RESUMO
The F-actin crosslinker filamin from Dictyostelium discoideum (ddFLN) has a rod domain consisting of six structurally similar immunoglobulin domains. When subjected to a stretching force, domain 4 unfolds at a lower force than all the other domains in the chain. Moreover, this domain shows a stable intermediate along its mechanical unfolding pathway. We have developed a mechanical single-molecule analogue to a double-jump stopped-flow experiment to investigate the folding kinetics and pathway of this domain. We show that an obligatory and productive intermediate also occurs on the folding pathway of the domain. Identical mechanical properties suggest that the unfolding and refolding intermediates are closely related. The folding process can be divided into two consecutive steps: in the first step 60 C-terminal amino acids form an intermediate at the rate of 55 s(-1); and in the second step the remaining 40 amino acids are packed on this core at the rate of 179 s(-1). This division increases the overall folding rate of this domain by a factor of ten compared with all other homologous domains of ddFLN that lack the folding intermediate.
Assuntos
Proteínas Contráteis/química , Proteínas Contráteis/metabolismo , Imunoglobulinas/química , Imunoglobulinas/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Dobramento de Proteína , Animais , Proteínas Contráteis/genética , Dictyostelium/química , Filaminas , Cinética , Proteínas dos Microfilamentos/genética , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Many F-actin crosslinking proteins consist of two actin-binding domains separated by a rod domain that can vary considerably in length and structure. In this study, we used single-molecule force spectroscopy to investigate the mechanics of the immunoglobulin (Ig) rod domains of filamin from Dictyostelium discoideum (ddFLN). We find that one of the six Ig domains unfolds at lower forces than do those of all other domains and exhibits a stable unfolding intermediate on its mechanical unfolding pathway. Amino acid inserts into various loops of this domain lead to contour length changes in the single-molecule unfolding pattern. These changes allowed us to map the stable core of approximately 60 amino acids that constitutes the unfolding intermediate. Fast refolding in combination with low unfolding forces suggest a potential in vivo role for this domain as a mechanically extensible element within the ddFLN rod.
Assuntos
Actinas/metabolismo , Proteínas Contráteis/química , Proteínas dos Microfilamentos/química , Proteínas de Protozoários/química , Animais , Proteínas Contráteis/genética , Proteínas Contráteis/metabolismo , Reagentes de Ligações Cruzadas , Dictyostelium/genética , Dictyostelium/metabolismo , Dimerização , Filaminas , Técnicas In Vitro , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia de Força Atômica , Mutagênese Sítio-Dirigida , Desnaturação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Coiled-coils occur in a variety of proteins involved in mechanical and structural tasks in the cell. Their mechanical properties are important in various contexts ranging from hair elasticity to synaptic fusion. Beyond their importance in biology, coiled-coils have also attracted interest as programmable protein sequences for the design of novel hydrogels and materials. We have studied the elastic properties of the myosin coiled-coil at the single molecule level. The coiled-coil undergoes a massive structural transition at forces between 20 and 25 pN where the coil extends to about 2.5 times its original length. Unlike all other proteins investigated mechanically so far, this transition is reversible on a timescale of less than a second, making the coiled-coil a truly elastic protein.