Single-Molecule FRET

Abstract

Single molecule studies have revolutionized our understanding of how biomolecules work. The ability to watch one molecule at a time reveals not only the average properties detected in ensemble measurements, but can yield the entire distribution of relevant properties, including subpopulations and rare events. Single molecule studies also reveal the time evolution of biochemical reactions, which, if asynchronous, are not observable via ensemble measurements. In the 1970s, for example, the development of the patch-clamp technique enabled the electrical current through a single ion channel to be measured, revealing many properties, including that ion channels are on/off devices.More recently, single molecule techniques applicable beyond ion channels have been developed. Methods such as optical (or magnetic) tweezers and scanning force microscopy are making it possible to follow, in real-time, the movements, forces and strains that develop during the course of a reaction (reviewed in [1]). These methods can be used to measure directly the forces that hold together molecular structures. New developments in single molecule fluorescence methodology are also broadening the scope of single molecule studies. Single biomolecules are labeled with an extrinsic fluorophore, and the fluorescent properties of the fluorophore can report on conformational changes occurring in the biomolecules. For example, intensity fluctuations of a single or small number of molecules can also measure reaction rates as well as the number of molecules present. Single enzyme reaction kinetics, for example, were measured in this way and found to have both static and dynamic disorder, as well as a rate which depended on the enzyme's history, properties which were undetectable via ensemble methods [2]. In addition, single molecule fluorescence polarization can be used to infer local rotation or changes in flexibility of biomolecules (reviewed in [3,4]).Perhaps the most general approach is the use of two fluorophores rather than one, in the form of fluorescence resonance energy transfer (FRET). In FRET, a "donor" dye transfers energy to an "acceptor" dye, and the donor intensity decreases and the acceptor intensity increases as the two dyes approach each other (reviewed in [5]). Importantly, it is sensitive to sub-nanometer distance changes in the range of 20-80 Å, a sensitivity and range well suited for studying biomolecules. Excitation energy of the donor is transferred to the acceptor via an induceddipole, induced-dipole interaction. The efficiency of energy transfer, E, is given by:where R is the distance between the donor and acceptor and R 0 is the distance at which 50% of the energy is transferred and is a function of the properties of the dyes. Because of this strong distance dependence, structural changes of biomolecules or relative motion and interaction between two different molecules can be detected via FRET. These conformational changes are often difficult to

Single‐molecule studies of conformational states and dynamics in the ABC importer OpuA

“…Over the past years [13], various laboratories have introduced single-molecule tools to investigate the structural dynamics of active membrane transporters [14][15][16][17][18][19][20][21][22][23][24][25]. F€ orster resonance energy transfer in combination with single-molecule detection (smFRET [26][27][28]) has proven to be a particularly useful tool for the validation of structural models [29][30][31] and for revealing functional features of transporters, which are mechanistically important, such as conformational heterogeneity [28,[32][33][34]. While the laboratories of Shimon Weiss [14], Scott Blanchard [15][16][17] and Antoine van Oijen [18] have published the first smFRET studies of secondary active transporters in a detergent environment [14][15][16] and within liposomes [17,18], we introduced smFRET for studies of ABC transporters in collaboration with the groups of Bert Poolman, Konstantinos Beis and Robert Tamp e. These transporters included importers (GlnPQ [19,20]) and their substrate-binding proteins and domains [21] and the exporter McjD [23].…”

Single‐molecule studies of conformational states and dynamics in the ABC importer OpuA

“…Over the past years [13], various laboratories have introduced single-molecule tools to investigate the structural dynamics of active membrane transporters [14][15][16][17][18][19][20][21][22][23][24][25]. F€ orster resonance energy transfer in combination with single-molecule detection (smFRET [26][27][28]) has proven to be a particularly useful tool for the validation of structural models [29][30][31] and for revealing functional features of transporters, which are mechanistically important, such as conformational heterogeneity [28,[32][33][34]. While the laboratories of Shimon Weiss [14], Scott Blanchard [15][16][17] and Antoine van Oijen [18] have published the first smFRET studies of secondary active transporters in a detergent environment [14][15][16] and within liposomes [17,18], we introduced smFRET for studies of ABC transporters in collaboration with the groups of Bert Poolman, Konstantinos Beis and Robert Tamp e. These transporters included importers (GlnPQ [19,20]) and their substrate-binding proteins and domains [21] and the exporter McjD [23].…”

Branching Out of Single‐Molecule Fluorescence Spectroscopy: Challenges for Chemistry and Influence on Biology

“…The groups of Ha and Weiss in particular, demonstrated the potential of spFRET to reveal conformational dynamics in single biomolecules on the millisecond time scale showing, for example, unzipping of DNA by a single helicase molecule, magnesium dependent RNA dynamics, and protein folding. [116][117][118][119][120][121][122][123][124] Deniz et al [117] used spFRET to study protein folding of chymotrypsin inhibitor 2 (CI2), a small, single-domain protein, at the single-molecule level. CI2 was selected as a model system to study protein folding at the single-molecule level because it shows two-state folding.…”

Single-Molecule Fluorescence Resonance Energy Transfer in Nanopipets: Improving Distance Resolution and Concentration Range

“…The efficiency of this dipole−dipole interaction depends on the distance between the fluorophores, the spectral overlap of the donor emission and the acceptor absorption (the so-called overlap integral), the refractive index of the medium, the donor fluorescence quantum yield, and the relative orientation of the fluorophores. As sample heterogeneity and the visualization of (bio)molecular dynamics are key issues to achieve quantifiable information, single-molecule FRET (smFRET) has become one of the most widespread tools of optical single-molecule methods. − Single-molecule techniques enable the selection of the population of interest that can further be analyzed, circumventing the need for 100% pure samples that are often not accessible because of limitations in purification procedures due to interconversion between different states or due to the transient nature of interactions. These advantages of the single-molecule approach are however partly compromised by specific requirements of smFRET measurements such as sophisticated equipment and by the lower resolution caused by the inherent lower signal-to-noise (S/N) ratio obtained in single molecule experiments.…”

Single‐molecule studies of conformational states and dynamics in the ABC importer OpuA

“…Over the past years [13], various laboratories have introduced single-molecule tools to investigate the structural dynamics of active membrane transporters [14][15][16][17][18][19][20][21][22][23][24][25]. F€ orster resonance energy transfer in combination with single-molecule detection (smFRET [26][27][28]) has proven to be a particularly useful tool for the validation of structural models [29][30][31] and for revealing functional features of transporters, which are mechanistically important, such as conformational heterogeneity [28,[32][33][34]. While the laboratories of Shimon Weiss [14], Scott Blanchard [15][16][17] and Antoine van Oijen [18] have published the first smFRET studies of secondary active transporters in a detergent environment [14][15][16] and within liposomes [17,18], we introduced smFRET for studies of ABC transporters in collaboration with the groups of Bert Poolman, Konstantinos Beis and Robert Tamp e. These transporters included importers (GlnPQ [19,20]) and their substrate-binding proteins and domains [21] and the exporter McjD [23].…”

Branching Out of Single‐Molecule Fluorescence Spectroscopy: Challenges for Chemistry and Influence on Biology

“…The groups of Ha and Weiss in particular, demonstrated the potential of spFRET to reveal conformational dynamics in single biomolecules on the millisecond time scale showing, for example, unzipping of DNA by a single helicase molecule, magnesium dependent RNA dynamics, and protein folding. [116][117][118][119][120][121][122][123][124] Deniz et al [117] used spFRET to study protein folding of chymotrypsin inhibitor 2 (CI2), a small, single-domain protein, at the single-molecule level. CI2 was selected as a model system to study protein folding at the single-molecule level because it shows two-state folding.…”

Single-Molecule Fluorescence Resonance Energy Transfer in Nanopipets: Improving Distance Resolution and Concentration Range

“…The efficiency of this dipole−dipole interaction depends on the distance between the fluorophores, the spectral overlap of the donor emission and the acceptor absorption (the so-called overlap integral), the refractive index of the medium, the donor fluorescence quantum yield, and the relative orientation of the fluorophores. As sample heterogeneity and the visualization of (bio)molecular dynamics are key issues to achieve quantifiable information, single-molecule FRET (smFRET) has become one of the most widespread tools of optical single-molecule methods. − Single-molecule techniques enable the selection of the population of interest that can further be analyzed, circumventing the need for 100% pure samples that are often not accessible because of limitations in purification procedures due to interconversion between different states or due to the transient nature of interactions. These advantages of the single-molecule approach are however partly compromised by specific requirements of smFRET measurements such as sophisticated equipment and by the lower resolution caused by the inherent lower signal-to-noise (S/N) ratio obtained in single molecule experiments.…”

Single‐molecule studies of conformational states and dynamics in the ABC importer OpuA

“…Over the past years [13], various laboratories have introduced single-molecule tools to investigate the structural dynamics of active membrane transporters [14][15][16][17][18][19][20][21][22][23][24][25]. F€ orster resonance energy transfer in combination with single-molecule detection (smFRET [26][27][28]) has proven to be a particularly useful tool for the validation of structural models [29][30][31] and for revealing functional features of transporters, which are mechanistically important, such as conformational heterogeneity [28,[32][33][34]. While the laboratories of Shimon Weiss [14], Scott Blanchard [15][16][17] and Antoine van Oijen [18] have published the first smFRET studies of secondary active transporters in a detergent environment [14][15][16] and within liposomes [17,18], we introduced smFRET for studies of ABC transporters in collaboration with the groups of Bert Poolman, Konstantinos Beis and Robert Tamp e. These transporters included importers (GlnPQ [19,20]) and their substrate-binding proteins and domains [21] and the exporter McjD [23].…”

Branching Out of Single‐Molecule Fluorescence Spectroscopy: Challenges for Chemistry and Influence on Biology

“…The groups of Ha and Weiss in particular, demonstrated the potential of spFRET to reveal conformational dynamics in single biomolecules on the millisecond time scale showing, for example, unzipping of DNA by a single helicase molecule, magnesium dependent RNA dynamics, and protein folding. [116][117][118][119][120][121][122][123][124] Deniz et al [117] used spFRET to study protein folding of chymotrypsin inhibitor 2 (CI2), a small, single-domain protein, at the single-molecule level. CI2 was selected as a model system to study protein folding at the single-molecule level because it shows two-state folding.…”

Single-Molecule Fluorescence Resonance Energy Transfer in Nanopipets: Improving Distance Resolution and Concentration Range

“…The efficiency of this dipole−dipole interaction depends on the distance between the fluorophores, the spectral overlap of the donor emission and the acceptor absorption (the so-called overlap integral), the refractive index of the medium, the donor fluorescence quantum yield, and the relative orientation of the fluorophores. As sample heterogeneity and the visualization of (bio)molecular dynamics are key issues to achieve quantifiable information, single-molecule FRET (smFRET) has become one of the most widespread tools of optical single-molecule methods. − Single-molecule techniques enable the selection of the population of interest that can further be analyzed, circumventing the need for 100% pure samples that are often not accessible because of limitations in purification procedures due to interconversion between different states or due to the transient nature of interactions. These advantages of the single-molecule approach are however partly compromised by specific requirements of smFRET measurements such as sophisticated equipment and by the lower resolution caused by the inherent lower signal-to-noise (S/N) ratio obtained in single molecule experiments.…”