scholarly journals LSSmScarlet, dCyRFP2s, dCyOFP2s and CRISPRed2s, Genetically Encoded Red Fluorescent Proteins with a Large Stokes Shift

2021 ◽  
Vol 22 (23) ◽  
pp. 12887
Author(s):  
Oksana M. Subach ◽  
Anna V. Vlaskina ◽  
Yuliya K. Agapova ◽  
Pavel V. Dorovatovskii ◽  
Alena Y. Nikolaeva ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Rational Design ◽  
Fluorescent Proteins ◽  
Stokes Shift ◽  
Confocal Imaging ◽  
Large Stokes Shift ◽  
Two Photon ◽  
Dual Color ◽  
Red Fluorescent Proteins

Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the right LSSRFP for a given application is hampered by the lack of a side-by-side comparison of the LSSRFPs’ performance. In this study, we employed rational design and random mutagenesis to convert conventional bright RFP mScarlet into LSSRFP, called LSSmScarlet, characterized by excitation/emission maxima at 470/598 nm. In addition, we utilized the previously reported LSSRFPs mCyRFP1, CyOFP1, and mCRISPRed as templates for directed molecular evolution to develop their optimized versions, called dCyRFP2s, dCyOFP2s and CRISPRed2s. We performed a quantitative assessment of the developed LSSRFPs and their precursors in vitro on purified proteins and compared their brightness at 488 nm excitation in the mammalian cells. The monomeric LSSmScarlet protein was successfully utilized for the confocal imaging of the structural proteins in live mammalian cells and multicolor confocal imaging in conjugation with other FPs. LSSmScarlet was successfully applied for dual-color two-photon imaging in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet protein at the resolution of 1.4 Å that revealed a hydrogen bond network supporting excited-state proton transfer (ESPT). Quantum mechanics/molecular mechanics molecular dynamic simulations confirmed the ESPT mechanism of a large Stokes shift. Structure-guided mutagenesis revealed the role of R198 residue in ESPT that allowed us to generate a variant with improved pH stability. Finally, we showed that LSSmScarlet protein is not appropriate for STED microscopy as a consequence of LSSRed-to-Red photoconversion with high-power 775 nm depletion light.

Rational Design of a Two-Photon Ratiometric Fluorescent Probe for Hypochlorous Acid with a Large Stokes Shift

2020 ◽  
Vol 92 (16) ◽  
pp. 11029-11034 ◽  
Author(s):  
Long He ◽  
Haiqing Xiong ◽  
Benhua Wang ◽  
Yun Zhang ◽  
Jingpei Wang ◽  
...  
Keyword(s):  
Fluorescent Probe ◽  
Rational Design ◽  
Hypochlorous Acid ◽  
Stokes Shift ◽  
Large Stokes Shift ◽  
Two Photon ◽  
Ratiometric Fluorescent Probe

scholarly journals Engineering ESPT Pathways Based on Structural Analysis of LSSmKate Red Fluorescent Proteins with Large Stokes Shift

2010 ◽  
Vol 132 (31) ◽  
pp. 10762-10770 ◽  
Author(s):  
Kiryl D. Piatkevich ◽  
Vladimir N. Malashkevich ◽  
Steven C. Almo ◽  
Vladislav V. Verkhusha
Keyword(s):  
Structural Analysis ◽  
Fluorescent Proteins ◽  
Stokes Shift ◽  
Large Stokes Shift ◽  
Red Fluorescent Proteins

scholarly journals Monomeric red fluorescent proteins with a large Stokes shift

2010 ◽  
Vol 107 (12) ◽  
pp. 5369-5374 ◽  
Author(s):  
K. D. Piatkevich ◽  
J. Hulit ◽  
O. M. Subach ◽  
B. Wu ◽  
A. Abdulla ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Stokes Shift ◽  
Large Stokes Shift ◽  
Red Fluorescent Proteins

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.

scholarly journals Extracellular DNA and Type IV Pilus Expression Regulate the Structure and Kinetics of Biofilm Formation by Nontypeable Haemophilus influenzae

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Jayajit Das ◽  
Elaine Mokrzan ◽  
Vinal Lakhani ◽  
Lucia Rosas ◽  
Joseph A. Jurcisek ◽  
...  
Keyword(s):  
Middle Ear ◽  
In Silico ◽  
Biofilm Formation ◽  
Rational Design ◽  
Bacterial Biofilm ◽  
Confocal Imaging ◽  
Extracellular Dna ◽  
Fractal Structures ◽  
Biofilm Development

ABSTRACT Biofilms formed in the middle ear by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and refractive nature of otitis media (OM). However, mechanisms that underlie the emergence of specific NTHI biofilm structures are unclear. We combined computational analysis tools and in silico modeling rooted in statistical physics with confocal imaging of NTHI biofilms formed in vitro during static culture in order to identify mechanisms that give rise to distinguishing morphological features. Our analysis of confocal images of biofilms formed by NTHI strain 86-028NP using pair correlations of local bacterial densities within sequential planes parallel to the substrate showed the presence of fractal structures of short length scales (≤10 μm). The in silico modeling revealed that extracellular DNA (eDNA) and type IV pilus (Tfp) expression played important roles in giving rise to the fractal structures and allowed us to predict a substantial reduction of these structures for an isogenic mutant (ΔcomE) that was significantly compromised in its ability to release eDNA into the biofilm matrix and had impaired Tfp function. This prediction was confirmed by analysis of confocal images of in vitro ΔcomE strain biofilms. The fractal structures potentially generate niches for NTHI survival in the hostile middle ear microenvironment by dramatically increasing the contact area of the biofilm with the surrounding environment, facilitating nutrient exchange, and by generating spatial positive feedback to quorum signaling. IMPORTANCE NTHI is a major bacterial pathogen for OM, which is a common ear infection in children worldwide. Chronic OM is associated with bacterial biofilm formation in the middle ear; therefore, knowledge of the mechanisms that underlie NTHI biofilm formation is important for the development of therapeutic strategies for NTHI-associated OM. Our combined approach using confocal imaging of NTHI biofilms formed in vitro and mathematical tools for analysis of pairwise density correlations and agent-based modeling revealed that eDNA and Tfp expression were important factors in the development of fractal structures in NTHI biofilms. These structures may help NTHI survive in hostile environments, such as the middle ear. Our in silico model can be used in combination with laboratory or animal modeling studies to further define the mechanisms that underlie NTHI biofilm development during OM and thereby guide the rational design of, and optimize time and cost for, benchwork and preclinical studies. IMPORTANCE NTHI is a major bacterial pathogen for OM, which is a common ear infection in children worldwide. Chronic OM is associated with bacterial biofilm formation in the middle ear; therefore, knowledge of the mechanisms that underlie NTHI biofilm formation is important for the development of therapeutic strategies for NTHI-associated OM. Our combined approach using confocal imaging of NTHI biofilms formed in vitro and mathematical tools for analysis of pairwise density correlations and agent-based modeling revealed that eDNA and Tfp expression were important factors in the development of fractal structures in NTHI biofilms. These structures may help NTHI survive in hostile environments, such as the middle ear. Our in silico model can be used in combination with laboratory or animal modeling studies to further define the mechanisms that underlie NTHI biofilm development during OM and thereby guide the rational design of, and optimize time and cost for, benchwork and preclinical studies.

scholarly journals Rational design of small indolic squaraine dyes with large two-photon absorption cross section

2015 ◽  
Vol 6 (1) ◽  
pp. 761-769 ◽  
Author(s):  
Chun-Lin Sun ◽  
Qing Liao ◽  
Ting Li ◽  
Jun Li ◽  
Jian-Qiao Jiang ◽  
...  
Keyword(s):  
Cross Section ◽  
Rational Design ◽  
Photon Absorption ◽  
Absorption Cross ◽  
Two Photon Absorption ◽  
Squaraine Dyes ◽  
Two Photon ◽  
Bio Imaging

Assisted by theoretical analysis, we designed a small indolic squaraine with δ > 8000 GM at 780 nm, which is ideal for both in vitro and in vivo bio-imaging applications.

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