Real-time assay for monitoring membrane association of lipid-binding domains.
The C2 domain is a common protein module which mediates calcium-dependent phospholipid binding. Several assays have previously been developed to measure membrane association. However, these assays either have technical drawbacks or are laborious to carry out. We now present a simple solution-based turbidity method for rapidly assaying membrane association of single lipid-binding domains in real time. We used the first C2 domain of synaptotagmin 1 (C2A) as a model lipid-binding moiety. Our use of the common dimeric glutathione-S-transferase (GST) fusion tag allowed two C2A domains to be brought into close proximity. Consequently, calcium-triggered phospholipid binding by this artificially dimerized C2A resulted in liposomal aggregation, easily assayed by following absorbance of the solution at 350 nm. The assay is simple and sensitive and can be scaled up conveniently for use in a multiwell plate format, allowing high-throughput screening. In our screens, we identified nickel as a novel activator of synaptotagmin 1 C2A domain membrane association. Finally, we show that the turbidity method can be applied to the study of other GST-tagged lipid-binding proteins such as epsin, protein kinase C-beta, and synaptobrevin.
Connell
Emma
E
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 OQH, UK.
Scott
Phillip
P
Davletov
Bazbek
B
D000042:Absorption; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D009391:Nephelometry and Turbidimetry / Q000379:methods*; D009532:Nickel / Q000378:metabolism / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D013053:Spectrophotometry; D055672:Static Electricity; D050863:Synaptotagmin I / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
D008081:Liposomes; D008565:Membrane Proteins; D010743:Phospholipids; D050863:Synaptotagmin I; D009532:Nickel; D005982:Glutathione Transferase; D002118:Calcium