RESUMO
Encapsulation of guest molecules into the vacant space of biomacromolecular crystals has been utilized for various purposes including functioning as a protein container to protect against physical stress and structural determination of the guest. Todarodes pacificus hemocyanin (TpHc) is a hollow cylindrical decameric protein complex with an inner space 110â¯Å in diameter and 160â¯Å in height. In the crystal, TpHc forms a straw-like bundle and contains one reactive Cys (Cys3246) in the inner domain of each protomer. Here, we conjugated biotin onto Cys3246 of TpHc followed by incubation with streptavidin. The streptavidin was immobilized into the inner space of TpHc due to its interaction with biotin. Moreover, the complex containing TpHc and streptavidin was crystallized under the same conditions used for unmodified TpHc. In order to expand this methodology for a variety of proteins, we conjugated the ligand nitrilotriacetic acid (NTA) chelated to a Ni2+ ion (Ni2+-NTA) to TpHc. We found that His-tagged green fluorescent protein (GFP) was encapsulated into the Ni2+-NTA-conjugated TpHc via the interaction between the His-tag and the Ni2+-NTA group. X-ray crystallography demonstrated that the crystal packing of the complex containing TpHc and GFP was identical to that of the unmodified TpHc. Our guest immobilization method is distinct from previous approaches that are dependent on diffusion of the guest into the host crystal. Thus, our findings may accelerate the development of proteinaceous crystal engineering.
Assuntos
Decapodiformes/química , Hemocianinas/química , Proteínas Imobilizadas/química , Animais , Biotina/química , Quelantes/química , Cristalização , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Níquel/química , Ácido Nitrilotriacético/química , Multimerização Proteica , Estreptavidina/químicaRESUMO
A short, monodisperse additive (octa(ethylene glycol) monophenyl ether) functions to suppress aggregation of thermally and chemically denatured lysozyme. Control studies with shorter and non-amphiphilic derivatives revealed that the amphiphilic structure is essential, and octa(ethylene glycol) is nearly the minimum chain length for amphiphilic poly(ethylene glycol)s to stabilize proteins.
Assuntos
Muramidase/química , Polietilenoglicóis/química , Varredura Diferencial de Calorimetria , Dicroísmo CircularRESUMO
The chemical modification of proteins to provide desirable functions and/or structures broadens their possibilities for use in various applications. Usually, proteins can acquire new functions and characteristics, in addition to their original ones, via the introduction of synthetic functional moieties. Here, we adopted a more radical approach to protein modification, i.e., the replacement of a functional domain of proteins with alternative chemical compounds to build "cyborg proteins." As a proof of concept model, we chose staphylococcal α-hemolysin (Hla), which is a well-studied, pore-forming toxin. The hemolytic activity of Hla mutants was dramatically decreased by truncation of the stem domain, which forms a ß-barrel pore in the membrane. However, the impaired hemolytic activity was significantly restored by attaching a pyrenyl-maleimide unit to the cysteine residue that was introduced in the remaining stem domain. In contrast, negatively charged fluorescein-maleimide completely abolished the remaining activity of the mutants.
Assuntos
Proteínas de Bactérias/química , Toxinas Bacterianas/química , Proteínas Hemolisinas/química , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Cisteína/química , Eritrócitos , Fluoresceínas/química , Regulação da Expressão Gênica , Proteínas Hemolisinas/genética , Hemólise , Concentração de Íons de Hidrogênio , Maleimidas/química , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Conformação Proteica , Ovinos , Staphylococcus aureusRESUMO
The mechanism by which anti-ciguatoxin antibody 10C9Fab recognizes a fragment of ciguatoxin CTX3C (CTX3C-ABCDE) was investigated by mutational analysis based on structural data. 10C9Fab has an extraordinarily large and deep antigen-binding pocket at the center of its variable region. We mutated several residues located at the antigen-binding pocket to Ala, and kinetic analysis of the interactions between the mutant proteins and the antigen fragment was performed. The results indicate that some residues associated with the rigid antigen-binding pocket are structural hot-spots and that L-N94 is an energetic hot-spot for association of the antibody with the antigen fragment CTX3C-ABCDE, suggesting the importance of structural complementarity and energetic hot-spot interactions for specific recognition of polycyclic ethers.