Based on the mechanism for chromophore formation in red fluorescent proteins, we developed three mCherry-derived monomeric variants, called fluorescent timers (FTs), that change their fluorescence from the blue to red over time. These variants exhibit distinctive fast, medium and slow blue-to-red chromophore maturation rates that depend on the temperature. At 37 °C, the maxima of the blue fluorescence are observed at 0.25, 1.2 and 9.8 h for the purified fast-FT, medium-FT and slow-FT, respectively. The half-maxima of the red fluorescence are reached at 7.1, 3.9 and 28 h, respectively. The FTs show similar timing behavior in bacteria, insect and mammalian cells. Medium-FT allowed for tracking of the intracellular dynamics of the lysosome-associated membrane protein type 2A (LAMP-2A) and determination of its age in the targeted compartments. The results indicate that LAMP-2A transport through the plasma membrane and early or recycling endosomes to lysosomes is a major pathway for LAMP-2A trafficking.Monomeric fluorescent proteins of various emission wavelengths have become invaluable tools for studying the spatial behavior of intracellular molecules, including their localization and interaction 1 . To visualize temporal and spatial molecular events, FTs 2 , which change their emission wavelengths over time, could be especially valuable. The only currently available FT is DsRed-Timer FT (also known as DsRed-E5) 3 ; however, it is a tetramer, which prevents its application as a protein fusion tag. Nevertheless, the tetrameric state of the DsRed-Timer does not limit its use to study gene activities 4 , relative age of organelles 5 and cell differentiation 3 .It has been suggested that a red DsRed-like chromophore in the red fluorescent proteins (RFPs) results from an oxidation of a protonated blue form of the GFP-like chromophore, not from the green anionic form, which is a dead-end product 6 . This suggested scheme for red chromophore maturation provides a basis for developing monomeric FTs that change their color from blue to red. The most suitable templates for this appear to be the monomeric variants of DsRed 7 . One of these variants, mCherry, was chosen for a directed molecular evolution to develop three monomeric FTs with different maturation rates between the protonated blue GFP-like and the anionic red DsRed-like chromophore states.FTs can be used as molecular genetically encoded tools to study trafficking of different cellular proteins and to provide accurate insight into the timing of intracellular processes. The sequence NIH Public Access Author ManuscriptNat Chem Biol. Author manuscript; available in PMC 2010 February 1. Published in final edited form as:Nat Chem Biol. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript of events during trafficking of different cellular proteins before they reach their final compartment has often been the subject of contradictory investigations. An example of a longstanding dilemma is the contribution of different pathways to trafficking a...

Far-red fluorescent proteins are required for deep-tissue and whole-animal imaging and multicolor labeling in the red wavelength range, as well as probes excitable with standard red lasers in flow cytometry and fluorescence microscopy. Rapidly evolving superresolution microscopy based on the stimulated emission depletion approach also demands genetically encoded monomeric probes to tag intracellular proteins at the molecular level. Based on the monomeric mKate variant, we have developed a far-red TagRFP657 protein with excitation/emission maxima at 611/657 nm. TagRFP657 has several advantages over existing monomeric far-red proteins including higher photostability, better pH stability, lower residual green fluorescence, and greater efficiency of excitation with red lasers. The red-shifted excitation and emission spectra, as compared to other far-red proteins, allows utilizing TagRFP657 in flow cytometry and fluorescence microscopy simultaneously with orange or near-red fluorescence proteins. TagRFP657 is shown to be an efficient protein tag for the superresolution fluorescence imaging using a commercially available stimulated emission depletion microscope.

Summary Maturation of red fluorescent proteins has been shown to proceed through a blue intermediate. We determined the 2.10 Å crystal structure of the red fluorescent protein TagRFP and the 1.95 Å crystal structure of its derivative, the blue fluorescent protein mTagBFP. The crystallographic analysis is consistent with a model in which TagRFP has the trans coplanar anionic chromophore with the conjugated π-electron system, similar to that of DsRed-like chromophores. Refined conformation of mTagBFP suggests the presence of an N-acylimine functionality in its chromophore and single Cα-Cβ bond in the Tyr64 side chain. Mass spectrum of mTagBFP chromophore-bearing peptide indicates a loss of 20 Da upon maturation, while tandem mass spectrometry reveals that the Cα-N bond in Leu63 is oxidized. These data, together with mutagenesis of mTagBFP, indicate that mTagBFP has a new type of the chromophore, N-[(5-hydroxy-1H-imidazole-2-yl)methylidene]acetamide.

Most GFP-like fluorescent proteins exhibit small Stokes shifts (10–45 nm) due to rigidity of the chromophore environment that excludes non-fluorescent relaxation to a ground state. An unusual near-infrared derivative of the red fluorescent protein mKate, named TagRFP675, exhibits the Stokes shift, which is 30 nm extended comparing to that of the parental protein. In physiological conditions, TagRFP675 absorbs at 598 nm and emits at 675 nm that makes it the most red-shifted protein of the GFP-like protein family. In addition, its emission maximum strongly depends on the excitation wavelength. Structures of TagRFP675 revealed the common DsRed-like chromophore, which, however, interacts with the protein matrix via an extensive network of hydrogen bonds capable of large flexibility. Based on the spectroscopic, biochemical, and structural analysis we suggest that the rearrangement of the hydrogen bond interactions between the chromophore and the protein matrix is responsible for the TagRFP675 spectral properties.

A hallmark of aging is an imbalance between production and clearance of reactive oxygen species and increased levels of oxidatively damaged biomolecules. Herein we demonstrate that splenic and nodal antigen presenting cells purified from old mice accumulate oxidatively modified proteins with side chain carbonylation, advanced glycation end products and lipid peroxidation. We show further that the endosomal accumulation of oxidatively modified proteins interferes with the efficient processing of exogenous antigens and degradation of macroautophagy-delivered proteins. In support of a causative role for oxidized products in the inefficient immune response, a decrease in oxidative stress improved the adaptive immune response to immunizing antigens. These findings underscore a previously unrecognized negative effect of age-dependent changes in cellular proteostasis on the immune response.