| dc.description.abstract | Abstract
In this study, we attempted to use electrospray ionization mass spectrometry (ESI-MS) to investigate the noncovalent assembly of protein quaternary structure. Two proteins with different sizes, sialic acid synthases (monomer ~ 38 kDa) from E. coli and Streptococcus agalactiae, and the vaccinia virus envelope protein A27L (monomer ~ 10.3 kDa) were studied as models for noncovalent assembly. Optimization of various critical parameters in interface was studied in detail to preserve the integrity of noncovalent assemblies. Furthermore, variations in solution pH were found to induce dramatic changes on the ESI mass spectra, which can be used as a readout for characterization of noncovalent protein assemblies. Moreover, collision-induced dissociation technique was applied to probe dissociation pathway and stability of noncovalent complexes. In addition, to characterize different degree of association, the Gibbs free energy of these oligomers was determined accordingly
E. coli sialic acid synthase (EcNeuB) and Streptococcus agalactiae sialic acid synthase (SaneuB) have high sequence homology. Tetrameric form was observed in ESI-MS at as the highest order quaternary structure for both enzymes at pH 7.5. Tetrameric form only exist at physiological range (pH 6.5 ~ pH 8.5), demonstrating that quaternary structure of sialic acid synthases is pH-sensitive. The dependence of structure on solution pH also provides a high level of confidence for “structural-specific” evidence.
Previous studies have revealed that recombinant vaccinia virus protein, A27L-aa, may facilitate vaccinia virus entry into host cell. The chemical shift index studies also strongly indicated that the three hydrophobic leucine residues (L47, L51, and L54) may play an important role in self-assembly of A27L-aa. In this investigation, we demonstrated that the quaternary structure of A27L-aa is a hexameric form. Besides, the dramatic change of quaternary structure of A27L-aa from hexamer to tetramer on mass spectrum by mutation of leucine 51 to alanine 51 demonstrated that leucine 51 play crucial role in contributing hydrophobic interaction of A27L-aa self-assembly. Furthermore, CID experiment showed that both tetramer and hexamers of A27L-aa and its mutants will directly dissociated to monomers without any intermediates. Gibbs free energy of hexamer and tetramer can be calculated from CID curves for hexamer and tetramer respectively. The Gibbs free energy of A27L-aa is 4.89kcal/mol and triple mutation is 3.98kcal/mol. The result demonstrate that the subunit-subunit interaction of L47,51,54A is weaker than A27L-aa.
ESI-MS may have great potentials for study noncovalent protein assembly. Moreover, the more detail structural information can also be provided by using the ESI-MS. | en_US |