Red fluorescent proteins engineered from green fluorescent proteins.

Fluorescent proteins (FPs) form a fluorophore through autocatalysis from three consecutive amino acid residues within a polypeptide chain. The two major groups, green FPs (GFPs) and red FPs (RFPs), have distinct fluorophore structures; RFPs have an extended π-conjugation system with an additional double bond. However, due to the low sequence homology between the two groups, amino acid residues essential for determining the different fluorophore structures were unclear. Therefore, engineering a GFP into an RFP has been challenging, and the exact mechanism of how GFPs and RFPs achieve different autocatalytic reactions remained elucidated. Here, we show the conversion of two coral GFPs, AzamiGreen (AG) and mcavGFP, into RFPs by defined mutations. Structural comparison of AG and AzamiRed1.0, an AG-derived RFP, revealed that the mutations triggered drastic rearrangements in the interaction networks between amino acid residues around the fluorophore, suggesting that coordinated multisite mutations are required for the green-to-red conversion. As a result of the structural rearrangements, a cavity suitable for the entry of an oxygen molecule, which is necessary for the double bond formation of the red fluorophores, is created in the proximity of the fluorophore. We also show that a monomeric variant of AzamiRed1.0 can be used for labeling organelles and proteins in mammalian cells. Our results provide a structural basis for understanding the red fluorophore formation mechanism and demonstrate that protein engineering of GFPs is a promising way to create RFPs suitable for fluorescent tags.

Prevalence of Autoantibodies and the Efficacy of Immunotherapy for Autoimmune Cerebellar Ataxia.

OBJECTIVE: Autoimmune cerebellar ataxias were recently reported to be treatable. However, the proportion of patients with cortical cerebellar atrophy of unknown etiology with autoimmune-associated cerebellar ataxia and the actual effectiveness of immunotherapy in these diseases remain unknown. METHODS: We measured the level of autoantibodies (including anti-gliadin antibody, anti-glutamic acid decarboxylase (GAD) antibody, and anti-thyroid antibody) in 58 Japanese patients with cerebellar ataxia, excluding those with multiple system atrophy, hereditary spinocerebellar ataxia, cancer, or those who were receiving phenytoin, and the efficacy of immunotherapy was assessed. RESULTS: Thirty-one of 58 (53%) patients were positive for anti-GAD antibody, anti-gliadin antibody, or anti-thyroid antibody. Seven of the 12 anti-gliadin antibody-positive patients, three of the four anti-GAD antibody-positive patients, and three of the six anti-thyroid antibody-positive patients responded well to immunotherapy, indicating that 59% of patients with ataxia-associated antibody-positive cerebellar ataxia undergoing immunotherapy responded well. CONCLUSION: Some patients with cerebellar ataxia have autoimmune conditions and diagnosing autoimmune cerebellar ataxia is therefore an important component in the care of patients with this disease entity.