scholarly journals Generation of bright monomeric red fluorescent proteins via computational design of enhanced chromophore packing

2022 ◽  
Author(s):  
Sandrine Legault ◽  
Derek Paco Fraser-Halberg ◽  
Ralph McAnelly ◽  
Matthew G Eason ◽  
Michael Thompson ◽  
...  
Keyword(s):  
Quantum Yield ◽  
Protein Design ◽  
Fluorescent Proteins ◽  
Computational Design ◽  
Computational Protein Design ◽  
Biological Research ◽  
Widespread Application ◽  
Red Fluorescent Proteins

Red fluorescent proteins (RFPs) have found widespread application in chemical and biological research due to their longer emission wavelengths. Here, we use computational protein design to increase the quantum yield...

scholarly journals Quantitative Determination of Dark Chromophore Population Explains the Apparent Low Quantum Yield of Red Fluorescent Proteins

2020 ◽  
Vol 124 (8) ◽  
pp. 1383-1391 ◽  
Author(s):  
Jord C. Prangsma ◽  
Robert Molenaar ◽  
Laura van Weeren ◽  
Daphne S. Bindels ◽  
Lindsay Haarbosch ◽  
...  
Keyword(s):  
Quantum Yield ◽  
Quantitative Determination ◽  
Fluorescent Proteins ◽  
Red Fluorescent Proteins

scholarly journals Challenges in the computational design of proteins

2009 ◽  
Vol 6 (suppl_4) ◽  
Author(s):  
María Suárez ◽  
Alfonso Jaramillo
Keyword(s):  
Amino Acid ◽  
Structural Biology ◽  
Protein Design ◽  
Current Knowledge ◽  
Computational Design ◽  
Amino Acid Sequences ◽  
Computational Protein Design ◽  
Energy Functions ◽  
Physical Description ◽  
Atomic Interactions

Protein design has many applications not only in biotechnology but also in basic science. It uses our current knowledge in structural biology to predict, by computer simulations, an amino acid sequence that would produce a protein with targeted properties. As in other examples of synthetic biology, this approach allows the testing of many hypotheses in biology. The recent development of automated computational methods to design proteins has enabled proteins to be designed that are very different from any known ones. Moreover, some of those methods mostly rely on a physical description of atomic interactions, which allows the designed sequences not to be biased towards known proteins. In this paper, we will describe the use of energy functions in computational protein design, the use of atomic models to evaluate the free energy in the unfolded and folded states, the exploration and optimization of amino acid sequences, the problem of negative design and the design of biomolecular function. We will also consider its use together with the experimental techniques such as directed evolution. We will end by discussing the challenges ahead in computational protein design and some of their future applications.

scholarly journals Quantification of reactive oxygen species production by the red fluorescent proteins KillerRed, SuperNova and mCherry

2019 ◽  
Author(s):  
John O. Onukwufor ◽  
Adam J. Trewin ◽  
Timothy M. Baran ◽  
Anmol Almast ◽  
Thomas H. Foster ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Quantum Yield ◽  
Singlet Oxygen ◽  
Fluorescent Proteins ◽  
Reactive Oxygen ◽  
Quantum Yields ◽  
Type I ◽  
Oxygen Species ◽  
Redox Biology ◽  
Red Fluorescent Proteins

ABSTRACTFluorescent proteins can generate reactive oxygen species (ROS) upon absorption of photons via type I and II photosensitization mechanisms. The red fluorescent proteins KillerRed and SuperNova are phototoxic proteins engineered to generate ROS and are used in a variety of biological applications. However, their relative quantum yields and rates of ROS production are unclear, which has limited the interpretation of their effects when used in biological systems. We cloned and purified KillerRed, SuperNova, and mCherry - a related red fluorescent protein not typically considered a photosensitizer - and measured the superoxide (O2•-) and singlet oxygen (1O2) quantum yields with irradiation at 561 nm. The formation of the O2•--specific product 2-hydroxyethidium (2-OHE+) was quantified via HPLC separation with fluorescence detection. Relative to a reference photosensitizer, Rose Bengal, the O2•- quantum yield (ΦO2•-) of SuperNova was determined to be 0.00150, KillerRed was 0.00097, and mCherry 0.00120. At an excitation fluence of 916.5 J/cm2 and matched absorption at 561 nm, SuperNova, KillerRed and mCherry made 3.81, 2.38 and 1.65 μM O2•-/min, respectively. Using the probe Singlet Oxygen Sensor Green (SOSG), we ascertained the 1O2 quantum yield (Φ1O2) for SuperNova to be 0.0220, KillerRed 0.0076, and mCherry 0.0057. These photosensitization characteristics of SuperNova, KillerRed and mCherry improve our understanding of fluorescent proteins and are pertinent for refining their use as tools to advance our knowledge of redox biology.GRAPHICAL ABSTRACT

scholarly journals Breakthroughs in computational design methods open up new frontiers for de novo protein engineering

2021 ◽  
Vol 34 ◽  
Author(s):  
Ben A Meinen ◽  
Christopher D Bahl
Keyword(s):  
Protein Engineering ◽  
Chemical Reactions ◽  
Protein Design ◽  
De Novo ◽  
Computational Design ◽  
Design Methods ◽  
Computational Protein Design ◽  
The Past ◽  
New Methods ◽  
De Novo Protein Design

Abstract Proteins catalyze the majority of chemical reactions in organisms, and harnessing this power has long been the focus of the protein engineering field. Computational protein design aims to create new proteins and functions in silico, and in doing so, accelerate the process, reduce costs and enable more sophisticated engineering goals to be accomplished. Challenges that very recently seemed impossible are now within reach thanks to several landmark advances in computational protein design methods. Here, we summarize these new methods, with a particular emphasis on de novo protein design advancements occurring within the past 5 years.

Ultrafast spectroscopy of biliverdin dimethyl ester in solution: pathways of excited-state depopulation

2020 ◽  
Vol 22 (35) ◽  
pp. 19903-19912
Author(s):  
Yangyi Liu ◽  
Zhuang Chen ◽  
Xueli Wang ◽  
Simin Cao ◽  
Jianhua Xu ◽  
...  
Keyword(s):  
Excited State ◽  
Quantum Yield ◽  
Quantum Efficiency ◽  
Fluorescence Quantum Yield ◽  
Ultrafast Spectroscopy ◽  
Fluorescent Proteins ◽  
Dimethyl Ester ◽  
Bile Pigments ◽  
Enhanced Fluorescence ◽  
Red Fluorescent Proteins

Biliverdin and its dimethyl ester derivatives are bile pigments with very low fluorescence quantum yield in solution, but naturally serve as chromophores in far-red fluorescent proteins with three orders of magnitude enhanced fluorescence quantum efficiency.

POSSIBLE APPLICATION OF METAL SENSITIVE RED FLUORESCENT PROTEINS IN ENVIRONMENTAL MONITORING

2012 ◽  
Vol 11 (1) ◽  
pp. 193-198 ◽  
Author(s):  
Laszlo Szilagyi ◽  
Maria Szabo (Palfi) ◽  
Judit Petres ◽  
Ildiko Miklossy ◽  
Beata Abraham ◽  
...  
Keyword(s):  
Environmental Monitoring ◽  
Fluorescent Proteins ◽  
Red Fluorescent Proteins

scholarly journals Split-wrmScarlet and split-sfGFP: Tools for faster, easier fluorescent l abeling of endogenous proteins in Caenorhabditis elegans

Genetics ◽  
2021 ◽  
Author(s):  
Jérôme Goudeau ◽  
Catherine S Sharp ◽  
Jonathan Paw ◽  
Laura Savy ◽  
Manuel D Leonetti ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Large Fragment ◽  
C Elegans ◽  
High Affinity ◽  
Red Fluorescent Proteins ◽  
Invaluable Tool ◽  
Endogenous Proteins ◽  
Fluorescent Tags

Abstract We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663

Sign in / Sign up

Export Citation Format

Share Document