Characterization of multimodal hydrophobic interaction chromatography media useful for isolation of green fluorescent proteins with small structural differences

2009 ◽  
Vol 22 (2) ◽  
pp. 104-109 ◽  
Author(s):  
Kristian Becker ◽  
Elisabeth Hallgren ◽  
Enrique Carredano ◽  
Ronnie Palmgren ◽  
Leif Bülow
Keyword(s):  
Hydrophobic Interaction ◽  
Fluorescent Proteins ◽  
Hydrophobic Interaction Chromatography ◽  
Green Fluorescent Proteins ◽  
Structural Differences ◽  
Green Fluorescent

Partitioning and Characterization of Tyrosine-Tagged Green Fluorescent Proteins in Aqueous Two-Phase Systems

2004 ◽  
Vol 20 (3) ◽  
pp. 793-798 ◽  
Author(s):  
S. Fexby ◽  
A. Nilsson ◽  
G. Hambraeus ◽  
F. Tjerneld ◽  
L. Bulow
Keyword(s):  
Fluorescent Proteins ◽  
Green Fluorescent Proteins ◽  
Two Phase ◽  
Aqueous Two Phase Systems ◽  
Phase Systems ◽  
Green Fluorescent

scholarly journals Characterization of green fluorescent proteins by 193 nm ultraviolet photodissociation mass spectrometry

PROTEOMICS ◽  
2014 ◽  
Vol 14 (10) ◽  
pp. 1165-1173 ◽  
Author(s):  
Joe R. Cannon ◽  
Christien Kluwe ◽  
Andrew Ellington ◽  
Jennifer S. Brodbelt
Keyword(s):  
Mass Spectrometry ◽  
Fluorescent Proteins ◽  
Green Fluorescent Proteins ◽  
Ultraviolet Photodissociation ◽  
Green Fluorescent ◽  
193 Nm

scholarly journals Coherent Dynamics of Photoexcited Green Fluorescent Proteins

2001 ◽  
Vol 86 (15) ◽  
pp. 3439-3442 ◽  
Author(s):  
Riccardo A. G. Cinelli ◽  
Valentina Tozzini ◽  
Vittorio Pellegrini ◽  
Fabio Beltram ◽  
Giulio Cerullo ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Green Fluorescent Proteins ◽  
Coherent Dynamics ◽  
Green Fluorescent

scholarly journals Shedding light on the dark and weakly fluorescent states of green fluorescent proteins

1999 ◽  
Vol 96 (11) ◽  
pp. 6177-6182 ◽  
Author(s):  
W. Weber ◽  
V. Helms ◽  
J. A. McCammon ◽  
P. W. Langhoff
Keyword(s):  
Fluorescent Proteins ◽  
Green Fluorescent Proteins ◽  
Green Fluorescent

scholarly journals Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

2017 ◽  
Vol 114 (11) ◽  
pp. E2146-E2155 ◽  
Author(s):  
Chi-Yun Lin ◽  
Johan Both ◽  
Keunbong Do ◽  
Steven G. Boxer
Keyword(s):  
Quantum Yield ◽  
Protein Interactions ◽  
Fluorescent Proteins ◽  
Point Mutations ◽  
Photoactive Yellow Protein ◽  
Green Fluorescent Proteins ◽  
Protein Protein Interactions ◽  
Low Efficiency ◽  
Green Fluorescent ◽  
Rate Limiting

Split GFPs have been widely applied for monitoring protein–protein interactions by expressing GFPs as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another. Although this complementation is typically irreversible, it has been shown previously that light accelerates dissociation of a noncovalently attached β-strand from a circularly permuted split GFP, allowing the interaction to be reversible. Reversible complementation is desirable, but photodissociation has too low of an efficiency (quantum yield <1%) to be useful as an optogenetic tool. Understanding the physical origins of this low efficiency can provide strategies to improve it. We elucidated the mechanism of strand photodissociation by measuring the dependence of its rate on light intensity and point mutations. The results show that strand photodissociation is a two-step process involving light-activated cis-trans isomerization of the chromophore followed by light-independent strand dissociation. The dependence of the rate on temperature was then used to establish a potential energy surface (PES) diagram along the photodissociation reaction coordinate. The resulting energetics–function model reveals the rate-limiting process to be the transition from the electronic excited-state to the ground-state PES accompanying cis-trans isomerization. Comparisons between split GFPs and other photosensory proteins, like photoactive yellow protein and rhodopsin, provide potential strategies for improving the photodissociation quantum yield.

scholarly journals Multiphoton Bleaching of Red Fluorescent Proteins and the Ways to Reduce It

2022 ◽  
Vol 23 (2) ◽  
pp. 770
Author(s):  
Mikhail Drobizhev ◽  
Rosana S. Molina ◽  
Jacob Franklin
Keyword(s):  
Chemical Structure ◽  
Fluorescent Proteins ◽  
Multiphoton Ionization ◽  
Excitation Power ◽  
Photon Absorption ◽  
Green Fluorescent Proteins ◽  
Two Photon Absorption ◽  
Two Photon ◽  
Red Fluorescent Proteins ◽  
Green Fluorescent

Red fluorescent proteins and biosensors built upon them are potentially beneficial for two-photon laser microscopy (TPLM) because they can image deeper layers of tissue, compared to green fluorescent proteins. However, some publications report on their very fast photobleaching, especially upon excitation at 750–800 nm. Here we study the multiphoton bleaching properties of mCherry, mPlum, tdTomato, and jREX-GECO1, measuring power dependences of photobleaching rates K at different excitation wavelengths across the whole two-photon absorption spectrum. Although all these proteins contain the chromophore with the same chemical structure, the mechanisms of their multiphoton bleaching are different. The number of photons required to initiate a photochemical reaction varies, depending on wavelength and power, from 2 (all four proteins) to 3 (jREX-GECO1) to 4 (mCherry, mPlum, tdTomato), and even up to 8 (tdTomato). We found that at sufficiently low excitation power P, the rate K often follows a quadratic power dependence, that turns into higher order dependence (K~Pα with α > 2) when the power surpasses a particular threshold P*. An optimum intensity for TPLM is close to the P*, because it provides the highest signal-to-background ratio and any further reduction of laser intensity would not improve the fluorescence/bleaching rate ratio. Additionally, one should avoid using wavelengths shorter than a particular threshold to avoid fast bleaching due to multiphoton ionization.

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