RESUMO
Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Most of the cross linkers available have flexible spacers. Because of this, the linked hybrids have significant structural flexibility and the relative structure between their two components is largely unpredictable. Here we describe a method of connecting two proteins via a 'fusion α helix' formed by joining two pre-existing helices into a single extended helix. Because simple ligation of two helices does not guarantee the formation of a continuous helix, we used EY-CBS, a synthetic cross linker that has been shown to react selectively with cysteines in α-helices, to stabilize the connecting helix. Formation and stabilization of the fusion helix was confirmed by determining the crystal structures of the fusion proteins with and without bound EY-CBS. Our method should be widely applicable for linking protein building blocks to generate predictable structures.
Assuntos
Anquirinas/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Proteína Estafilocócica A/efeitos dos fármacos , Anquirinas/química , Cristalização , Cristalografia por Raios X , Cisteína/química , Cisteína/efeitos dos fármacos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Proteína Estafilocócica A/químicaRESUMO
A novel benzimidazole molecule that was identified in a small-molecule screen and is known as antibiofilm compound 1 (ABC-1) has been found to prevent bacterial biofilm formation by multiple bacterial pathogens, including Staphylococcus aureus, without affecting bacterial growth. Here, the biofilm inhibiting ability of 156 µM ABC-1 was tested in various biofilm-forming strains of S. aureus. It was demonstrated that ABC-1 inhibits biofilm formation by these strains at micromolar concentrations regardless of the strains' dependence on Polysaccharide Intercellular Adhesin (PIA), cell wall-associated protein dependent or cell wall- associated extracellular DNA (eDNA). Of note, ABC-1 treatment primarily inhibited Protein A (SpA) expression in all strains tested. spa gene disruption showed decreased biofilm formation; however, the mutants still produced more biofilm than ABC-1 treated strains, implying that ABC-1 affects not only SpA but also other factors. Indeed, ABC-1 also attenuated the accumulation of PIA and eDNA on cell surface. Our results suggest that ABC-1 has pleotropic effects on several biofilm components and thus inhibits biofilm formation by S. aureus.
Assuntos
Antibacterianos/farmacologia , Benzimidazóis/farmacologia , Biofilmes/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Aminoaciltransferases/genética , Antibacterianos/síntese química , Antibacterianos/química , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Benzimidazóis/química , Biofilmes/crescimento & desenvolvimento , Parede Celular/metabolismo , Cisteína Endopeptidases/genética , Regulação para Baixo , Polissacarídeos Bacterianos/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteína Estafilocócica A/biossíntese , Proteína Estafilocócica A/efeitos dos fármacos , Proteína Estafilocócica A/genética , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMO
A recombinant VH single-domain antibody recognizing staphylococcal protein A was functionalized on reactive lysine residues with N-hydroxysuccimidyl-activated 4-cyanobenzoate. Surface plasmon resonance analysis of antibody-antigen binding revealed that modified and unmodified antibodies bound protein A with similar affinities. Raman imaging of the modified antibodies indicated that the benzonitrile group provides vibrational contrast enhancement in a region of the electromagnetic spectrum that is transparent to cellular materials. Thus, the modified single-domain antibody may be amenable to detecting protein A from samples of the human pathogen Staphylococcus aureus using vibronic detection schemes such as Raman and coherent anti-Stokes Raman scattering. The generality of this labeling strategy should make it applicable to modifying an array of proteins with varied structure and function.
