RESUMO
SNARE proteins mediate fusion of intracellular eukaryotic membranes and their alpha-helical transmembrane domains are known to contribute to lipid bilayer mixing. Synthetic transmembrane domain peptides were previously shown to mimic the function of SNARE proteins in that they trigger liposome fusion in a sequence-specific fashion. Here, we performed a detailed investigation of the conformational dynamics of the transmembrane helices of the presynaptic SNAREs synaptobrevin II and syntaxin 1a. To this end, we recorded deuterium/hydrogen-exchange kinetics in isotropic solution as well as in the membrane-embedded state. In solution, the exchange kinetics of each peptide can be described by three different classes of amide deuteriums that exchange with different rate constants. These are likely to originate from exchange at different domains of the helices. Interestingly, the rate constants of each class vary with the TMD sequence. Thus, the exchange rate is position-specific and sequence-specific. Further, the rate constants correlate with the previously determined membrane fusogenicities. In membranes, exchange is retarded and a significant proportion of amide hydrogens are protected from exchange. We conclude that the conformational dynamics of SNARE TMD helices is mechanistically linked to their ability to drive lipid mixing.
Assuntos
Membrana Celular/química , Medição da Troca de Deutério , Proteínas de Membrana/química , Proteínas de Membrana/ultraestrutura , Modelos Químicos , Modelos Moleculares , Proteínas SNARE/química , Proteínas SNARE/ultraestrutura , Sequência de Aminoácidos , Simulação por Computador , Dados de Sequência Molecular , Conformação ProteicaRESUMO
The transmembrane segments of soluble N-ethylmaleimide-sensitive factor (SNARE) proteins or viral envelope proteins drive membrane fusion, which suggests that simple synthetic biology constructs for fusion exist and can be evaluated. We describe the high-yield synthesis of a set of de novo designed fusogenic peptides for use in functional investigations, which are highly enriched in 13C and 15N using three equivalents of labelled amino acids and optimized reaction conditions minimizing aggregation. The biomimetic peptides have a high purity >90% and show reproducible and fusogenic activity that correlates well with the intended functional design characteristics, from strongly fusogenic to almost non-fusogenic.
Assuntos
Isótopos de Carbono , Marcação por Isótopo/métodos , Isótopos de Nitrogênio , Peptídeos/síntese química , Sequência de Aminoácidos , Exocitose , Lipossomos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/síntese química , Proteínas do Tecido Nervoso/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transmissão SinápticaRESUMO
The ancestral galectin from the sponge Geodia cydonium (GCG) is classified on a structural basis to the prototype subfamily, whereas its carbohydrate-binding specificity is related to that of the mammalian chimera-type galectin-3. This dual coordination reveals GCG as a potential precursor of the later evolved galectin subfamilies, which is reflected in the primary structure of the protein. This study provides evidence that GCG is the LECT1 gene product, while neither a previously described LECT2 gene nor a functional LECT2 gene product was found in the specimen under investigation. The electrophoretically separated protein isomers with apparent molecular masses of 13, 15, and 16 kDa correspond to variants of the LECT1 protein-exhibiting peptide sequence polymorphisms that concern critical positions of the carbohydrate recognition domain (13 kDa: Leu51, Asn55, His130, Gly137; 15 kDa: Ser51, Asn55, Asn130, Gly137; 16 kDa: Ser51, Tyr55, Asn130, Glu137). Four residues, highly conserved in the galectin family, are substituted. None of the residues claimed to be involved in interactions with GalNAcalpha1-3 moieties at an extended binding subsite of galectin-3 was identified in the corresponding positions of GCG. Apparently, the substitutions do not confer distinct binding characteristics to the GCG variants as evidenced by binding studies with a recombinantly expressed 15-kDa isoform. The natural isoforms as well as the recombinant 15-kDa isoform oligomerize by the formation of non-covalent heteromeric or homomeric complexes. A phosphorylation of the galectin was confirmed neither by mass spectrometry nor by alkaline phosphatase treatment combined with isoelectric focusing.
Assuntos
Galectinas/química , Galectinas/genética , Polimorfismo Genético , Poríferos/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência de Carboidratos , Sequência Conservada , Galectinas/metabolismo , Dados de Sequência Molecular , Peso Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Fusion of biological membranes is mediated by distinct integral membrane proteins, e.g., soluble N-ethylmaleimide-sensitive factor attachment protein receptors and viral fusion proteins. Previous work has indicated that the transmembrane segments (TMSs) of such integral membrane proteins play an important role in fusion. Furthermore, peptide mimics of the transmembrane part can drive the fusion of liposomes, and evidence had been obtained that fusogenicity depends on their conformational flexibility. To test this hypothesis, we present a series of unnatural TMSs that were designed de novo based on the structural properties of hydrophobic residues. We find that the fusogenicity of these peptides depends on the ratio of alpha-helix-promoting Leu and beta-sheet-promoting Val residues and is enhanced by helix-destabilizing Pro and Gly residues within their hydrophobic cores. The ability of these peptides to refold from an alpha-helical state to a beta-sheet conformation and backwards was determined under different conditions. Membrane fusogenic peptides with mixed Leu/Val sequences tend to switch more readily between different conformations than a nonfusogenic peptide with an oligo-Leu core. We propose that structural flexibility of these TMSs is a prerequisite of fusogenicity.