A Guide to Fluorescent Protein FRET Pairs

Bajar, Bryce T.; Wang, Emily S.; Zhang, Shu; Lin, Michael Z.; Chu, Jun

doi:10.3390/s16091488

Cited by 377 publications

(388 citation statements)

References 122 publications

Supporting

Mentioning

364

Contrasting

Unclassified

Order By: Relevance

“…For reference, EC of YGFP and GFPuv calculated in our laboratory were 53,300 M -1 cm -1 and 27,800 M -1 cm -1 , respectively; both ECs were slightly lower than the published values of 56,000 M -1 cm -1 [3] and 30,500 M -1 cm -1 [21]. On the other hand, the QY of GFPuv calculated in our laboratory was 8.0, which was almost identical to the published value of 0.79 [21].…”

Section: Resultsmentioning

confidence: 66%

Molecular evolution of versatile derivatives from a GFP-like protein in the marine copepod Chiridius poppei

Shimizu¹,

Shiratori²,

Horii³

et al. 2017

PLoS ONE

View full text Add to dashboard Cite

Fluorescent proteins are now indispensable tools in molecular research. They have also been adapted for a wide variety of uses in cases involving creative applications, including textiles, aquarium fish, and ornamental plants. Our colleagues have previously cloned a yellow GFP-like protein derived from the marine copepod Chiridius poppei (YGFP), and moreover, succeeded in generating transgenic flowers with clearly visible fluorescence, without the need for high-sensitivity imaging equipment. However, due to the low Stokes shift of YGFP (10 nm), it is difficult to separate emitted light of a labeled object from the light used for excitation; hence, limitations for various applications remain. In this study, which was aimed at developing YGFP mutants with increased Stokes shifts, we conducted stepwise molecular evolution experiments on YGFP by screening random mutations at three key amino acids, based on their fluorescent characteristics and structural stabilities, followed by optimization of their fluorescence output by DNA shuffling of the entire coding sequence. We successfully identified an eYGFPuv that had an excitation maximum in UV wavelengths and a 24-fold increase in fluorescence intensity compared to the previously reported YGFP mutant (H52D). In addition, eYGFPuv exhibited almost 9-fold higher fluorescence intensity compared to the commercially available GFPuv when expressed in human colon carcinoma HCT116 cells and without any differences in cytotoxicity. Thus, this novel mutant with the desirable characteristics of bright fluorescence, long Stokes shift, and low cytotoxity, may be particularly well suited to a variety of molecular and biological applications.

show abstract

Section: Resultsmentioning

confidence: 66%

Molecular evolution of versatile derivatives from a GFP-like protein in the marine copepod Chiridius poppei

Shimizu¹,

Shiratori²,

Horii³

et al. 2017

PLoS ONE

View full text Add to dashboard Cite

show abstract

“…apFRET is dependent on emission energy transfer from the co‐expressed fluorescent donor to the acceptor, such that excitation of the acceptor will quench donor emission when the proteins are in close proximity (Bajar et al. ; Martin et al. ).…”

Section: Resultsmentioning

confidence: 99%

“…FRET efficiency was measured via increased donor (GFP) emission following acceptor photobleaching (mCh), using the formula:

E = 1 - (I_{normalpre} / I_{normalpost})

where I pre and I post are the fluorescent intensities of the donor before and after photobleaching (Bajar et al. ). Mean fluorescent intensities within the ROI were determined using Fiji (Schindelin et al.…”

Section: Methodsmentioning

confidence: 99%

The sigma‐1 receptor behaves as an atypical auxiliary subunit to modulate the functional characteristics of Kv1.2 channels expressed in HEK293 cells

et al. 2019

View full text Add to dashboard Cite

Expression of Kv1.2 within Kv1.x potassium channel complexes is critical in maintaining appropriate neuronal excitability and determining the threshold for action potential firing. This is attributed to the interaction of Kv1.2 with a hitherto unidentified protein that confers bimodal channel activation gating, allowing neurons to adapt to repetitive trains of stimulation and protecting against hyperexcitability. One potential protein candidate is the sigma‐1 receptor (Sig‐1R), which regulates other members of the Kv1.x channel family; however, the biophysical nature of the interaction between Sig‐1R and Kv1.2 has not been elucidated. We hypothesized that Sig‐1R may regulate Kv1.2 and may further act as the unidentified modulator of Kv1.2 activation. In transiently transfected HEK 293 cells, we found that ligand activation of the Sig‐1R modulates Kv1.2 current amplitude. More importantly, Sig‐1R interacts with Kv1.2 in baseline conditions to influence bimodal activation gating. These effects are abolished in the presence of the auxiliary subunit Kv β 2 and when the Sig‐1R mutation underlying ALS 16 (Sig‐1R‐E102Q), is expressed. These data suggest that Kv β 2 occludes the interaction of Sig‐1R with Kv1.2, and that E102 may be a residue critical for Sig‐1R modulation of Kv1.2. The results of this investigation describe an important new role for Sig‐1R in the regulation of neuronal excitability and introduce a novel mechanism of pathophysiology in Sig‐1R dysfunction.

show abstract

“…1a). These proteins are bright and photostable 17 , and Clover is better separated from mTurquoise2 than other GFPs due to its sharper and more red-shifted spectra 18 . To confirm orthogonality, we expressed mTurquoise2, Clover, mKO2, or mMaroon1 fused to a nuclear protein and imaged cells in cyan, green, orange, and far-red channels.…”

mentioning

confidence: 99%

Fluorescent indicators for simultaneous reporting of all four cell cycle phases

et al. 2016

Self Cite

View full text Add to dashboard Cite

A robust method for simultaneous visualization of all four cell cycle phases in living cells would be highly desirable. We developed an intensiometric reporter of the S/G2 transition and engineered a far-red fluorescent protein, mMaroon1, to visualize chromatin condensation in mitosis. We combined these new reporters with the previously described Fucci system to create Fucci4, a set of four orthogonal fluorescent indicators that together resolve all cell cycle phases.

show abstract

A Guide to Fluorescent Protein FRET Pairs

Cited by 377 publications

References 122 publications

Molecular evolution of versatile derivatives from a GFP-like protein in the marine copepod Chiridius poppei

Molecular evolution of versatile derivatives from a GFP-like protein in the marine copepod Chiridius poppei

The sigma‐1 receptor behaves as an atypical auxiliary subunit to modulate the functional characteristics of Kv1.2 channels expressed in HEK293 cells

Fluorescent indicators for simultaneous reporting of all four cell cycle phases

Contact Info

Product

Resources

About