RESUMO
Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are evolutionarily conserved machines that couple their folding/assembly to membrane fusion. However, it is unclear how these processes are regulated and function. To determine these mechanisms, we characterized the folding energy and kinetics of four representative SNARE complexes at a single-molecule level using high-resolution optical tweezers. We found that all SNARE complexes assemble by the same step-wise zippering mechanism: slow N-terminal domain (NTD) association, a pause in a force-dependent half-zippered intermediate, and fast C-terminal domain (CTD) zippering. The energy release from CTD zippering differs for yeast (13 kBT) and neuronal SNARE complexes (27 kBT), and is concentrated at the C-terminal part of CTD zippering. Thus, SNARE complexes share a conserved zippering pathway and polarized energy release to efficiently drive membrane fusion, but generate different amounts of zippering energy to regulate fusion kinetics.
Assuntos
Complexos Multiproteicos/química , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas SNARE/química , Sequência de Aminoácidos , Animais , Transferência de Energia , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Pinças Ópticas , Estrutura Quaternária de Proteína , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/metabolismo , Ratos , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Homologia de Sequência de Aminoácidos , Termodinâmica , Proteína 2 Associada à Membrana da Vesícula/química , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismoRESUMO
We reported recently that certain beta-peptides self-assemble spontaneously into cooperatively folded bundles whose kinetic and thermodynamic metrics mirror those of natural helix bundle proteins. The structures of four such beta-peptide bundles are known in atomic detail. These structures reveal a solvent-sequestered, hydrophobic core stabilized by a unique arrangement of leucine side chains and backbone methylene groups. Here we report that this hydrophobic core can be re-engineered to contain a fluorous subdomain while maintaining the characteristic beta-peptide bundle fold. Like alpha-helical bundles possessing fluorous cores, fluorous beta-peptide bundles are stabilized relative to hydrocarbon analogues and undergo cold denaturation. Beta-peptide bundles with fluorous cores represent the essential first step in the synthesis of orthogonal protein assemblies that can sequester selectively in an interstitial membrane environment.
