RESUMO
Supercharged proteins exhibit high solubility and other desirable properties, but no engineered superpositively charged enzymes have previously been made. Superpositively charged variants of proteins such as green fluorescent protein have been efficiently encapsulated within Archaeoglobus fulgidus thermophilic ferritin (AfFtn). Encapsulation by supramolecular ferritin can yield systems with a variety of sequestered cargo. To advance applications in enzymology and green chemistry, we sought a general method for supercharging an enzyme that retains activity and is compatible with AfFtn encapsulation. The zinc metalloenzyme human carbonic anhydrase II (hCAII) is an attractive encapsulation target based on its hydrolytic activity and physiologic conversion of carbon dioxide to bicarbonate. A computationally designed variant of hCAII contains positively charged residues substituted at 19 sites on the protein's surface, resulting in a shift of the putative net charge from -1 to +21. This designed hCAII(+21) exhibits encapsulation within AfFtn without the need for fusion partners or additional reagents. The hCAII(+21) variant retains esterase activity comparable to the wild type and spontaneously templates the assembly of AfFtn 24mers around itself. The AfFtn-hCAII(+21) host-guest complex exhibits both greater activity and thermal stability when compared to hCAII(+21). Upon immobilization on a solid support, AfFtn-hCAII(+21) retains enzymatic activity and exhibits an enhancement of activity at elevated temperatures.
Assuntos
Proteínas Arqueais/química , Anidrase Carbônica II/química , Enzimas Imobilizadas/química , Ferritinas/química , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Archaeoglobus fulgidus/enzimologia , Anidrase Carbônica II/genética , Anidrase Carbônica II/isolamento & purificação , Anidrase Carbônica II/metabolismo , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/isolamento & purificação , Enzimas Imobilizadas/metabolismo , Ferritinas/genética , Ferritinas/isolamento & purificação , Ferritinas/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
Photoresponsive materials afford spatiotemporal control over desirable physical, chemical and biological properties. For advanced applications, there is need for molecular phototriggers that are readily incorporated within larger structures, and spatially-sequentially addressable with different wavelengths of visble light, enabling multiplexing. Here we describe spectrally tunable (λmax = 420-530 nm) ruthenium polypyridyl complexes functionalized with two photolabile nitrile ligands that present terminal alkynes for subsequent crosslinking reactions, including hydrogel formation. Two Ru crosslinkers were incorporated within a PEG-hydrogel matrix, and sequentially degraded by irradiation with 592 nm and 410 nm light.
