Gas phase dimerization of neuropeptide head activator analogs useful for the noncovalent constraint of peptides

Abstract

We have used electrospray mass spectrometry to examine the dimerization of EFLIVKS, a reversed sequence analog of part of the neuropeptide head activator, and other similar analogs. Observation of the EFLIVKS gas phase dimer is concentration-dependent, with a half-saturation concentration for relative dimer formation of 7.8 microM, similar to that of SKVILFE of 12 microM. The lowest energy conformers from quenched molecular dynamics simulations suggest EFLIVKS may dimerize in the gas phase by formation of multiple ion pairs across the dimer interface. Alanine-scan mutants also dimerize in the gas phase, with replacement of the interior residues FLIVK diminishing dimerization. The concentration-dependence of the EFLIVKS circular dichroism spectrum at pH 7.5 suggests the existence of different conformation states at different concentrations, but does not provide evidence supporting the saturable dimer formation in solution. Different analogs of EFLIVKS, when fused to each end of a 18mer unfolded peptide, induce solution structures with T(m)s of 42-50 degrees C. These peptides and analogs may thus be useful for the noncovalent constraint of peptides and peptide library members used in cellular screens.

Discovery of Undefined Protein Cross-Linking Chemistry: A Comprehensive Methodology Utilizing 18O-Labeling and Mass Spectrometry

“…Each peak was evaluated for false positives. For instance, gas-phase dimerization, which is commonly observed in mass spectrometry, , was readily determined based on retention time (same as the monomers) and mass (exactly double that of monomers). In addition, weak precursor ions (typically with a peak intensity of 50 000 counts or lower) with poor or no MS/MS data were excluded.…”