Intramolecular photostabilization via triplet-state quenching: design principles to make organic fluorophores “self-healing”

Covalent linkage of fluorophores and photostabilizers was recently revived as a strategy to make organic fluorophores “self-healing” via triplet-state quenching. Although Lüttke and co-workers pioneered this strategy already in the 1980s, the general design principles still remain elusive. In this contribution, we combine experiments and theory to understand what determines the photostabilization efficiency in dye–photostabilizer conjugates. Our results from single-molecule microscopy and molecular dynamics simulations of different Cy5-derivatives suggest that the distance and relative geometry between the fluorophore and photostabilizer are more important than the chemical nature of the photostabilizer, e.g. its redox potential, which is known to influence electron-transfer rates. We hypothesize that the efficiency of photostabilization scales directly with the contact rate of the fluorophore and photostabilizer. This study represents an important step in the understanding of the molecular mechanism of intramolecular photostabilization and can pave the way for further development of stable emitters for various applications.

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Tuning the Baird aromatic triplet-state energy of cyclooctatetraene to maximize the self-healing mechanism in organic fluorophores

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2006517117 ◽

2020 ◽

Vol 117 (39) ◽

pp. 24305-24315

Author(s):

Avik K. Pati ◽

Ouissam El Bakouri ◽

Steffen Jockusch ◽

Zhou Zhou ◽

Roger B. Altman ◽

...

Keyword(s):

Triplet State ◽

Large Scale ◽

State Energy ◽

Conformational Dynamics ◽

The Self ◽

Detection Methods ◽

Wide Field ◽

Self Healing ◽

Organic Fluorophores ◽

Triplet State Energy

Bright, photostable, and nontoxic fluorescent contrast agents are critical for biological imaging. “Self-healing” dyes, in which triplet states are intramolecularly quenched, enable fluorescence imaging by increasing fluorophore brightness and longevity, while simultaneously reducing the generation of reactive oxygen species that promote phototoxicity. Here, we systematically examine the self-healing mechanism in cyanine-class organic fluorophores spanning the visible spectrum. We show that the Baird aromatic triplet-state energy of cyclooctatetraene can be physically altered to achieve order of magnitude enhancements in fluorophore brightness and signal-to-noise ratio in both the presence and absence of oxygen. We leverage these advances to achieve direct measurements of large-scale conformational dynamics within single molecules at submillisecond resolution using wide-field illumination and camera-based detection methods. These findings demonstrate the capacity to image functionally relevant conformational processes in biological systems in the kilohertz regime at physiological oxygen concentrations and shed important light on the multivariate parameters critical to self-healing organic fluorophore design.

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Tuning the Baird Aromatic Triplet State Energy of Cyclooctatetraene to Maximize the Self-Healing Mechanism in Organic Fluorophores

Biophysical Journal ◽

10.1016/j.bpj.2020.11.2271 ◽

2021 ◽

Vol 120 (3) ◽

pp. 114a

Author(s):

Avik K. Pati ◽

Ouissam El Bakouri ◽

Steffen Jockusch ◽

Zhou Zhou ◽

Roger B. Altman ◽

...

Keyword(s):

Triplet State ◽

State Energy ◽

The Self ◽

Self Healing ◽

Organic Fluorophores ◽

Triplet State Energy

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On the impact of competing intra- and intermolecular triplet-state quenching on photobleaching and photoswitching kinetics of organic fluorophores

Physical Chemistry Chemical Physics ◽

10.1039/c8cp05063e ◽

2019 ◽

Vol 21 (7) ◽

pp. 3721-3733 ◽

Cited By ~ 8

Author(s):

Jochem H. Smit ◽

Jasper H. M. van der Velde ◽

Jingyi Huang ◽

Vanessa Trauschke ◽

Sarah S. Henrikus ◽

...

Keyword(s):

Triplet State ◽

Single Molecule ◽

Photophysical Properties ◽

Super Resolution ◽

Organic Fluorophores ◽

Resolution Imaging ◽

The Impact ◽

Kinetics Of

How photostabilizer molecules influence the photophysical properties of various organic fluorophores used for single-molecule and super-resolution imaging.

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Structural dynamics of P-type ATPase ion pumps

Biochemical Society Transactions ◽

10.1042/bst20190124 ◽

2019 ◽

Vol 47 (5) ◽

pp. 1247-1257 ◽

Cited By ~ 17

Author(s):

Mateusz Dyla ◽

Sara Basse Hansen ◽

Poul Nissen ◽

Magnus Kjaergaard

Keyword(s):

Structural Dynamics ◽

Single Molecule ◽

New Technologies ◽

Atp Hydrolysis ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Time Resolved ◽

Dynamics Simulations ◽

Types Of Information ◽

P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

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Optimal Relabeling of Water Molecules and Single-Molecule Entropy Estimation

Biophysica ◽

10.3390/biophysica1030021 ◽

2021 ◽

Vol 1 (3) ◽

pp. 279-296

Author(s):

Federico Fogolari ◽

Gennaro Esposito

Keyword(s):

Single Molecule ◽

Degrees Of Freedom ◽

Water Molecules ◽

Maximum Information ◽

Nearest Neighbours ◽

Biomolecular Simulations ◽

Solvent Molecules ◽

Solvation Entropy ◽

Dynamics Simulations ◽

Internal Degrees Of Freedom

Estimation of solvent entropy from equilibrium molecular dynamics simulations is a long-standing problem in statistical mechanics. In recent years, methods that estimate entropy using k-th nearest neighbours (kNN) have been applied to internal degrees of freedom in biomolecular simulations, and for the rigorous computation of positional-orientational entropy of one and two molecules. The mutual information expansion (MIE) and the maximum information spanning tree (MIST) methods were proposed and used to deal with a large number of non-independent degrees of freedom, providing estimates or bounds on the global entropy, thus complementing the kNN method. The application of the combination of such methods to solvent molecules appears problematic because of the indistinguishability of molecules and of their symmetric parts. All indistiguishable molecules span the same global conformational volume, making application of MIE and MIST methods difficult. Here, we address the problem of indistinguishability by relabeling water molecules in such a way that each water molecule spans only a local region throughout the simulation. Then, we work out approximations and show how to compute the single-molecule entropy for the system of relabeled molecules. The results suggest that relabeling water molecules is promising for computation of solvation entropy.

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Advances in the study of the mechanochemical coupling of kinesin

International Journal of Modern Physics B ◽

10.1142/s0217979218400015 ◽

2018 ◽

Vol 32 (18) ◽

pp. 1840001 ◽

Cited By ~ 1

Author(s):

Ming Li ◽

Zhong-Can Ou-Yang ◽

Yao-Gen Shu

Keyword(s):

Molecular Dynamics ◽

Single Molecule ◽

Intracellular Transport ◽

Coordination Mechanism ◽

Personal Perspective ◽

Long Distance ◽

Future Studies ◽

Mechanochemical Coupling ◽

Dynamics Simulations ◽

Made In

Kinesin is a two-headed linear motor for intracellular transport. It can walk a long distance in a hand-over-hand manner along the track before detaching (i.e., high processivity), and it consumes one ATP molecule for each step (i.e., tight mechanochemical coupling). The mechanisms of the coordination of its two heads and the mechanochemical coupling are the central issues of numerous researches. A few advances have been made in recent decades, thanks to the development of single-molecule technologies and molecular dynamics simulations. In this paper, we review some progress of the studies on the kinematics, energetics, coordination mechanism, mechanochemical mechanism of kinesin. We also present a personal perspective on the future studies of kinesin.

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A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization

Nature Communications ◽

10.1038/ncomms10144 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 62

Author(s):

Jasper H. M. van der Velde ◽

Jens Oelerich ◽

Jingyi Huang ◽

Jochem H. Smit ◽

Atieh Aminian Jazi ◽

...

Keyword(s):

Single Molecule ◽

Unnatural Amino Acids ◽

Photophysical Properties ◽

Super Resolution ◽

Single Step ◽

General Strategy ◽

Self Healing ◽

Chemical Transformations ◽

Scaffolding Strategy ◽

Organic Fluorophore

Abstract Intramolecular photostabilization via triple-state quenching was recently revived as a tool to impart synthetic organic fluorophores with ‘self-healing’ properties. To date, utilization of such fluorophore derivatives is rare due to their elaborate multi-step synthesis. Here we present a general strategy to covalently link a synthetic organic fluorophore simultaneously to a photostabilizer and biomolecular target via unnatural amino acids. The modular approach uses commercially available starting materials and simple chemical transformations. The resulting photostabilizer–dye conjugates are based on rhodamines, carbopyronines and cyanines with excellent photophysical properties, that is, high photostability and minimal signal fluctuations. Their versatile use is demonstrated by single-step labelling of DNA, antibodies and proteins, as well as applications in single-molecule and super-resolution fluorescence microscopy. We are convinced that the presented scaffolding strategy and the improved characteristics of the conjugates in applications will trigger the broader use of intramolecular photostabilization and help to emerge this approach as a new gold standard.

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Hard–Soft Chemistry Design Principles for Predictive Assembly of Single Molecule-Metal Junctions

Journal of the American Chemical Society ◽

10.1021/jacs.1c05142 ◽

2021 ◽

Author(s):

Hannah E. Skipper ◽

Claire V. May ◽

Arnold L. Rheingold ◽

Linda H. Doerrer ◽

Maria Kamenetska

Keyword(s):

Single Molecule ◽

Design Principles ◽

Soft Chemistry

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'Self-healing' dyes: intramolecular stabilization of organic fluorophores

Nature Methods ◽

10.1038/nmeth.1977 ◽

2012 ◽

Vol 9 (5) ◽

pp. 426-427 ◽

Cited By ~ 50

Author(s):

Philip Tinnefeld ◽

Thorben Cordes

Keyword(s):

Self Healing ◽

Organic Fluorophores

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Planar Boronic Graphene and Nitrogenized Graphene Heterostructure for Protein Stretch and Confinement

Biomolecules ◽

10.3390/biom11121756 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1756

Author(s):

Xuchang Su ◽

Zhi He ◽

Lijun Meng ◽

Hong Liang ◽

Ruhong Zhou

Keyword(s):

Single Molecule ◽

Intrinsically Disordered Proteins ◽

Electron Tunneling ◽

Amyloid Β ◽

Protein Sequencing ◽

Disordered Proteins ◽

Force Microscopy ◽

Intrinsically Disordered ◽

Atomic Force ◽

Dynamics Simulations

Single-molecule techniques such as electron tunneling and atomic force microscopy have attracted growing interests in protein sequencing. For these methods, it is critical to refine and stabilize the protein sample to a “suitable mode” before applying a high-fidelity measurement. Here, we show that a planar heterostructure comprising boronic graphene (BC3) and nitrogenized graphene (C3N) sandwiched stripe (BC3/C3N/BC3) is capable of the effective stretching and confinement of three types of intrinsically disordered proteins (IDPs), including amyloid-β (1–42), polyglutamine (Q42), and α-Synuclein (61–95). Our molecular dynamics simulations demonstrate that the protein molecules interact more strongly with the C3N stripe than the BC3 one, which leads to their capture, elongation, and confinement along the center C3N stripe of the heterostructure. The conformational fluctuations of IDPs are substantially reduced after being stretched. This design may serve as a platform for single-molecule protein analysis with reduced thermal noise.

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