RESUMO
The ability of molecular chaperones to overcome the misfolding and aggregation of proteins is essential for the maintenance of proper protein homeostasis in all cells. Thus far, the best studied disaggregase systems are the Clp/Hsp100 family of "ATPases associated with various cellular activities" (AAA(+)) ATPases, which use mechanical forces powered by ATP hydrolysis to remodel protein aggregates. An alternative system to disassemble large protein aggregates is provided by the 38-kDa subunit of the chloroplast signal recognition particle (cpSRP43), which uses binding energy with its substrate proteins to drive disaggregation. The mechanism of this novel chaperone remains unclear. Here, molecular genetics and structure-activity analyses show that the action of cpSRP43 can be dissected into two steps with distinct molecular requirements: (i) initial recognition, during which cpSRP43 binds specifically to a recognition motif displayed on the surface of the aggregate; and (ii) aggregate remodeling, during which highly adaptable binding interactions of cpSRP43 with hydrophobic transmembrane domains of the substrate protein compete with the packing interactions within the aggregate. This establishes a useful framework to understand the molecular mechanism by which binding interactions from a molecular chaperone can be used to overcome protein aggregates in the absence of external energy input from ATP.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis , Complexos de Proteínas Captadores de Luz/química , Partícula de Reconhecimento de Sinal/química , Sequência de Aminoácidos , Polarização de Fluorescência , Interações Hidrofóbicas e Hidrofílicas , Cinética , Complexos de Proteínas Captadores de Luz/genética , Modelos Moleculares , Chaperonas Moleculares/química , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Desdobramento de Proteína , Deleção de Sequência , Especificidade por Substrato , Propriedades de Superfície , TermodinâmicaRESUMO
Understanding how the folding of proteins establishes their functional characteristics at the molecular level challenges both theorists and experimentalists. The simplest test beds for confronting this issue are provided by electron transfer proteins. The environment provided by the folded protein to the cofactor tunes the metal's electron transport capabilities as envisioned in the entatic hypothesis. To see how the entatic state is achieved one must study how the folding landscape affects and in turn is affected by the metal. Here, we develop a coarse-grained functional to explicitly model how the coordination of the metal (which results in a so-called entatic or rack-induced state) modifies the folding of the metallated Pseudomonas aeruginosa azurin. Our free-energy functional-based approach directly yields the proper nonlinear extra-thermodynamic free energy relationships for the kinetics of folding the wild type and several point-mutated variants of the metallated protein. The results agree quite well with corresponding laboratory experiments. Moreover, our modified free-energy functional provides a sufficient level of detail to explicitly model how the geometric entatic state of the metal modifies the dynamic folding nucleus of azurin.