Molecular and spectroscopic characterization of green and red cyanine fluorophores from the Alexa Fluor and AF series

Gebhardt, Christian; Lehmann, Martin; Reif, Maria M.; Zacharias, Martin; Cordes, Thorben

doi:10.1101/2020.11.13.381152

Cited by 5 publications

(8 citation statements)

References 112 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…All variants showed very good agreement between experimental and simulated values in smFRET experiments. Ligand binding was confirmed using microscale thermophoresis as shown previously 63 .…”

Section: Resultsmentioning

confidence: 98%

“… (1) Maleimide-thiol adducts of Alexa Fluor 555 63 (2) TMR (tetramethylrhodamin-5-maleimide) 64 and (3) Alexa Fluor 647 63 were used in the present study. (4) The MTSSL- 65 , (5) DOTA-Gd- 66 and (6) trityl-spin labels 67 attached to a cysteine residue via a disulfide bridge.…”

Section: Resultsmentioning

confidence: 99%

“…The distance distributions were determined using DeerAnalysis 40 and the distance predictions were performed with mtsslWizard 68 . smFRET experiments of diffusing protein molecules were performed in buffer at room temperature using standard procedures suitable for microsecond alternating laser excition (ALEX) as described before 63 . The experiments were performed with Alexa Fluor 555 (donor) and Alexa Fluor 647 (acceptor) using a homebuilt confocal microscopy setup with 2-colour detection 63 .…”

Section: Resultsmentioning

confidence: 99%

“…smFRET experiments of diffusing protein molecules were performed in buffer at room temperature using standard procedures suitable for microsecond alternating laser excition (ALEX) as described before 63 . The experiments were performed with Alexa Fluor 555 (donor) and Alexa Fluor 647 (acceptor) using a homebuilt confocal microscopy setup with 2-colour detection 63 . For smFRET assay design, we used a unpublished label analyzer script to identify residues for labelling with optimal dynamic range within the smFRET assay (Gebhardt, Bawidamann, Lipfert & Cordes, unpublished).…”

Section: Resultsmentioning

confidence: 99%

“…98 (ch. 11) on a homebuilt setup 63 : 400 μl of sample is measured in a 1.5×10 mm cuvette at a concentration of around 100 nM. The sample is excited by a pulsed laser (LDH-P-FA-530B for green fluorophores/LDH-D-C-640 for red fluorophores with PDL 828 “Sepia II” controller, Picoquant).…”

Section: Methodsmentioning

confidence: 99%

See 4 more Smart Citations

Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET

Gebhardt

Mächtel

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Pulsed electron-electron double resonance spectroscopy (PELDOR or DEER) and single molecule Förster resonance energy transfer spectroscopy (smFRET) are recent additions to the toolbox of integrative structural biology. Both methods are frequently used to visualize conformational changes and to determine nanometer-scale distances in biomacromolecules including proteins and nucleic acids. A prerequisite for the application of PELDOR/DEER and smFRET is the presence of suitable spin centers or fluorophores in the target molecule, which are usually introduced via chemical biology methods. The application portfolio of the two methods is overlapping: each allows determination of distances, to monitor distance changes and to visualize conformational heterogeneity and -dynamics. Both methods can provide qualitative information that facilitates mechanistic understanding, for instance on conformational changes, as well as quantitative data for structural modelling. Despite their broad application, a comprehensive comparison of the accuracy of PELDOR/DEER and smFRET is still missing and we set out here to fill this gap. For this purpose, we prepared a library of double cysteine mutants of three well-studied substrate binding proteins that undergo large-scale conformational changes upon ligand binding. The distances between the introduced spin- or fluorescence labels were determined via PELDOR/DEER and smFRET, using established standard experimental protocols and data analysis routines. The experiments were conducted in the presence and absence of the natural ligands to investigate how well the ligand-induced conformational changes could be detected by the two methods. Overall, we found good agreement for the determined distances, yet some surprising inconsistencies occurred. In our set of experiments, we identified the source of discrepancies as the use of cryoprotectants for PELDOR/DEER and label-protein interactions for smFRET. Our study highlights strength and weaknesses of both methods and paves the way for a higher confidence in quantitative comparison of PELDOR/DEER and smFRET results in the future.

show abstract

Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET

Gebhardt

Mächtel

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

Molecular structure, DNA binding mode, photophysical properties and recommendations for use of SYBR Gold

Kolbeck

Vanderlinden

Gemmecker

et al. 2021

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

SYBR Gold is a commonly used and particularly bright fluorescent DNA stain, however, its chemical structure is unknown and its binding mode to DNA remains controversial. Here, we solve the structure of SYBR Gold by NMR and mass spectrometry to be [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium] and determine its extinction coefficient. We quantitate SYBR Gold binding to DNA using two complementary approaches. First, we use single-molecule magnetic tweezers (MT) to determine the effects of SYBR Gold binding on DNA length and twist. The MT assay reveals systematic lengthening and unwinding of DNA by 19.1° ± 0.7° per molecule upon binding, consistent with intercalation, similar to the related dye SYBR Green I. We complement the MT data with spectroscopic characterization of SYBR Gold. The data are well described by a global binding model for dye concentrations ≤2.5 μM, with parameters that quantitatively agree with the MT results. The fluorescence increases linearly with the number of intercalated SYBR Gold molecules up to dye concentrations of ∼2.5 μM, where quenching and inner filter effects become relevant. In summary, we provide a mechanistic understanding of DNA-SYBR Gold interactions and present practical guidelines for optimal DNA detection and quantitative DNA sensing applications using SYBR Gold.

show abstract

Topology-dependent DNA binding

Kolbeck,

Tišma,

Analikwu

et al. 2023

Preprint

View full text Add to dashboard Cite

DNA stores our genetic information and is ubiquitous in biological and biotechnological applications, where it interacts with binding partners ranging from small molecules to large macromolecular complexes. Binding is modulated by mechanical strains in the molecule and, in turn, can change the local DNA structure. Frequently, DNA occurs in closed topological forms where topology and supercoiling add a global constraint to the interplay of binding-induced deformations and strain-modulated binding. Here, we present a quantitative model of how the global constraints introduced by DNA topology modulate binding and create a complex interplay between topology and affinity. We focus on fluorescent intercalators, which unwind DNA and enable direct quantification via fluorescence detection. Using bulk measurements, we show that DNA supercoiling can increase or decrease intercalation relative to an open topology depending on ligand concentration and the initial topology. Our model quantitatively accounts for observations obtained using psoralen for UV-induced DNA crosslinking, which is frequently used to quantify supercoiling in vivo. Finally, we observe topology-dependent binding in a single-molecule assay, which provides direct access to binding kinetics and DNA supercoil dynamics. Our results have broad implications for the detection and quantification of DNA and for the modulation of DNA binding in cellular contexts.

show abstract

Molecular and spectroscopic characterization of green and red cyanine fluorophores from the Alexa Fluor and AF series

Cited by 5 publications

References 112 publications

Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET

Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET

Molecular structure, DNA binding mode, photophysical properties and recommendations for use of SYBR Gold

Topology-dependent DNA binding

Contact Info

Product

Resources

About