Because only the limited population of photoactivated molecules exhibit noticeable fluorescence, their lifetime and behavior can be followed independently of other proteins that are newly synthesized.
These effects result in the direct and controlled highlighting of distinct molecular pools within the cell. Photoconversion optical highlighters, on the other hand, undergo a change in the fluorescence emission bandwidth profile upon optically-induced changes to the chromophore. Appropriately termed molecular or optical highlighters, photoactivated fluorescent proteins generally display little or no initial fluorescence under excitation at the imaging wavelength, but dramatically increase their fluorescence intensity after activation by irradiation at a different (usually lower) wavelength. Protein chromophores that can be activated to initiate fluorescence emission from a quiescent state (a process known as photoactivation), or are capable of being optically converted from one fluorescence emission bandwidth to another ( photoconversion), represent perhaps the most promising approach to the in vivo investigation of protein lifetimes, transport, and turnover rates. This ratio can then be used to gather temporal data for gene expression investigations. The advancing age of a chimeric protein fused with the timer moiety can be determined by a continual decrease in the observed ratio of green to red fluorescence. Initially, the timer protein produces a green-emitting fluorophore (peak at 500 nanometers similar to green fluorescent protein), but over a period of several hours, the fluorophore is slowly converted to a species that emits in the yellow-red spectral region (with a maximum at 580 nanometers). One of the first and most basic examples of probes in this class is a fluorescent timer protein that was generated by random mutagenesis of a red-emitting coral reef fluorescent protein derived from Discosoma striata (first designated as mutant drFP583, but now commonly referred to as DsRed). Unfortunately, the rapid decay of fluorescence results in very low signal levels, which presents a compromise between accurately monitoring protein dynamics and acquiring suitable digital images.Ī more useful solution is to employ fluorescent proteins whose spectral properties change with time, illumination wavelength, or a similar variable that can be controlled by the investigator. Thus, a chimeric fluorescent protein that is rapidly turned over by proteolysis can be segregated into classes of younger, freshly synthesized fluorescent molecules, and older protein chimeras that have lost their fluorescence due to degradation of the chromophore.
As a result, a large ensemble of proteins at different stages in their lifetime are being observed at any particular moment with the traditional fluorescent proteins that exhibit stable emission spectral profiles.Ī simple approach to characterize protein expression timing or lifetime is to utilize destabilized fluorescent protein variants that can be readily differentiated by fluorescence intensity. However, as useful as fluorescent proteins have been for investigations of sub-cellular targets and the functional properties of gene products in living cells, many applications designed to determine protein turnover rates or the analysis of temporal expression patterns are virtually impossible with conventional fluorescent proteins due to the fact that they are continuously being recycled (synthesized, folded, and subsequently degraded) within the cell.
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