a marked preference for GTP with ribose ring-conjugated BODIPY FL. The more commonly used gamma-NH-conjugated BODIPY FL GTP analogue failed to bind to GST-Rab7. In contrast, both BODIPY analogues bound equally well to GST-RhoA and GST-RhoC. Comparisons of the GST-Rab7 and GST-RhoA GTP binding pockets provide a structural basis for the observed binding differences. In sum, the flow cytometric assay can be used to measure nucleotide binding properties of GTPases in real time and to quantitatively assess differences between GTPases.
Schwartz
Samantha L
SL
Department of Pathology and Cancer Research and Treatment Center, University of New Mexico, Albuquerque, NM 87131, USA.
Tessema
Mathewos
M
Buranda
Tione
T
Pylypenko
Olena
O
Rak
Alexey
A
Simons
Peter C
PC
Surviladze
Zurab
Z
Sklar
Larry A
LA
Wandinger-Ness
Angela
A
D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008274:Magnesium / Q000737:chemistry; D020559:Monomeric GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins; D020741:rho GTP-Binding Proteins / Q000378:metabolism
C486958:BODIPY FL GTPgammaS; D005456:Fluorescent Dyes; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020559:Monomeric GTP-Binding Proteins; D020691:rab GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D008274:Magnesium