RESUMEN
Mutants of human cellular retinol-binding proteinâ II (hCRBPII) were engineered to bind a julolidine retinal analogue for the purpose of developing a ratiometric pH sensor. The design relied on the electrostatic influence of a titratable amino acid side chain, which affects the absorption and, thus, the emission of the protein/fluorophore complex. The ratio of emissions obtained at two excitation wavelengths that correspond to the absorption of the two forms of the protein/fluorophore complex, leads to a concentration-independent measure of pH.
Asunto(s)
Técnicas Biosensibles/métodos , Colorantes Fluorescentes/metabolismo , Retinaldehído/metabolismo , Proteínas Celulares de Unión al Retinol/metabolismo , Fluorescencia , Colorantes Fluorescentes/química , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Mutagénesis Sitio-Dirigida/métodos , Conformación Proteica , Retinaldehído/análogos & derivados , Proteínas Celulares de Unión al Retinol/química , Proteínas Celulares de Unión al Retinol/genética , Espectrometría de Fluorescencia/métodosRESUMEN
Some members of the class A ß-lactamase family are capable of conferring resistance to the last resort antibiotics, carbapenems. A unique structural feature of these clinically important enzymes, collectively referred to as class A carbapenemases, is a disulfide bridge between invariant Cys69 and Cys238 residues. It was proposed that this conserved disulfide bridge is responsible for their carbapenemase activity, but this has not yet been validated. Here we show that disruption of the disulfide bridge in the GES-5 carbapenemase by the C69G substitution results in only minor decreases in the conferred levels of resistance to the carbapenem imipenem and other ß-lactams. Kinetic and circular dichroism experiments with C69G-GES-5 demonstrate that this small drop in antibiotic resistance is due to a decline in the enzyme activity caused by a marginal loss of its thermal stability. The atomic resolution crystal structure of C69G-GES-5 shows that two domains of this disulfide bridge-deficient enzyme are held together by an intensive hydrogen-bonding network. As a result, the protein architecture and imipenem binding mode remain unchanged. In contrast, the corresponding hydrogen-bonding networks in NMCA, SFC-1, and SME-1 carbapenemases are less intensive, and as a consequence, disruption of the disulfide bridge in these enzymes destabilizes them, which causes arrest of bacterial growth. Our results demonstrate that the disulfide bridge is essential for stability but does not play a direct role in the carbapenemase activity of the GES family of ß-lactamases. This would likely apply to all other class A carbapenemases given the high degree of their structural similarity.
Asunto(s)
Proteínas Bacterianas/química , Disulfuros/química , Mutación Missense , beta-Lactamasas/química , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Cisteína/química , Dominios Proteicos , beta-Lactamasas/genéticaRESUMEN
Cellular retinol-binding proteins (CRBPs) I and II, which are members of the intracellular lipid-binding protein (iLBP) family, are retinoid chaperones that are responsible for the intracellular transport and delivery of both retinol and retinal. Although structures of retinol-bound CRBPI and CRBPII are known, no structure of a retinal-bound CRBP has been reported. In addition, the retinol-bound human CRBPII (hCRBPII) structure shows partial occupancy of a noncanonical conformation of retinol in the binding pocket. Here, the structure of retinal-bound hCRBPII and the structure of retinol-bound hCRBPII with retinol fully occupying the binding pocket are reported. It is further shown that the retinoid derivative seen in both the zebrafish CRBP and the hCRBPII structures is likely to be the product of flux-dependent and wavelength-dependent X-ray damage during data collection. The structures of retinoid-bound CRBPs are compared and contrasted, and rationales for the differences in binding affinities for retinal and retinol are provided.
Asunto(s)
Retinaldehído/metabolismo , Proteínas Celulares de Unión al Retinol/química , Proteínas Celulares de Unión al Retinol/metabolismo , Vitamina A/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Retinaldehído/química , Vitamina A/químicaRESUMEN
Human Cellular Retinol Binding Protein II (hCRBPII), a member of the intracellular lipid-binding protein family, is a monomeric protein responsible for the intracellular transport of retinol and retinal. Herein we report that hCRBPII forms an extensive domain-swapped dimer during bacterial expression. The domain-swapped region encompasses almost half of the protein. The dimer represents a novel structural architecture with the mouths of the two binding cavities facing each other, producing a new binding cavity that spans the length of the protein complex. Although wild-type hCRBPII forms the dimer, the propensity for dimerization can be substantially increased via mutation at Tyr60. The monomeric form of the wild-type protein represents the thermodynamically more stable species, making the domain-swapped dimer a kinetically trapped entity. Hypothetically, the wild-type protein has evolved to minimize dimerization of the folding intermediate through a critical hydrogen bond (Tyr60-Glu72) that disfavors the dimeric form.
Asunto(s)
Sustitución de Aminoácidos , Ácido Glutámico/química , Proteínas Celulares de Unión al Retinol/química , Tirosina/química , Secuencias de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Expresión Génica , Ácido Glutámico/metabolismo , Humanos , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios Proteicos , Pliegue de Proteína , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Celulares de Unión al Retinol/genética , Proteínas Celulares de Unión al Retinol/metabolismo , Termodinámica , Tirosina/metabolismoRESUMEN
Protein-chromophore interactions are a central component of a wide variety of critical biological processes such as color vision and photosynthesis. To understand the fundamental elements that contribute to spectral tuning of a chromophore inside the protein cavity, we redesigned human cellular retinol binding protein II (hCRBPII) to fully encapsulate all-trans-retinal and form a covalent bond as a protonated Schiff base. This system, using rational mutagenesis designed to alter the electrostatic environment within the binding pocket of the host protein, enabled regulation of the absorption maximum of the pigment in the range of 425 to 644 nanometers. With only nine point mutations, the hCRBPII mutants induced a systematic shift in the absorption profile of all-trans-retinal of more than 200 nanometers across the visible spectrum.