RESUMO
C2 domains are widespread protein modules that often occur as tandem repeats in many membrane-trafficking proteins such as synaptotagmin and rabphilin. The first and second C2 domains (C2A and C2B, respectively) have a high degree of homology but also specific differences. The structure of the C2A domain of synaptotagmin I has been extensively studied but little is known about the C2B domains. We have used NMR spectroscopy to determine the solution structure of the C2B domain of rabphilin. The overall structure of the C2B domain is very similar to that of other C2 domains, with a rigid beta-sandwich core and loops at the top (where Ca2+ binds) and the bottom. Surprisingly, a relatively long alpha-helix is inserted at the bottom of the domain and is conserved in all C2B domains. Our results, together with the Ca(2+)-independent interactions observed for C2B domains, indicate that these domains have a Janus-faced nature, with a Ca(2+)-binding top surface and a Ca(2+)-independent bottom surface.
Assuntos
Proteínas do Tecido Nervoso/química , Estrutura Secundária de Proteína , Proteínas rab de Ligação ao GTP/química , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ligação ao Cálcio/química , Sequência Conservada , Glicoproteínas de Membrana/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Ratos , Sequências Repetitivas de Aminoácidos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência/métodos , Sinaptotagmina I , Sinaptotagminas , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP/metabolismo , Rabfilina-3ARESUMO
The interaction of bilirubin, biliverdin, bilirubin dimethyl ester, biliverdin dimethyl ester, xanthobilirubic acid, and xanthobilirubin methyl ester with trihydroxy and dihydroxy bile salt solutions is investigated by micellar electrokinetic chromatography (MEKC). The capacity factor of each compound is measured in solutions of the different bile salts over the pH range of 6.5-9.0. The capacity factor of bilirubin increases with pH below 7 in all bile salt solutions. Biliverdin and xanthobilirubin show essentially identical capacity factors for all bile salts. Biliverdin dimethyl ester and xanthobilirubin methyl ester also have very similar capacity factors, which are greater than those of the carboxy analogs, in trihydroxy bile salts. The capacity factors of these esters are higher in the dihydroxy bile salts, with the capacity factor of biliverdin dimethyl ester being twice that of xanthobilirubin methyl ester. Factors involved in the MEKC analysis of these compounds are discussed.
