RESUMO
Members of the p24 family of type I transmembrane proteins are involved in budding of coat protein type I (COPI)-coated vesicles. They serve as coat protein receptors, binding via their cytoplasmic domains to coatomer, a stable cytosolic protein complex that represents the major coat component of these vesicles. Experimental evidence suggest that p23, a member of the p24 family, binds to coatomer in an oligomeric state and that this binding triggers polymerization of the coat protein. Toward an understanding of this process at the molecular level, formation of noncovalent complexes and their relative stabilities were analyzed by Fourier transform ion cyclotron resonance mass spectrometry using nanoelectrospray ionization. Specificity and stability of oligomers formed were established to depend on characteristic peptide sequence motifs and were confirmed by mass spectrometric competition experiments with control peptides. Mutations in the peptide sequence caused decreased interaction and destabilization of the noncovalent complexes. The formation and relative stabilities of dimeric and tetrameric complexes were assessed to be formed by cytoplasmic tails of coatomer receptors. The direct molecular identification provided by mass spectrometry correlates well with biochemical results. Thus, electrospray ionization mass spectrometry proves to be a powerful tool to investigate physiologically relevant peptide complexes.
Assuntos
Complexo I de Proteína do Envoltório/química , Complexo I de Proteína do Envoltório/metabolismo , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Sequência de Aminoácidos , Complexo I de Proteína do Envoltório/genética , Proteína Coatomer/química , Proteína Coatomer/genética , Proteína Coatomer/metabolismo , Estabilidade de Medicamentos , Espectrometria de Massas/métodos , Oligopeptídeos/genética , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
The feasibility of nanoelectrospray mass spectrometry (nanoESI) for the direct analysis of protein chemical reactions and structural changes of proteins has been evaluated. Taking advantage of the long spraying time and the capability of nanoESI for employing a wide range of solvent conditions such as buffers and detergents, applications of monitoring reaction pathways, and dynamics have been carried out with several peptides and proteins. The time course of proteolytic digestions with trypsin and pepsin was investigated for several model polypeptides, and nanoESI showed to provide an efficient tool for optimising digestion conditions for the mass spectrometric peptide mapping analysis. Examples of specific protein chemical modification reactions at arginine and tyrosine residues illustrate the feasibility of nanoESI to monitoring reaction yields and modification sites for more than 180 min. Furthermore, changes of the pattern of protonated molecules caused by temperature effects and by protein unfolding due to disulfide bond reduction have been studied with the model proteins cytochrome c and hen eggwhite lysozyme. The results indicate that nanoESI is an efficient technique for the direct, molecular characterisation of protein-chemical reactions in solution.
Assuntos
Proteínas/química , Sequência de Aminoácidos , Dissulfetos/química , Hidrólise , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/químicaRESUMO
Mass spectrometry has significantly extended its applicability in the area of characterization of protein structures. Electrospray ionization enables on-line coupling with liquid chromatography which has become a powerful tool for the characterization of peptide and protein mixtures. The most recent development of a nanoelectrospray source, using capillary forces for a particularly mild analyte transport and ionization into the mass spectrometer, opens a wide field for applications to protein structure analysis. In this paper, the analytical development of liquid chromatography-electrospray ionization mass spectrometry and nano-electrospray ionization mass spectrometry, adapted to an electrospray ionization quadrupole mass spectrometer and its application to the characterization of noncovalent protein complexes are described.