Confocal fluorescence resonance energy transfer microscopy study of protein-protein interactions of lens crystallins in living cells.
Year: 2007
Type: article
Abstract: PURPOSE: To determine protein-protein interactions among lens crystallins in living cells. METHODS: Fluorescence resonance energy transfer (FRET) microscopy was used to visualize interactions in living cells directly. Two genes, one (alphaA-crystallin) fused with green fluorescence protein (GFP) and the other (each of the following genes: alphaB-, betaB2-, gammaC-crystallin, and R120G alphaB-crystallin mutant) fused with GFP variant red fluorescence protein (RED), were cotransfected into HeLa cells. After culture, confocal microscopy images were taken and FRET values were calculated. RESULTS: FRET occurs when the two proteins interact. The data show strong interactions between alphaA- and alphaB-crystallin and weak interactions between alphaA- and betaB2- or gammaC-crystallin, which is consistent with our previous two-hybrid system study. The R120G alphaB-crystallin mutant, however, showed significantly less FRET than wild-type alphaB-crystallin. There are also more R120G alphaB-crystallin transfected cells with protein aggregates than wild-type alphaB-crystallin transfected cells. Cotransfection with alphaA-crystallin could not rescue R120G alphaB-crystallin from aggregation. CONCLUSIONS: FRET microscopy gave excellent results on the protein-protein interactions among crystallins. It supports many previous studies and provides a novel technique for further study of protein-protein interactions among lens proteins including membrane and cytoskeletal proteins.
Source: PubMed Central
Institutions: Brigham and Women's Hospital, Harvard University
Language: English
Topic: Heat shock proteins research
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2294579860