RESUMO
The unfolding enthalpy of the native state of ubiquitin in solution is 5 to 8 times that of its gaseous ions, as determined by electron capture dissociation (ECD) mass spectrometry. Although two-state folding occurs in solution, the three-state gaseous process proposed for this by Clemmer and co-workers based on ion mobility data is supported in general by ECD mass spectra, including relative product yields, distinct Delta H(unfolding) values between states, site-specific melting temperatures, and folding kinetics indicating a cooperative process. ECD also confirms that the 13+ ions represent separate conformers, possibly with side-chain solvated alpha-helical structures. However, the ECD data on the noncovalent bonding in the 5+ to 13+ ions, determined overall in 69 of the 75 interresidue sites, shows that thermal unfolding proceeds via a diversity of intermediates whose conformational characteristics also depend on charge site locations. As occurs with increased acidity in solution, adding 6 protons to the 5+ ions completely destroys their tertiary noncovalent bonding. However, solvation of the newly protonated sites to the backbone instead increases the stability of the secondary structure (possibly an alpha-helix) of these gaseous ions, while in solution these new sites aid denaturation by solvation in the aqueous medium. Extensive ion equilibration can lead to even more compact and diverse conformers. The three-state unfolding of gaseous ubiquitin appears to involve ensembles of individual chain conformations in a "folding funnel" of parallel reaction paths. This also provides a further caution for characterizing solution conformers from their gas-phase behavior.
Assuntos
Ubiquitina/química , Sequência de Aminoácidos , Gases , Íons , Cinética , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , TermodinâmicaRESUMO
The relative alkali metal ion (M(+)) affinities (binding energies) between seventeen different amino acids (AA) and the corresponding methyl esters (AAOMe) were determined in the gas phase by the kinetic method based on the dissociation of AA-M(+)-AAOMe heterodimers (M=Li, Na, K, Cs). With the exception of proline, the Li(+), Na(+), and K(+) affinities of the other aliphatic amino acids increase in the order AA
Assuntos
Aminoácidos/química , Metais Alcalinos/química , Sítios de Ligação , Ésteres/química , Gases , Modelos Moleculares , Estrutura MolecularRESUMO
Although a poly(ethylene/propylene glycol) (PEG/PPG) copolymer mixture is far too complex (approximately 150 oligomeric formulas) for conventional purification, oligomer ion compositions of <1% abundance can be separated by Fourier transform mass spectrometry and dissociated into sequence-specific fragment ions. Using collisionally activated dissociation (CAD) or other conventional energetic methods, we found that misleading rearrangements are common; however, these are negligible with electron capture dissociation (ECD), consistent with its nonergodic mechanism. Despite the lack of reference compounds, ECD of five oligomers ranging from PEG(1)PPG(18) to PEG(9)PPG(15) shows that approximately 80% of their isomers have all PEG units at one end, while CAD gave lower values because of an approximately 21% rearrangement loss of internal monomer units. In contrast to the indicated triblock "PEG/PPG/PEG" sample designation of this commercial surfactant, all of these oligomers are found to consist primarily of diblock PEG/PPG structures, so that their termini differ significantly in hydrophobicity, as expected for a surfactant.