RESUMEN
A NADPH-dependent blue fluorescent protein from Vibrio vulnificus CKM-1 (BFPvv) emits blue fluorescence under UV-exposure. Previously, the BFPvvD7 mutant generated by directed evolution displayed a fourfold enhancement in fluorescent intensity. Herein, a further increase in fluorescence in the new BFPvvD8 mutant, with three additional mutations from BFPvvD7, was made. To understand the underlying mechanism of the increased fluorescent intensity of BFPvv, we solved the BFPvvD8-NADPH complex structure. Accompanied with lifetime detection, we proposed that the enhanced intensity is related to the conformational change caused by a glycine residue (Gly176) mutated to other non-glycine residues at a turn close to the NADPH binding site. We also observed the Förster resonance energy transfer (FRET) from our BFPvvD8 to each of the GFP-like fluorescent proteins, mTFP1 and EGFP, joined by an eight-residue linker between the N-terminal of BFPvvD8 and the C-terminal of GFPs. Taken together, with the newly solved BFPvvD8 structure, our results not only provide new considerations within the rational-based protein engineering of this NADPH-dependent BFP, but also suggest that BFPvvD8 could be a potential candidate in FRET-based biosensor techniques.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Glicina/química , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , NADP/química , Conformación Proteica , Secuencia de Aminoácidos , Técnicas Biosensibles , Dominio Catalítico , Color , Fluorescencia , Transferencia Resonante de Energía de Fluorescencia/métodos , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Vibrio vulnificus/química , Difracción de Rayos XRESUMEN
Elimination of redundant synapses formed early in development and strengthening of necessary connections are crucial for shaping functional neural circuits. Purkinje cells (PCs) in the neonatal cerebellum are innervated by multiple climbing fibers (CFs) with similar strengths. A single CF is strengthened whereas the other CFs are eliminated in each PC during postnatal development. The underlying mechanisms, particularly for the strengthening of single CFs, are poorly understood. Here we report that progranulin, a multi-functional growth factor implicated in the pathogenesis of frontotemporal dementia, strengthens developing CF synaptic inputs and counteracts their elimination from postnatal day 11 to 16. Progranulin derived from PCs acts retrogradely onto its putative receptor Sort1 on CFs. This effect is independent of semaphorin 3A, another retrograde signaling molecule that counteracts CF synapse elimination. We propose that progranulin-Sort1 signaling strengthens and maintains developing CF inputs, and may contribute to selection of single "winner" CFs that survive synapse elimination.