Dynamic Changes in Molecular Interactions along the Circadian Rhythm (Orr-LDRD Membrane Biology GC, PNNL Scope #90001)
EMSL Project ID
16714
Abstract
One of the most challenging tasks in cell biology is the study of the molecular interactions in the natural cellular environment in order to gain information about their dynamic changes in time and space. Currently, only a limited number of approaches are available for investigating molecular interactions in the living cell. Unlike in vitro biochemical methods, fluorescence resonance energy transfer (FRET) allows us to identify interactions among full-length proteins in their native cell, and quantify the spatial and temporal distribution of these interactions. FRET efficiency is proportional to the sixth power of the distance between two fluorescent molecules, thus leading to relatively large changes in FRET efficiency with subtle changes (Å resolution) in the distance between the two dyes. This property makes FRET a highly sensitive and reliable reporter of molecular interactions, which occur within less than 100 Å. Whereas conventional fluorescence imaging can identify spatiotemporal distributions of cellular proteins, and even suggest the occurrence of molecular interactions by fluorescence colocalization, only FRET can unambiguously report the existence of true molecular interactions. To study the molecular interactions in the living intact cyanobacterium as it goes through cycles of carbon and nitrogen fixations, and identify the spatial and temporal patterns of the interactions underlying circadian rhythm, we will establish FRET to report on molecular interaction dynamics in the intact cell. This effort is especially challenging when dealing with cyanobacteria, which contain their own pigments.
Project Details
Project type
Grand Challenge
Start Date
2005-10-17
End Date
2008-10-20
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Xu M, T. Ogawa, HB Pakrasi, and H Mi. 2008. Identification and localization of the CupB protein involved in constitutive CO2-uptake in the cyanobacterium, Synechocystis sp. strain PCC 6803. Plant and Cell Physiology 49(6): 994-997