The 2.1Å Crystal Structure of the Far-red Fluorescent Protein HcRed: Inherent Conformational Flexibility of the Chromophore

Wilmann, P.G.; Petersen, Jan; Pettikiriarachchi, Anne; Buckle, Ashley M.; Smith, Sean C.; Olsen, Seth; Perugini, Matthew A.; Devenish, Rodney J.; Prescott, Mark; Rossjohn, Jamie

doi:10.1016/j.jmb.2005.03.020

Cited by 82 publications

(145 citation statements)

References 43 publications

Supporting

Mentioning

134

Contrasting

Order By: Relevance

“…No similar hydrogen bond has previously been observed or reported in the other red fluorescent proteins such as eqFP611, 9 HcRed, 23 DsRed, 6,7 zRFP574, 14 and other mFruits. 12 Indeed, the residue corresponding to Glu16 in mPlum is hydrophobic in all known red fluorescent proteins, that is valine, leucine, isoleucine, and phenolalanine.…”

Section: Unique Hydrogen Bondsupporting

confidence: 73%

Unique interactions between the chromophore and glutamate 16 lead to far‐red emission in a red fluorescent protein

et al. 2009

View full text Add to dashboard Cite

mPlum is a far-red fluorescent protein with emission maximum at~650 nm and was derived by directed evolution from DsRed. Two residues near the chromophore, Glu16 and Ile65, were previously revealed to be indispensable for the far-red emission. Ultrafast time-resolved fluorescence emission studies revealed a time dependent shift in the emission maximum, initially about 625 nm, to about 650 nm over a period of 500 ps. This observation was attributed to rapid reorganization of the residues solvating the chromophore within mPlum. Here, the crystal structure of mPlum is described and compared with those of two blue shifted mutants mPlum-E16Q and -I65L. The results suggest that both the identity and precise orientation of residue 16, which forms a unique hydrogen bond with the chromophore, are required for far-red emission. Both the far-red emission and the time dependent shift in emission maximum are proposed to result from the interaction between the chromophore and Glu16. Our findings suggest that significant red shifts might be achieved in other fluorescent proteins using the strategy that led to the discovery of mPlum.

show abstract

Section: Unique Hydrogen Bondsupporting

confidence: 73%

Unique interactions between the chromophore and glutamate 16 lead to far‐red emission in a red fluorescent protein

et al. 2009

View full text Add to dashboard Cite

show abstract

“…Different geometric restraint schemes were tested to determine the optimal geometry of the group (63)C ␣ ϭN-C(O)-C ␣ (62) bridging the C ␣ atoms of the first chromophore residue Met 63 and the preceding Phe 62 . Similar to HcRed (38), it exhibits, at optimal fit to electron density, considerable deviation from planarity with torsion angle around the quasi-peptide N-C(O) bond in a range 20 -35°. Moreover, similarly to other red and far-red fluorescent proteins (18,19,22,38), the C(O)-N-C ␣ bond angle of the linkage in both chromophore isomers is strongly linearized, in the range of 140 -160°.…”

Section: Resultsmentioning

confidence: 94%

A Crystallographic Study of Bright Far-Red Fluorescent Protein mKate Reveals pH-induced cis-trans Isomerization of the Chromophore

Pletnev

Shcherbo

Chudakov

et al. 2008

Journal of Biological Chemistry

101

159

View full text Add to dashboard Cite

The far-red fluorescent protein mKate ( ex , 588 nm; em , 635 nm; chromophore-forming triad Met 63 -Tyr 64 -Gly 65 ), originating from wild-type red fluorescent progenitor eqFP578 (sea anemone Entacmaea quadricolor), is monomeric and characterized by the pronounced pH dependence of fluorescence, relatively high brightness, and high photostability. The protein has been crystallized at a pH ranging from 2 to 9 in three space groups, and four structures have been determined by x-ray crystallography at the resolution of 1.75-2.6 Å . The pH-dependent fluorescence of mKate has been shown to be due to reversible cis-trans isomerization of the chromophore phenolic ring. In the non-fluorescent state at pH 2.0, the chromophore of mKate is in the trans-isomeric form. Green fluorescent proteins (GFP)2 and GFP-like proteins (FP) have become important noninvasive tools for visualization and monitoring of the internal processes within cells or whole organisms, such as gene expression, monitoring the cellular pH, ion concentration, embryogenesis, inflammatory processes, tracking protein trafficking, the migration of parasites within a host, etc (1-13). Fluorescent proteins can be used to visualize many types of cancer processes, including primary tumor growth, tumor cell motility and invasion, metastatic seeding and colonization, angiogenesis, and interactions between the tumor and its host microenvironment (14 -16). FPs might be very useful in real-time testing of the efficacy of cancer drugs in animal models of human cancer.The extensive spectral diversity of fluorescent proteins arises mostly from variations in the chemical structure of the mature chromophore and in the stereochemistry of its adjacent environment. The FP chromophore forms autocatalytically in vivo and in vitro from three residues, Xxx-Tyr-Gly, without need for any cofactors or enzymes, except for molecular oxygen (17). In most cases, the post-translational modification results in a blue/green emitting state, characterized by formation of an imidazolinone heterocycle with a p-hydroxybenzylidene substituent. Often, the reaction chain propagates further with formation of an additional N-acylimine double bond, which extends the conjugation of the chromophore electronic system and results in a bathochromic shift in spectra (18 -22).Proteins that emit red, and especially far-red light, are of particular interest (13). The longer wavelength light extends the range of fluorescence resonance energy transfer (FRET)-based applications and causes fewer damaging events to proteins and DNA because of its lower energy. The most favorable "optical window" for the visualization in living tissues is ϳ650 -1100 nm (23). Light with wavelength longer than 1100 nm is absorbed by water. Detection of fluorescence from proteins with emission peaks much shorter than 650 nm encounters the problem of interfering cellular autofluorescence. At present the brightest red fluorescent proteins have emission maxima too far from the preferred "optical window." Besides, their excitation maxima...

show abstract

“…The latter cells do not express endogenous MAL. The tandem dimer of the far-red FP HcRED (DiHcRED) (Wilmann et al, 2005), mCFP, or mYFP was attached to the cytosolic N terminus of human MAL, its homologue MAL2 (de Marco et al, 2002) (Marazuela et al, 2004b).…”

Section: Resultsmentioning

confidence: 99%

Clustering and Lateral Concentration of Raft Lipids by the MAL Protein

Magal

Yaffe

Shepshelovich

et al. 2009

MBoC

View full text Add to dashboard Cite

MAL, a compact hydrophobic, four-transmembrane-domain apical protein that copurifies with detergent-resistant membranes is obligatory for the machinery that sorts glycophosphatidylinositol (GPI)-anchored proteins and others to the apical membrane in epithelia. The mechanism of MAL function in lipid-raft-mediated apical sorting is unknown. We report that MAL clusters formed by two independent procedures-spontaneous clustering of MAL tagged with the tandem dimer DiHcRED (DiHcRED-MAL) in the plasma membrane of COS7 cells and antibody-mediated cross-linking of FLAG-tagged MAL-laterally concentrate markers of sphingolipid rafts and exclude a fluorescent analogue of phosphatidylethanolamine. Site-directed mutagenesis and bimolecular fluorescence complementation analysis demonstrate that MAL forms oligomers via xx intramembrane protein-protein binding motifs. Furthermore, results from membrane modulation by using exogenously added cholesterol or ceramides support the hypothesis that MAL-mediated association with raft lipids is driven at least in part by positive hydrophobic mismatch between the lengths of the transmembrane helices of MAL and membrane lipids. These data place MAL as a key component in the organization of membrane domains that could potentially serve as membrane sorting platforms.

show abstract

The 2.1Å Crystal Structure of the Far-red Fluorescent Protein HcRed: Inherent Conformational Flexibility of the Chromophore

Cited by 82 publications

References 43 publications

Unique interactions between the chromophore and glutamate 16 lead to far‐red emission in a red fluorescent protein

Unique interactions between the chromophore and glutamate 16 lead to far‐red emission in a red fluorescent protein

A Crystallographic Study of Bright Far-Red Fluorescent Protein mKate Reveals pH-induced cis-trans Isomerization of the Chromophore

Clustering and Lateral Concentration of Raft Lipids by the MAL Protein

Contact Info

Product

Resources

About