HIV-1 TAT PEPTIDE BINDING DO TO TAR RNA BY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

Electrospray ionization mass spectrometry (ESI-MS) has been used to st udy the noncovalent complexes formed from the interaction between HIV- 1 Tat peptide and Tat protein with TAR RNA. Both positive ion and nega tive ion ESI mass spectra showed a maximum stoichiometry of 3:1 betwee n Tat peptide and TAR RNA. However, the higher order complexes only oc curred at high relative concentrations of Tat peptide. The 1:1 Tat pep tide-TAR RNA complex is believed to involve only specific interactions , whereas the higher order complexes involve non-specific interactions . Relative binding affinities between Tat peptide and TAR RNA and its various mutants (TAR missing the three-nucleotide bulge, TAR with a po ly(ethylene glycol) linker in the bulge region, and TAR with a poly(et hylene glycol) linker in the loop region) can be differentiated by com petitive binding experiments and ESI-MS measurements. The gas phase ma ss spectrometry experiments are consistent with solution phase studies , as they show that mutations in the bulge region reduce TAR RNA affin ity to Tat peptide.