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Article ; Online: Folding pathway of a discontinuous two-domain protein.

Agam, Ganesh / Barth, Anders / Lamb, Don C

2024 Volume 15, Issue 1, Page(s) 690

Abstract: It is estimated that two-thirds of all proteins in higher organisms are composed of multiple domains, many of them containing discontinuous folds. However, to date, most in vitro protein folding studies have focused on small, single-domain proteins. As a ...

Abstract	It is estimated that two-thirds of all proteins in higher organisms are composed of multiple domains, many of them containing discontinuous folds. However, to date, most in vitro protein folding studies have focused on small, single-domain proteins. As a model system for a two-domain discontinuous protein, we study the unfolding/refolding of a slow-folding double mutant of the maltose binding protein (DM-MBP) using single-molecule two- and three-color Förster Resonance Energy Transfer experiments. We observe a dynamic folding intermediate population in the N-terminal domain (NTD), C-terminal domain (CTD), and at the domain interface. The dynamic intermediate fluctuates rapidly between unfolded states and compact states, which have a similar FRET efficiency to the folded conformation. Our data reveals that the delayed folding of the NTD in DM-MBP is imposed by an entropic barrier with subsequent folding of the highly dynamic CTD. Notably, accelerated DM-MBP folding is routed through the same dynamic intermediate within the cavity of the GroEL/ES chaperone system, suggesting that the chaperonin limits the conformational space to overcome the entropic folding barrier. Our study highlights the subtle tuning and co-dependency in the folding of a discontinuous multi-domain protein.
MeSH term(s)	Maltose-Binding Proteins ; Entropy ; Fluorescence Resonance Energy Transfer ; Protein Folding ; Research Design
Chemical Substances	Maltose-Binding Proteins
Language	English
Publishing date	2024-01-23
Publishing country	England
Document type	Journal Article
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-024-44901-3
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Article ; Online: Diameter dependence of transport through nuclear pore complex mimics studied using optical nanopores.

Klughammer, Nils / Barth, Anders / Dekker, Maurice / Fragasso, Alessio / Onck, Patrick R / Dekker, Cees

eLife

2024 Volume 12

Abstract: The nuclear pore complex (NPC) regulates the selective transport of large biomolecules through the nuclear envelope. As a model system for nuclear transport, we construct NPC mimics by functionalizing the pore walls of freestanding palladium zero-mode ... ...

Abstract	The nuclear pore complex (NPC) regulates the selective transport of large biomolecules through the nuclear envelope. As a model system for nuclear transport, we construct NPC mimics by functionalizing the pore walls of freestanding palladium zero-mode waveguides with the FG-nucleoporin Nsp1. This approach enables the measurement of single-molecule translocations through individual pores using optical detection. We probe the selectivity of Nsp1-coated pores by quantitatively comparing the translocation rates of the nuclear transport receptor Kap95 to the inert probe BSA over a wide range of pore sizes from 35 nm to 160 nm. Pores below 55 ± 5 nm show significant selectivity that gradually decreases for larger pores. This finding is corroborated by coarse-grained molecular dynamics simulations of the Nsp1 mesh within the pore, which suggest that leakage of BSA occurs by diffusion through transient openings within the dynamic mesh. Furthermore, we experimentally observe a modulation of the BSA permeation when varying the concentration of Kap95. The results demonstrate the potential of single-molecule fluorescence measurements on biomimetic NPCs to elucidate the principles of nuclear transport.
MeSH term(s)	Humans ; Nuclear Pore ; Nanopores ; Nuclear Envelope ; Biomimetics ; Diffusion ; Translocation, Genetic
Language	English
Publishing date	2024-02-20
Publishing country	England
Document type	Journal Article
ZDB-ID	2687154-3
ISSN	2050-084X ; 2050-084X
ISSN (online)	2050-084X
ISSN	2050-084X
DOI	10.7554/eLife.87174
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Article ; Online: Zero-Mode Waveguide Nanowells for Single-Molecule Detection in Living Cells.

Yang, Sora / Klughammer, Nils / Barth, Anders / Tanenbaum, Marvin E / Dekker, Cees

ACS nano

2023 Volume 17, Issue 20, Page(s) 20179–20193

Abstract: Single-molecule fluorescence imaging experiments generally require sub-nanomolar protein concentrations to isolate single protein molecules, which makes such experiments challenging in live cells due to high intracellular protein concentrations. Here, we ...

Abstract	Single-molecule fluorescence imaging experiments generally require sub-nanomolar protein concentrations to isolate single protein molecules, which makes such experiments challenging in live cells due to high intracellular protein concentrations. Here, we show that single-molecule observations can be achieved in live cells through a drastic reduction in the observation volume using overmilled zero-mode waveguides (ZMWs- subwavelength-size holes in a metal film). Overmilling of the ZMW in a palladium film creates a nanowell of tunable size in the glass layer below the aperture, which cells can penetrate. We present a thorough theoretical and experimental characterization of the optical properties of these nanowells over a wide range of ZMW diameters and overmilling depths, showing an excellent signal confinement and a 5-fold fluorescence enhancement of fluorescent molecules inside nanowells. ZMW nanowells facilitate live-cell imaging as cells form stable protrusions into the nanowells. Importantly, the nanowells greatly reduce the cytoplasmic background fluorescence, enabling the detection of individual membrane-bound fluorophores in the presence of high cytoplasmic expression levels, which could not be achieved with TIRF microscopy. Zero-mode waveguide nanowells thus provide great potential to study individual proteins in living cells.
MeSH term(s)	Nanotechnology/methods ; Microscopy ; Single Molecule Imaging ; Spectrometry, Fluorescence/methods
Language	English
Publishing date	2023-10-04
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1936-086X
ISSN (online)	1936-086X
DOI	10.1021/acsnano.3c05959
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Article: Fundamental photophysics of isomorphic and expanded fluorescent nucleoside analogues

Dziuba, Dmytro / Didier, Pascal / Ciaco, Stefano / Barth, Anders / Seidel, Claus A. M. / Mély, Yves

Chemical Society reviews. 2021 June 21, v. 50, no. 12

2021

Abstract: Fluorescent nucleoside analogues (FNAs) are structurally diverse mimics of the natural essentially non-fluorescent nucleosides which have found numerous applications in probing the structure and dynamics of nucleic acids as well as their interactions ... ...

Abstract	Fluorescent nucleoside analogues (FNAs) are structurally diverse mimics of the natural essentially non-fluorescent nucleosides which have found numerous applications in probing the structure and dynamics of nucleic acids as well as their interactions with various biomolecules. In order to minimize disturbance in the labelled nucleic acid sequences, the FNA chromophoric groups should resemble the natural nucleobases in size and hydrogen-bonding patterns. Isomorphic and expanded FNAs are the two groups that best meet the criteria of non-perturbing fluorescent labels for DNA and RNA. Significant progress has been made over the past decades in understanding the fundamental photophysics that governs the spectroscopic and environmentally sensitive properties of these FNAs. Herein, we review recent advances in the spectroscopic and computational studies of selected isomorphic and expanded FNAs. We also show how this information can be used as a rational basis to design new FNAs, select appropriate sequences for optimal spectroscopic response and interpret fluorescence data in FNA applications.
Keywords	DNA ; RNA ; fluorescence ; hydrogen bonding ; nucleobases ; nucleosides ; spectroscopy
Language	English
Dates of publication	2021-0621
Size	p. 7062-7107.
Publishing place	The Royal Society of Chemistry
Document type	Article
ZDB-ID	1472875-8
ISSN	1460-4744 ; 0306-0012
ISSN (online)	1460-4744
ISSN	0306-0012
DOI	10.1039/d1cs00194a
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Article: Early intervention with Kan Jang® to treat upper-respiratory tract infections: A randomized, quadruple-blind study.

Narimanyan, Mikayel / Jamalyan, Kristina / Balyan, Anna / Barth, Anders / Palm, Staffan / Wikman, Georg / Panossian, Alexander

Journal of traditional and complementary medicine

2021 Volume 11, Issue 6, Page(s) 552–562

Abstract: Background and aim: Andrographis paniculata: Experimental procedure: A total of 179 patients with URTI symptoms received six Kan Jang® (daily dose of andrographolides: 60 mg) or placebo capsules a day for five consecutive days in this randomized, ... ...

Abstract	Background and aim: Andrographis paniculata Experimental procedure: A total of 179 patients with URTI symptoms received six Kan Jang® (daily dose of andrographolides: 60 mg) or placebo capsules a day for five consecutive days in this randomized, quadruple-blinded, placebo-controlled, two-parallel-group phase II study. The primary efficacy outcomes were the decrease in the acute-phase duration and the mean URTI symptoms score (sore throat, runny nose, nasal congestion, hoarseness, cough, headache, and fatigue). Results: Early intervention with Kan Jang® significantly increased the recovery rate and reduced the number of sick leave days by >21% (0.64/day) relative to that observed in the placebo group (2.38 vs. 3.02 days, p = 0.0053). Kan Jang® significantly alleviated all URTI symptoms starting from the second day of treatment. A superior anti-inflammatory effect of Kan Jang® to that of placebo was also observed on the white blood cell count (p = 0.007) and erythrocyte sedimentation rate (p = 0.0258). Treatment with Kan Jang® was tolerated well. Conclusion: This study demonstrates that early intervention with Kan Jang® capsules reduces the recovery duration of patients by 21% and significantly relieves the severity of typical URTI symptoms.
Language	English
Publishing date	2021-06-11
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	2709698-1
ISSN	2225-4110
ISSN	2225-4110
DOI	10.1016/j.jtcme.2021.06.001
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Article ; Online: Fundamental photophysics of isomorphic and expanded fluorescent nucleoside analogues.

Dziuba, Dmytro / Didier, Pascal / Ciaco, Stefano / Barth, Anders / Seidel, Claus A M / Mély, Yves

Chemical Society reviews

2021 Volume 50, Issue 12, Page(s) 7062–7107

Abstract	Fluorescent nucleoside analogues (FNAs) are structurally diverse mimics of the natural essentially non-fluorescent nucleosides which have found numerous applications in probing the structure and dynamics of nucleic acids as well as their interactions with various biomolecules. In order to minimize disturbance in the labelled nucleic acid sequences, the FNA chromophoric groups should resemble the natural nucleobases in size and hydrogen-bonding patterns. Isomorphic and expanded FNAs are the two groups that best meet the criteria of non-perturbing fluorescent labels for DNA and RNA. Significant progress has been made over the past decades in understanding the fundamental photophysics that governs the spectroscopic and environmentally sensitive properties of these FNAs. Herein, we review recent advances in the spectroscopic and computational studies of selected isomorphic and expanded FNAs. We also show how this information can be used as a rational basis to design new FNAs, select appropriate sequences for optimal spectroscopic response and interpret fluorescence data in FNA applications.
MeSH term(s)	Fluorescence ; Fluorescent Dyes/chemistry ; Nucleosides/chemistry ; Photochemical Processes
Chemical Substances	Fluorescent Dyes ; Nucleosides
Language	English
Publishing date	2021-05-06
Publishing country	England
Document type	Journal Article ; Review
ZDB-ID	1472875-8
ISSN	1460-4744 ; 0306-0012
ISSN (online)	1460-4744
ISSN	0306-0012
DOI	10.1039/d1cs00194a
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Article ; Online: Unraveling multi-state molecular dynamics in single-molecule FRET experiments. II. Quantitative analysis of multi-state kinetic networks.

Opanasyuk, Oleg / Barth, Anders / Peulen, Thomas-Otavio / Felekyan, Suren / Kalinin, Stanislav / Sanabria, Hugo / Seidel, Claus A M

The Journal of chemical physics

2022 Volume 157, Issue 3, Page(s) 31501

Abstract: Single-molecule Förster Resonance Energy Transfer (smFRET) experiments are ideally suited to resolve the structural dynamics of biomolecules. A significant challenge to date is capturing and quantifying the exchange between multiple conformational states, ...

Abstract	Single-molecule Förster Resonance Energy Transfer (smFRET) experiments are ideally suited to resolve the structural dynamics of biomolecules. A significant challenge to date is capturing and quantifying the exchange between multiple conformational states, mainly when these dynamics occur on the sub-millisecond timescale. Many methods for quantitative analysis are challenged if more than two states are involved, and the appropriate choice of the number of states in the kinetic network is difficult. An additional complication arises if dynamically active molecules coexist with pseudo-static molecules in similar conformational states with undistinguishable Förster Resonance Energy Transfer (FRET) efficiencies. To address these problems, we developed a quantitative integrative analysis framework that combines the information from FRET-lines that relate average fluorescence lifetimes and intensities in two-dimensional burst frequency histograms, fluorescence decays obtained by time-correlated single-photon-counting, photon distribution analysis of the intensities, and fluorescence correlation spectroscopy. Individually, these methodologies provide ambiguous results for the characterization of dynamics in complex kinetic networks. However, the global analysis approach enables accurate determination of the number of states, their kinetic connectivity, the transition rate constants, and species fractions. To challenge the potential of smFRET experiments for studying multi-state kinetic networks, we apply our integrative framework using a set of synthetic data for three-state systems with different kinetic connectivity and exchange rates. Our methodology paves the way toward an integrated analysis of multiparameter smFRET experiments that spans all dimensions of the experimental data. Finally, we propose a workflow for the analysis and show examples that demonstrate the usefulness of this toolkit for dynamic structural biology.
MeSH term(s)	Fluorescence Resonance Energy Transfer/methods ; Molecular Conformation ; Molecular Dynamics Simulation ; Photons ; Spectrometry, Fluorescence
Language	English
Publishing date	2022-07-19
Publishing country	United States
Document type	Journal Article
ZDB-ID	3113-6
ISSN	1089-7690 ; 0021-9606
ISSN (online)	1089-7690
ISSN	0021-9606
DOI	10.1063/5.0095754
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Article ; Online: Unraveling multi-state molecular dynamics in single-molecule FRET experiments. I. Theory of FRET-lines.

Barth, Anders / Opanasyuk, Oleg / Peulen, Thomas-Otavio / Felekyan, Suren / Kalinin, Stanislav / Sanabria, Hugo / Seidel, Claus A M

The Journal of chemical physics

2022 Volume 156, Issue 14, Page(s) 141501

Abstract: Conformational dynamics of biomolecules are of fundamental importance for their function. Single-molecule studies of Förster Resonance Energy Transfer (smFRET) between a tethered donor and acceptor dye pair are a powerful tool to investigate the ... ...

Abstract	Conformational dynamics of biomolecules are of fundamental importance for their function. Single-molecule studies of Förster Resonance Energy Transfer (smFRET) between a tethered donor and acceptor dye pair are a powerful tool to investigate the structure and dynamics of labeled molecules. However, capturing and quantifying conformational dynamics in intensity-based smFRET experiments remains challenging when the dynamics occur on the sub-millisecond timescale. The method of multiparameter fluorescence detection addresses this challenge by simultaneously registering fluorescence intensities and lifetimes of the donor and acceptor. Together, two FRET observables, the donor fluorescence lifetime τ
MeSH term(s)	Fluorescence Resonance Energy Transfer/methods ; Molecular Conformation ; Molecular Dynamics Simulation
Language	English
Publishing date	2022-04-15
Publishing country	United States
Document type	Journal Article
ZDB-ID	3113-6
ISSN	1089-7690 ; 0021-9606
ISSN (online)	1089-7690
ISSN	0021-9606
DOI	10.1063/5.0089134
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Article ; Online: Quantitative Single-Molecule Three-Color Förster Resonance Energy Transfer by Photon Distribution Analysis.

Barth, Anders / Voith von Voithenberg, Lena / Lamb, Don C

The journal of physical chemistry. B

2019 Volume 123, Issue 32, Page(s) 6901–6916

Abstract: Single-molecule Förster resonance energy transfer (FRET) is a powerful tool to study conformational dynamics of biomolecules. Using solution-based single-pair FRET by burst analysis, conformational heterogeneities and fluctuations of fluorescently ... ...

Abstract	Single-molecule Förster resonance energy transfer (FRET) is a powerful tool to study conformational dynamics of biomolecules. Using solution-based single-pair FRET by burst analysis, conformational heterogeneities and fluctuations of fluorescently labeled proteins or nucleic acids can be studied by monitoring a single distance at a time. Three-color FRET is sensitive to three distances simultaneously and can thus elucidate complex coordinated motions within single molecules. While three-color FRET has been applied on the single-molecule level before, a detailed quantitative description of the obtained FRET efficiency distributions is still missing. Direct interpretation of three-color FRET data is additionally complicated by an increased shot noise contribution when converting photon counts to FRET efficiencies. However, to address the question of coordinated motion, it is of special interest to extract information about the underlying distance heterogeneity, which is not easily extracted from the FRET efficiency histograms directly. Here, we present three-color photon distribution analysis (3C-PDA), a method to extract distributions of interdye distances from three-color FRET measurements. We present a model for diffusion-based three-color FRET experiments and apply Bayesian inference to extract information about the physically relevant distance heterogeneity in the sample. The approach is verified using simulated data sets and experimentally applied to triple-labeled DNA duplexes. Finally, three-color FRET experiments on the Hsp70 chaperone BiP reveal conformational coordinated changes between individual domains. The possibility to address the co-occurrence of intramolecular distances makes 3C-PDA a powerful method to study the coordination of domain motions within biomolecules undergoing conformational dynamics.
MeSH term(s)	Adenosine Diphosphate/metabolism ; Adenosine Triphosphate/metabolism ; Bayes Theorem ; Computer Simulation ; DNA/chemistry ; DNA/metabolism ; Fluorescence Resonance Energy Transfer ; Heat-Shock Proteins/chemistry ; Heat-Shock Proteins/metabolism ; Humans ; Molecular Conformation ; Nucleic Acid Conformation ; Photons
Chemical Substances	Heat-Shock Proteins ; Adenosine Diphosphate (61D2G4IYVH) ; Adenosine Triphosphate (8L70Q75FXE) ; DNA (9007-49-2) ; molecular chaperone GRP78 (YCYIS6GADR)
Language	English
Publishing date	2019-06-04
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1520-5207
ISSN (online)	1520-5207
DOI	10.1021/acs.jpcb.9b02967
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Article ; Online: A DNA Origami Platform for Single-Pair Förster Resonance Energy Transfer Investigation of DNA-DNA Interactions and Ligation.

Bartnik, Kira / Barth, Anders / Pilo-Pais, Mauricio / Crevenna, Alvaro H / Liedl, Tim / Lamb, Don C

Journal of the American Chemical Society

2020 Volume 142, Issue 2, Page(s) 815–825

Abstract: DNA double-strand breaks (DSBs) pose an everyday threat to the conservation of genetic information and therefore life itself. Several pathways have evolved to repair these cytotoxic lesions by rejoining broken ends, among them the nonhomologous end- ... ...

Abstract	DNA double-strand breaks (DSBs) pose an everyday threat to the conservation of genetic information and therefore life itself. Several pathways have evolved to repair these cytotoxic lesions by rejoining broken ends, among them the nonhomologous end-joining mechanism that utilizes a DNA ligase. Here, we use a custom-designed DNA origami nanostructure as a model system to specifically mimic a DNA DSB, enabling us to study the end-joining of two fluorescently labeled DNA with the T4 DNA ligase on the single-molecule level. The ligation reaction is monitored by Förster resonance energy transfer (FRET) experiments both in solution and on surface-anchored origamis. Due to the modularity of DNA nanotechnology, DNA double strands with different complementary overhang lengths can be studied using the same DNA origami design. We show that the T4 DNA ligase repairs sticky ends more efficiently than blunt ends and that the ligation efficiency is influenced by both DNA sequence and the incubation conditions. Before ligation, dynamic fluctuations of the FRET signal are observed due to transient binding of the sticky overhangs. After ligation, the FRET signal becomes static. Thus, we can directly monitor the ligation reaction through the transition from dynamic to static FRET signals. Finally, we revert the ligation process using a restriction enzyme digestion and religate the resulting blunt ends. The here-presented DNA origami platform is thus suited to study complex multistep reactions occurring over several cycles of enzymatic treatment.
MeSH term(s)	DNA/chemistry ; DNA Ligases/chemistry ; DNA-Binding Proteins/chemistry ; Fluorescence Resonance Energy Transfer/methods
Chemical Substances	DNA-Binding Proteins ; DNA (9007-49-2) ; DNA Ligases (EC 6.5.1.-)
Language	English
Publishing date	2020-01-02
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	3155-0
ISSN	1520-5126 ; 0002-7863
ISSN (online)	1520-5126
ISSN	0002-7863
DOI	10.1021/jacs.9b09093
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