Quantitative FRET studies and integrative modeling unravel the structure and dynamics of biomolecular systems

Dimura, Mykola; Peulen, Thomas-Otavio; Hanke, Christian A.; Prakash, Aiswaria; Gohlke, Holger; Seidel, Claus A. M.

doi:10.1016/j.sbi.2016.11.012

Cited by 171 publications

(190 citation statements)

References 139 publications

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“…Therefore, the variance of the FRET efficiency distribution in the TD-A1 experiment is much smaller for the tetramer than for the dimer. Several groups have recently developed tools to calculate an accurate distribution of the distance between the donor and acceptor by appropriate modeling of fluorophores and linkers for macromolecules with well-defined structures (64)(65)(66)(67)(68)(69). This calculation may allow for a more quantitative comparison.…”

Section: Discussionmentioning

confidence: 99%

Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET

Chung

Meng

Kim

et al. 2017

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

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We describe a method that combines two-and three-color singlemolecule FRET spectroscopy with 2D FRET efficiency-lifetime analysis to probe the oligomerization process of intrinsically disordered proteins. This method is applied to the oligomerization of the tetramerization domain (TD) of the tumor suppressor protein p53. TD exists as a monomer at subnanomolar concentrations and forms a dimer and a tetramer at higher concentrations. Because the dissociation constants of the dimer and tetramer are very close, as we determine in this paper, it is not possible to characterize different oligomeric species by ensemble methods, especially the dimer that cannot be readily separated. However, by using single-molecule FRET spectroscopy that includes measurements of fluorescence lifetime and two-and three-color FRET efficiencies with corrections for submillisecond acceptor blinking, we show that it is possible to obtain structural information for individual oligomers at equilibrium and to determine the dimerization kinetics. From these analyses, we show that the monomer is intrinsically disordered and that the dimer conformation is very similar to that of the tetramer but the C terminus of the dimer is more flexible.single-molecule spectroscopy | three-color FRET | intrinsically disordered protein | p53 oligomerization | fluorescence lifetime I t is well known that intrinsically disordered proteins (IDPs) can fold into different structures when attaching to their binding targets. This structural flexibility and binding promiscuity are required for the formation of protein-protein interaction networks in various biological processes such as signal transduction and gene transcription (1-3). On the other hand, some IDPs selfassemble and form oligomers, many of which are implicated in the development of diseases such as Alzheimer's disease (amyloid-β protein) (4, 5) and Parkinson's disease (α-synuclein) (6). An ensemble of these oligomers with different sizes and conformations is not easy to separate, and therefore, their characterization is very difficult. However, single-molecule spectroscopy can be a very powerful tool because it can probe subpopulations in a mixture without the need for separation. Single-molecule spectroscopy has been successfully used for characterizing individual molecular states such as the folded and unfolded states of proteins (7-9), intermediate states (10, 11), and transition paths (12-17) and for identifying specific molecular species and complexes (18)(19)(20)(21)(22). In this paper, we describe the development of a singlemolecule fluorescence method that probes individual oligomers in a mixture, characterizes their conformations, and determines oligomerization kinetics.We have used two-and three-color Förster resonance energy transfer (FRET) spectroscopy. Compared with two-color FRET that monitors a single distance, three-color FRET can determine three distances and therefore has great potential to obtain 3D structural information for a molecule or a molecular complex. Multicolor FRET has been d...

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Section: Discussionmentioning

confidence: 99%

Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET

Chung

Meng

Kim

et al. 2017

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

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“…Several studies (117–119) have followed this route, reporting successful structure determination (120–122). The information retrieved from smFRET measurements of multiple distances can be used to directly triangulate a structure of a whole or part of a macromolecule, or can be used as experimental constraints for structural simulations (121). In the latter approach, each iteration produces a structural snapshot.…”

Section: Solving 3d Structures With Smfretmentioning

confidence: 99%

Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer

Lerner

Cordes

Ingargiola

et al. 2018

Science

479

449

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Classical structural biology can only provide static snapshots of biomacromolecules. Single-molecule Förster resonance energy transfer (smFRET) paved the way for studying dynamics in macromolecular structures under biologically relevant conditions. Since its first implementation in 1996, smFRET experiments have confirmed previously hypothesized mechanisms and provided new insights into many fundamental biological processes, such as DNA maintenance and repair, transcription, translation, and membrane transport. We review 22 years of contributions of smFRET to our understanding of basic mechanisms in biochemistry, molecular biology, and structural biology. Additionally, building on current state-of-the-art implementations of smFRET, we highlight possible future directions for smFRET in applications such as biosensing, high-throughput screening, and molecular diagnostics.

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“…New developments in cryo‐electron microscopy have propelled this technique to the forefront of the field and this will have a profound effect on the future of structural biology . The recent years have also witnessed the maturation of several other techniques such as single molecule Förster resonance energy transfer spectroscopy (smFRET) and small‐angle X‐ray scattering (SAXS), which have been established as important methods in structural biology. This growing box of tools has led to the concept of “integrative structural biology”, in which combinations of suitable methods are used to tackle complex structural problems that would be difficult or impossible to solve by one technique alone .…”

Section: Figurementioning

confidence: 99%

Inhibitor‐Directed Spin Labelling—A High Precision and Minimally Invasive Technique to Study the Conformation of Proteins in Solution

Yin

Hammler

Peter

et al. 2018

Chemistry A European J

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Pulsed electron–electron double resonance spectroscopy (known as PELDOR or DEER) has recently become a very popular tool in structural biology. The technique can be used to accurately measure distance distributions within macromolecules or macromolecular complexes, and has become a standard method to validate structural models and to study the conformational flexibility of macromolecules. It can be applied in solution, in lipid environments or even in cells. Because most biological macromolecules are diamagnetic, they are normally invisible for PELDOR spectroscopy. To render a particular target molecule accessible for PELDOR, it can be engineered to contain only one or two surface‐exposed cysteine residues, which can be efficiently spin‐labelled using thiol‐reactive nitroxide compounds. This method has been coined “site‐directed spin labelling” (SDSL) and is normally straight‐forward. But, SDSL can be very challenging for proteins with many native cysteines, or even a single functionally or structurally important cysteine residue. For such cases, alternative spin labelling techniques are needed. Here we describe the concept of “inhibitor‐directed spin labelling” (IDSL) as an approach to spin label suitable cysteine‐rich proteins in a site‐directed and highly specific manner by employing bespoke spin‐labelled inhibitors. Advantages and disadvantages of IDSL are discussed.

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Quantitative FRET studies and integrative modeling unravel the structure and dynamics of biomolecular systems

Cited by 171 publications

References 139 publications

Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET

Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET

Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer

Inhibitor‐Directed Spin Labelling—A High Precision and Minimally Invasive Technique to Study the Conformation of Proteins in Solution

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