Oligomerization of DsRed is required for the generation of a functional red fluorescent chromophore.
The coral red fluorescent protein (DsRed) absorbs and emits light at much higher wavelengths than the structurally homologous green fluorescent protein, raising questions about the properties of its chromophore. We have analyzed the relationship between the aggregation state and fluorescence of native, 6-histidine-tagged, or maltose-binding protein-fused DsRed. In all cases, newly synthesized DsRed molecules were largely monomeric and devoid of covalently closed chromophores. Maturation in vitro induces the expression of red fluorescent chromophores but only in oligomeric forms of the protein, whereas monomers are essentially devoid of fluorescence. NaOH-denatured samples demonstrated a generalized breakdown of the DsRed oligomers to monomers, which refolded after neutralization into weakly green fluorescent and still monomeric species. Red fluorescent chromophores were regenerated only upon oligomerization. These findings demonstrate that 'red' chromophores form and are functional only as oligomers, and suggest that the smallest red fluorescent functional unit is a dimer. A comparison of alkali-, acid- and guanidinium-denatured DsRed indicates that stabilization of the DsRed chromophore by concerted steps of folding and oligomerization may play a critical role in its maturation process.
Sacchetti
Andrea
A
Laboratory of Experimental Oncology, Department of Cell Biology and Oncology, Istituto di Ricerche Farmacologiche Mario Negri-Consorzio Mario Negri Sud, 66030, Chieti, Santa Maria Imbaro, Italy.
Subramaniam
Vinod
V
Jovin
Thomas M
TM
Alberti
Saverio
S
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins / Q000235:genetics; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins; D005453:Fluorescence; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000502:physiology; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013329:Structure-Activity Relationship
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C413663:fluorescent protein 583; C461599:maltose transport system, E coli