scholarly journals Direct single-molecule measurements of phycocyanobilin photophysics in monomeric C-phycocyanin

2017 ◽  
Vol 114 (37) ◽  
pp. 9779-9784 ◽  
Author(s):  
Allison H. Squires ◽  
W. E. Moerner
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Rational Design ◽  
Light Adaptation ◽  
De Novo ◽  
Photosynthetic Apparatus ◽  
Emission Spectra ◽  
Free Solution ◽  
Surface Attachment

Phycobilisomes are highly organized pigment–protein antenna complexes found in the photosynthetic apparatus of cyanobacteria and rhodophyta that harvest solar energy and transport it to the reaction center. A detailed bottom-up model of pigment organization and energy transfer in phycobilisomes is essential to understanding photosynthesis in these organisms and informing rational design of artificial light-harvesting systems. In particular, heterogeneous photophysical behaviors of these proteins, which cannot be predicted de novo, may play an essential role in rapid light adaptation and photoprotection. Furthermore, the delicate architecture of these pigment–protein scaffolds sensitizes them to external perturbations, for example, surface attachment, which can be avoided by study in free solution or in vivo. Here, we present single-molecule characterization of C-phycocyanin (C-PC), a three-pigment biliprotein that self-assembles to form the midantenna rods of cyanobacterial phycobilisomes. Using the Anti-Brownian Electrokinetic (ABEL) trap to counteract Brownian motion of single particles in real time, we directly monitor the changing photophysical states of individual C-PC monomers from Spirulina platensis in free solution by simultaneous readout of their brightness, fluorescence anisotropy, fluorescence lifetime, and emission spectra. These include single-chromophore emission states for each of the three covalently bound phycocyanobilins, providing direct measurements of the spectra and photophysics of these chemically identical molecules in their native protein environment. We further show that a simple Förster resonant energy transfer (FRET) network model accurately predicts the observed photophysical states of C-PC and suggests highly variable quenching behavior of one of the chromophores, which should inform future studies of higher-order complexes.

scholarly journals Marine Cyanobacteria Tune Energy Transfer Efficiency in their Light-harvesting Antennae by Modifying Pigment Coupling

2019 ◽  
Author(s):  
Yuval Kolodny ◽  
Hagit Zer ◽  
Mor Propper ◽  
Shira Yochelis ◽  
Yossi Paltiel ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Light Harvesting ◽  
Vertical Mixing ◽  
Photosynthetic Apparatus ◽  
Emission Spectra ◽  
Energy Transfer Efficiency ◽  
Transfer Efficiency ◽  
Transfer Energy ◽  
Harvesting Process

AbstractPhotosynthetic organisms regulate energy transfer to fit to changes in environmental conditions. The biophysical principles underlying the flexibility and efficiency of energy transfer in the light-harvesting process are still not fully understood. Here we examine how energy transfer is regulatedin-vivo. We compare different acclimation states of the photosynthetic apparatus in a marine cyanobacterial species that is well adapted to vertical mixing of the ocean water column and identify a novel acclimation strategy for photosynthetic life under low light intensities. Antennae rods extend, as expected, increasing light absorption. Surprisingly, in contrast to what was known for plants and predicted by classic calculations, these longer rods transfer energy fasteri.e.more efficiently. The fluorescence lifetime and emission spectra dependence on temperature, at the range of 4-300K, suggests that energy transfer efficiency is tuned by modifying the energetic coupling strength between antennae pigments.

scholarly journals Rational design of protein-specific folding modifiers

2020 ◽  
Author(s):  
Anirban Das ◽  
Anju Yadav ◽  
Mona Gupta ◽  
R Purushotham ◽  
Vishram L. Terse ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rational Design ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Force Measurements ◽  
Folding Nucleus ◽  
Dynamics Simulations ◽  
General Method

AbstractProtein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and the pharmaceutical industry, respectively. Here we propose a general design principle for constructing small peptide-based protein-specific folding modifiers. We construct a ‘xenonucleus’, which is a pre-folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Förster Resonance Energy transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the refolding faster by 33 ± 5% at 3 M GdnHCl. In principle, our approach provides a general method for constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined contiguous folding nucleus.

The architecture and function of the light-harvesting apparatus of purple bacteria: from single molecules to in vivo membranes

2006 ◽  
Vol 39 (3) ◽  
pp. 227-324 ◽  
Author(s):  
Richard J. Cogdell ◽  
Andrew Gall ◽  
Jürgen Köhler
Keyword(s):  
Quantum Mechanics ◽  
Energy Transfer ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Light Harvesting ◽  
Structural Information ◽  
Purple Bacteria ◽  
Theoretical Background ◽  
Single Molecule Spectroscopy

1. Introduction 2292. Structures 2342.1 The structure of LH2 2342.2 Natural variants of peripheral antenna complexes 2422.3 RC–LH1 complexes 2423. Spectroscopy 2493.1 Steady-state spectroscopy 2493.2 Factors which affect the position of the Qy absorption band of Bchla 2494. Regulation of biosynthesis and assembly 2574.1 Regulation 2574.1.1 Oxygen 2574.1.2 Light 2584.1.2.1 AppA: blue-light-mediated regulation 2594.1.2.2 Bacteriophytochromes 2594.1.3 From the RC to the mature PSU 2614.2 Assembly 2614.2.1 LH1 2624.2.2 LH2 2635. Frenkel excitons 2655.1 General 2655.2 B800 2675.3 B850 2675.4 B850 delocalization 2736. Energy-transfer pathways: experimental results 2746.1 Theoretical background 2746.2 ‘Follow the excitation energy’ 2766.2.1 Bchla→Bchla energy transfer 2776.2.1.1 B800→B800 2776.2.1.2 B800→B850 2786.2.1.3 B850→B850 2796.2.1.4 B850→B875 2806.2.1.5 B875→RC 2806.2.2 Car[harr ]Bchla energy transfer 2817. Single-molecule spectroscopy 2847.1 Introduction to single-molecule spectroscopy 2847.2 Single-molecule spectroscopy on LH2 2857.2.1 Overview 2857.2.2 B800 2867.2.2.1 General 2867.2.2.2 Intra- and intercomplex disorder of site energies 2877.2.2.3 Electron-phonon coupling 2897.2.2.4 B800→B800 energy transfer revisited 2907.2.3 B850 2938. Quantum mechanics and the purple bacteria LH system 2989. Appendix 2999.1 A crash course on quantum mechanics 2999.2 Interacting dimers 30510. Acknowledgements 30611. References 307This review describes the structures of the two major integral membrane pigment complexes, the RC–LH1 ‘core’ and LH2 complexes, which together make up the light-harvesting system present in typical purple photosynthetic bacteria. The antenna complexes serve to absorb incident solar radiation and to transfer it to the reaction centres, where it is used to ‘power’ the photosynthetic redox reaction and ultimately leads to the synthesis of ATP. Our current understanding of the biosynthesis and assembly of the LH and RC complexes is described, with special emphasis on the roles of the newly described bacteriophytochromes. Using both the structural information and that obtained from a wide variety of biophysical techniques, the details of each of the different energy-transfer reactions that occur, between the absorption of a photon and the charge separation in the RC, are described. Special emphasis is given to show how the use of single-molecule spectroscopy has provided a more detailed understanding of the molecular mechanisms involved in the energy-transfer processes. We have tried, with the help of an Appendix, to make the details of the quantum mechanics that are required to appreciate these molecular mechanisms, accessible to mathematically illiterate biologists. The elegance of the purple bacterial light-harvesting system lies in the way in which it has cleverly exploited quantum mechanics.

scholarly journals Guiding biomolecular interactions in cells using de novo protein-protein interfaces

2018 ◽  
Author(s):  
Abigail J Smith ◽  
Franziska Thomas ◽  
Deborah Shoemark ◽  
Derek N Woolfson ◽  
Nigel J Savery
Keyword(s):  
Rational Design ◽  
De Novo ◽  
Gene Activation ◽  
Low Complexity ◽  
Coiled Coils ◽  
Biomolecular Interactions ◽  
Biomolecular Systems ◽  
Binding Domains ◽  
In Cells

An improved ability to direct and control biomolecular interactions in living cells would impact on synthetic biology. A key issue is the need to introduce interacting components that act orthogonally to endogenous proteomes and interactomes. Here we show that low-complexity, de novo designed protein-protein-interaction (PPI) domains can substitute for natural PPIs and guide engineered protein-DNA interactions in Escherichia coli. Specifically, we use de novo homo- and hetero-dimeric coiled coils to reconstitute a cytoplasmic split adenylate cyclase; to recruit RNA polymerase to a promoter and activate gene expression; and to oligomerize both natural and designed DNA-binding domains to repress transcription. Moreover, the stabilities of the heterodimeric coiled coils can be modulated by rational design and, thus, adjust the levels of gene activation and repression in vivo. These experiments demonstrate the possibilities for using designed proteins and interactions to control biomolecular systems such as enzyme cascades and circuits in cells.

scholarly journals De novo synthetic biliprotein design, assembly and excitation energy transfer

2018 ◽  
Vol 15 (141) ◽  
pp. 20180021 ◽  
Author(s):  
Joshua A. Mancini ◽  
Molly Sheehan ◽  
Goutham Kodali ◽  
Brian Y. Chow ◽  
Donald A. Bryant ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Excitation Energy ◽  
De Novo ◽  
Excitation Energy Transfer ◽  
Fluorescence Yield ◽  
Conformational Restriction ◽  
Wide Range ◽  
Α Helix

Bilins are linear tetrapyrrole chromophores with a wide range of visible and near-visible light absorption and emission properties. These properties are tuned upon binding to natural proteins and exploited in photosynthetic light-harvesting and non-photosynthetic light-sensitive signalling. These pigmented proteins are now being manipulated to develop fluorescent experimental tools. To engineer the optical properties of bound bilins for specific applications more flexibly, we have used first principles of protein folding to design novel, stable and highly adaptable bilin-binding four-α-helix bundle protein frames, called maquettes, and explored the minimal requirements underlying covalent bilin ligation and conformational restriction responsible for the strong and variable absorption, fluorescence and excitation energy transfer of these proteins. Biliverdin, phycocyanobilin and phycoerythrobilin bind covalently to maquette Cys in vitro . A blue-shifted tripyrrole formed from maquette-bound phycocyanobilin displays a quantum yield of 26%. Although unrelated in fold and sequence to natural phycobiliproteins, bilin lyases nevertheless interact with maquettes during co-expression in Escherichia coli to improve the efficiency of bilin binding and influence bilin structure. Bilins bind in vitro and in vivo to Cys residues placed in loops, towards the amino end or in the middle of helices but bind poorly at the carboxyl end of helices. Bilin-binding efficiency and fluorescence yield are improved by Arg and Asp residues adjacent to the ligating Cys on the same helix and by His residues on adjacent helices.

scholarly journals Ferredoxin 1b (Fdx1b) Is the Essential Mitochondrial Redox Partner for Cortisol Biosynthesis in Zebrafish

Endocrinology ◽  
2016 ◽  
Vol 157 (3) ◽  
pp. 1122-1134 ◽  
Author(s):  
Aliesha Griffin ◽  
Silvia Parajes ◽  
Meltem Weger ◽  
Andreas Zaucker ◽  
Angela E. Taylor ◽  
...  
Keyword(s):  
Redox Regulation ◽  
Light Adaptation ◽  
De Novo ◽  
Transcription Activator ◽  
Dark Light ◽  
Cyp Enzymes ◽  
Redox Partner ◽  
The Impact

Abstract Mitochondrial cytochrome P450 (CYP) enzymes rely on electron transfer from the redox partner ferredoxin 1 (FDX1) for catalytic activity. Key steps in steroidogenesis require mitochondrial CYP enzymes and FDX1. Over 30 ferredoxin mutations have been explored in vitro; however, no spontaneously occurring mutations have been identified in humans leaving the impact of FDX1 on steroidogenesis in the whole organism largely unknown. Zebrafish are an important model to study human steroidogenesis, because they have similar steroid products and endocrine tissues. This study aimed to characterize the influence of ferredoxin on steroidogenic capacity in vivo by using zebrafish. Zebrafish have duplicate ferredoxin paralogs: fdx1 and fdx1b. Although fdx1 was observed throughout development and in most tissues, fdx1b was expressed after development of the zebrafish interrenal gland (counterpart to the mammalian adrenal gland). Additionally, fdx1b was restricted to adult steroidogenic tissues, such as the interrenal, gonads, and brain, suggesting that fdx1b was interacting with steroidogenic CYP enzymes. By using transcription activator-like effector nucleases, we generated fdx1b mutant zebrafish lines. Larvae with genetic disruption of fdx1b were morphologically inconspicuous. However, steroid hormone analysis by liquid chromatography tandem mass spectrometry revealed fdx1b mutants failed to synthesize glucocorticoids. Additionally, these mutants had an up-regulation of the hypothalamus-pituitary-interrenal axis and showed altered dark-light adaptation, suggesting impaired cortisol signaling. Antisense morpholino knockdown confirmed Fdx1b is required for de novo cortisol biosynthesis. In summary, by using zebrafish, we generated a ferredoxin knockout model system, which demonstrates for the first time the impact of mitochondrial redox regulation on glucocorticoid biosynthesis in vivo.

scholarly journals A convolutional neural network for the prediction and forward design of ribozyme-based gene-control elements

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Calvin M Schmidt ◽  
Christina D Smolke
Keyword(s):  
Machine Learning ◽  
Rational Design ◽  
De Novo ◽  
Regulation Of Gene Expression ◽  
Regulatory Activity ◽  
Rna Sequences ◽  
Genetic Switch ◽  
Gene Regulatory ◽  
Functional Rnas

Ribozyme switches are a class of RNA-encoded genetic switch that support conditional regulation of gene expression across diverse organisms. An improved elucidation of the relationships between sequence, structure, and activity can improve our capacity for de novo rational design of ribozyme switches. Here, we generated data on the activity of hundreds of thousands of ribozyme sequences. Using automated structural analysis and machine learning, we leveraged these large datasets to develop predictive models that estimate the in vivo gene-regulatory activity of a ribozyme sequence. These models supported the de novo design of ribozyme libraries with low mean basal gene-regulatory activities and new ribozyme switches that exhibit changes in gene-regulatory activity in the presence of a target ligand, producing functional switches for four out of five aptamers. Our work examines how biases in the model and the dataset that affect prediction accuracy can arise and demonstrates that machine learning can be applied to RNA sequences to predict gene-regulatory activity, providing the basis for design tools for functional RNAs.

scholarly journals Pathogenic Variants of Urolithiasis

2017 ◽  
Vol 8 (1) ◽  
pp. 95-105 ◽  
Author(s):  
Petr S Baketin ◽  
Rashid A Mollaev ◽  
Denis A Mazurenko ◽  
Vladislav E Grigoryev ◽  
Nariman K Gadzhiev ◽  
...  
Keyword(s):  
Clinical Practice ◽  
Kidney Stones ◽  
De Novo ◽  
Free Solution ◽  
Pathogenic Variants ◽  
Long Time ◽  
Randall’S Plaques

The essence of Urolithiasis - one of the oldest diseases known by the mankind - is still not understood completely. For a long time the comprehension of Urolithiasis was based on matrix, colloid, ionic, inhibitory and precipitation theories. In these cases it was impossible to single out separate pathogenetic patterns. Besides, in spite of the absence of in vivo proofs the fact that new concrement nucleus forming (de novo nucleation) is only possible outside of the stone-forming metastability range should be taken into consideration for metaphilactic purposes. Fortunately, certain progress in understanding Urolithiasis started with the onset of studies devoted to detailed scrutiny of stone-forming patients’ metabolic peculiarities as well as with the introduction into clinical practice of the up-to-date digital endoscopes. Based on existing publications one may classify pathogenesis of stone-forming into 4 major groups: growth of calcium-oxalic stones on Randall’s plaques in patients with hypercalciumuria; excrescences on the of Belliny’s ducts’ “gags”; microlites forming within the internal medullar layer discharging tubules’ lumen in patients with cystineuria; stones forming in free solution. There is no doubt this classification is not the final one, neither does it exclude mixed variants, however better understanding of the aforementioned pathogenic variants would facilitate a novel view at Urolithiasis and in patients with kidney stones would increase anti-relapse measures’ effectiveness.

Nitroxyl Modified Tobacco Mosaic Virus as a Metal-Free High-Relaxivity MRI and EPR Active Superoxide Sensor

2018 ◽  
Author(s):  
Madushani Dharmarwardana ◽  
André F. Martins ◽  
Zhuo Chen ◽  
Philip M. Palacios ◽  
Chance M. Nowak ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Single Molecule ◽  
Biological Fluids ◽  
Cellular Respiration ◽  
Organic Radical ◽  
Paramagnetic Resonance ◽  
Metal Free ◽  
Disease States

Superoxide overproduction is known to occur in multiple disease states requiring critical care yet non-invasive detection of superoxide in deep tissue remains a challenge. Herein, we report a metal-free magnetic resonance imaging (MRI) and electron paramagnetic resonance (EPR) active contrast agent prepared by “click conjugating” paramagnetic organic radical contrast agents (ORCAs) to the surface of tobacco mosaic virus (TMV). While ORCAs are known to be reduced <i>in vivo</i> to an MRI/EPR silent state, their oxidation is facilitated specifically by reactive oxygen species—in particular superoxide—and are largely unaffected by peroxides and molecular oxygen. Unfortunately, single molecule ORCAs typically offer weak MRI contrast. In contrast, our data confirm that the macromolecular ORCA-TMV conjugates show marked enhancement for <i>T<sub>1</sub></i> contrast at low field (<3.0 T), and <i>T<sub>2</sub></i> contrast at high field (9.4 T). Additionally, we demonstrated that the unique topology of TMV allows for “quenchless fluorescent” bimodal probe for concurrent fluorescence and MRI/EPR imaging, which was made possible by exploiting the unique inner and outer surface of the TMV nanoparticle. <a>Finally, we show TMV-ORCAs do not respond to normal cellular respiration, minimizing the likelihood for background, yet still respond to enzymatically produced superoxide in complicated biological fluids like serum.</a>

Supramolecular Enhancement of Aminoxyl ORCA Contrast Agents

2019 ◽  
Author(s):  
Hamilton Lee ◽  
Jenica Lumata ◽  
Michael A. Luzuriaga ◽  
Candace Benjamin ◽  
Olivia Brohlin ◽  
...  
Keyword(s):  
Magnetic Resonance Imaging ◽  
Side Effects ◽  
Magnetic Resonance ◽  
Tobacco Mosaic Virus ◽  
Contrast Agents ◽  
Single Molecule ◽  
Organic Radical ◽  
Resonance Imaging ◽  
Physiological Conditions

<div><div><div><p>Many contrast agents for magnetic resonance imaging are based on gadolinium, however side effects limit their use in some patients. Organic radical contrast agents (ORCAs) are potential alternatives, but are reduced rapidly in physiological conditions and have low relaxivities as single molecule contrast agents. Herein, we use a supramolecular strategy where cucurbit[8]uril binds with nanomolar affinities to ORCAs and protects them against biological reductants to create a stable radical in vivo. We further over came the weak contrast by conjugating this complex on the surface of a self-assembled biomacromolecule derived from the tobacco mosaic virus.</p></div></div></div>

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