Two-Dimensional Fluorescence Intensity Distribution Analysis: Theory and Applications

Kask, Peet; Palo, Kaupo; Fay, Nicolas; Brand, Leif; Mets, Ülo; Ullmann, Dirk; Jungmann, Joern; Pschorr, Johannes; Gall, Karsten

doi:10.1016/s0006-3495(00)76722-1

Cited by 156 publications

(142 citation statements)

References 17 publications

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“…The spectroscopic information can be used to classify molecules into the different subpopulations that compose an ensemble and to characterize their size, identity, diffusion times, or local environment. The use of multiple, simultaneously acquired parameters improves the resolution of distinct species significantly (14,15).To obtain structural information at the single-molecule level, spectroscopic techniques can be combined with fluorescence resonance energy transfer (FRET). In FRET, the energy from an excited donor dye, D, is nonradiatively transferred to an acceptor dye, A.…”

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confidence: 99%

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Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes

Rothwell

Berger

Kensch

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

217

244

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By using single-molecule multiparameter fluorescence detection, fluorescence resonance energy transfer experiments, and newly developed data analysis methods, this study demonstrates directly the existence of three structurally distinct forms of reverse transcriptase (RT):nucleic acid complexes in solution. Single-molecule multiparameter fluorescence detection also provides first information on the structure of a complex not observed by x-ray crystallography. This species did not incorporate nucleotides and is structurally distinct from the other two observed species. We determined that the nucleic acid substrate is bound at a site far removed from the nucleic acid-binding tract observed by crystallography. In contrast, the other two states are identified as being similar to the x-ray crystal structure and represent distinct enzymatically productive stages in DNA polymerization. These species differ by only a 5-Å shift in the position of the nucleic acid. Addition of nucleoside triphosphate or of inorganic pyrophosphate allowed us to assign them as the educt and product state in the polymerization reaction cycle; i.e., the educt state is a complex in which the nucleic acid is positioned to allow nucleotide incorporation. The second RT:nucleic acid complex is the product state, which is formed immediately after nucleotide incorporation, but before RT translates to the next nucleotide.H IV-1 reverse transcriptase (RT) is a key enzyme in the life cycle of HIV. This multifunctional enzyme is responsible for the complex process of transcribing viral RNA into doublestranded DNA for integration into the host cell genome. The enzyme is a heterodimer composed of subunits which share a common N terminus and have subdomains referred to as fingers, palm, thumb, and connection ( Fig. 1). Although the subdomains of each subunit are structurally similar, the overall folding within the two subunits is quite different (1). The large subunit, p66, contains RNA-and DNA-dependent DNA polymerase as well as RNase H activities. The p51 subunit, which is inactive in the heterodimer but active in homodimers (2, 3), is thought to play a role in stabilizing the conformation of p66.Crystallographic studies of RT:nucleic acid primer͞template (p͞t) complexes have provided valuable insights into the structural features and conformational changes induced by p͞t binding (1, 4-7). To date, these crystallographic models have indicated a single p͞t-binding mode. In contrast, recent solutionbased kinetic studies on p͞t binding and nucleotide incorporation by RT suggest a heterogeneous mixture of several different binding modes (8). To confirm directly the existence of several species, and to obtain structural and functional information about each, we have used single-molecule spectroscopy to investigate RT:p͞t complexes in solution.Single-molecule techniques have proven to be valuable tools for resolving static and dynamic heterogeneity of an ensemble (9-13). For this investigation, we use a newly developed multiparameter fluorescence detection (MF...

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confidence: 99%

Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes

Rothwell

Berger

Kensch

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

217

244

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“…Complex formation between the PS/PS-labelled hit compound and SAP-SH2 was monitored by recording the fluorescence fluctuation data for each compound in the presence and absence of SAP-SH2 (16 μM) and by determining the fluorescence anisotropy with 2D-FIDA [14]. The resulting values were then compared with the values predicted by the 3D plot in Fig.…”

Section: Single-point Solution Confirmation and Affinity Determinationmentioning

confidence: 99%

Towards mimicking short linear peptide motifs: identification of new mixed α,β-peptidomimetic ligands for SLAM-Associated Protein (SAP) by confocal on-bead screening

et al. 2012

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An array of chemical modifications have recently emerged, designed to improve the stability of natural peptides that inherently suffer from short in vivo half-lives, thereby preventing their use as therapeutics. The resultant peptidomimetics resemble native peptides; however, they contain synthetic elements (e.g. non-coded amino acids) which confer improved biophysical properties. An elegant approach towards the identification of peptidomimetics is through screening of large combinatorial chemical libraries incorporating both coded and non-coded amino acids (e.g. β amino acids). We apply here our recently developed Integrated Chemical Biophysics (ICB) platform, which combines microscale one-bead one-compound screening with fluorescence tagging of retrieved hit beads and subsequent affinity determination of hit compounds in homogenous solution, to the task of identifying novel mixed α, β peptidomimetic binders for the adaptor protein SLAM-associated protein (SAP), which acts as an intracellular adapter that transduces T and NK cell activation. An enhancement to the ICB process is introduced which enables ranking hit compounds from single-point measurements even if the library compound is <95% pure and without HPLC purification of single-bead-derived substance. Finally, a novel computational protocol enabling binding mode and SAR rationalisation of hit compounds is also described which we now utilise to inform future library design. Application of the full ICB process has allowed identification of a highly interesting motif, Ac-β 3 -Pro-α-pTyr, as a mimic for the −1 and −2 positions of the natural binding motif and provides a promising starting point for further optimization towards higher-affinity SAP inhibitors with enhanced metabolic stability.Electronic supplementary material The online version of this article

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“…Different schemes have been developed to acquire and utilize such macro-time information in single-molecule experiments (25)(26)(27)(28). Fluorescence correlation spectroscopy, photon-counting histograms and other related analyses are extremely powerful tools for analyzing molecular interactions and we refer the reader to the literature for details (29)(30)(31)(32)(33). One principal advantage of these approaches is that even when shot noise severely limits the usefulness of time traces at short time scales (where < 20 photons are counted on average), keeping the time stamp information of each photon allows correlation methods to reveal fluorescence dynamics at the ensemble level.…”

Section: Single-photon Time-stampingmentioning

confidence: 99%

Detectors for single-molecule fluorescence imaging and spectroscopy

Michalet

Siegmund

Vallerga

et al. 2007

Journal of Modern Optics

115

106

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Single-molecule observation, characterization and manipulation techniques have recently come to the forefront of several research domains spanning chemistry, biology and physics. Due to the exquisite sensitivity, specificity, and unmasking of ensemble averaging, single-molecule fluorescence imaging and spectroscopy have become, in a short period of time, important tools in cell biology, biochemistry and biophysics. These methods led to new ways of thinking about biological processes such as viral infection, receptor diffusion and oligomerization, cellular signaling, protein-protein or protein-nucleic acid interactions, and molecular machines. Such achievements require a combination of several factors to be met, among which detector sensitivity and bandwidth are crucial. We examine here the needed performance of photodetectors used in these types of experiments, the current state of the art for different categories of detectors, and actual and future developments of single-photon counting detectors for single-molecule imaging and spectroscopy.

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Two-Dimensional Fluorescence Intensity Distribution Analysis: Theory and Applications

Cited by 156 publications

References 17 publications

Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes

Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes

Towards mimicking short linear peptide motifs: identification of new mixed α,β-peptidomimetic ligands for SLAM-Associated Protein (SAP) by confocal on-bead screening

Detectors for single-molecule fluorescence imaging and spectroscopy

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