Article Information

• PDF (92 kb)
• Full Text with Large Figures
• Supporting Material
• Cited by in Scopus (4)

PubMed

• Articles by Peizhi Zhu
• Articles by Ashok A. Deniz

Related Articles

• Novel Two-Band Ratiometric Fluorescence Probes with Differen...

  Novel Two-Band Ratiometric Fluorescence Probes with Different Location and Orientation in Phospholipid Membranes Chemistry & Biology, Volume 9, Issue 11, 1 November 2002, Pages 1199-1208 Andrey S Klymchenko, Guy Duportail, Turan Ozturk, Vasyl G Pivovarenko, Yves Mély and Alexander P Demchenko Summary 3-hydroxyflavone (3-HF) derivatives are very attractive fluorescence sensors due to their ability to respond to small changes in their microenvironment via a dramatic alteration of the relative intensities of their two well-separated emission bands. We developed fluorescence probes with locations at different depths and orientations of 3-HF moiety in the phospholipid bilayer, which determine their fluorescence behavior. While the spectral shifts of the probes correlate with their binding site polarity, the intensity ratio is a complex parameter that is also sensitive to the local hydration. We demonstrate that even the deeply located probes sense this hydration effect, which can be modulated by the charge of the lipid heads and is anisotropic with respect to the bilayer plane. Thus the two-band ratiometric fluorescence probes can provide multiparametric information on the properties of lipid membranes at different depths. Summary | Full Text | PDF (216 kb)

• The Mechanism of Membrane Insertion for a Cholesterol-Depend...

  The Mechanism of Membrane Insertion for a Cholesterol-Dependent Cytolysin Cell, Volume 99, Issue 3, 29 October 1999, Pages 293-299 Oleg Shatursky, Alejandro P Heuck, Laura A Shepard, Jamie Rossjohn, Michael W Parker, Arthur E Johnson and Rodney K Tweten Summary Perfringolysin O (PFO), a water-soluble monomeric cytolysin secreted by pathogenic , oligomerizes and forms large pores upon encountering cholesterol-containing membranes. Whereas all pore-forming bacterial toxins examined previously have been shown to penetrate the membrane using a single amphipathic β hairpin per polypeptide, cysteine-scanning mutagenesis and multiple independent fluorescence techniques here reveal that each PFO monomer contains a second domain involved in pore formation, and that each of the two amphipathic β hairpins completely spans the membrane. In the soluble monomer, these transmembrane segments are folded into six α helices. The insertion of two transmembrane hairpins per toxin monomer and the major change in secondary structure are striking and define a novel paradigm for the mechanism of membrane insertion by a cytolytic toxin. Summary | Full Text | PDF (163 kb)

• Porphyrin Depth in Lipid Bilayers as Determined by Iodide an...

  Porphyrin Depth in Lipid Bilayers as Determined by Iodide and Parallax Fluorescence Quenching Methods and Its Effect on Photosensitizing Efficiency Biophysical Journal, Volume 87, Issue 2, 1 August 2004, Pages 1155-1164 Irena Bronshtein, Michal Afri, Hana Weitman, Aryeh A. Frimer, Kevin M. Smith and Benjamin Ehrenberg Abstract Photosensitization by porphyrins and other tetrapyrrole chromophores is used in biology and medicine to kill cells. This light-triggered generation of singlet oxygen is used to eradicate cancer cells in a process dubbed “photodynamic therapy,” or PDT. Most photosensitizers are of amphiphilic character and they partition into cellular lipid membranes. The photodamage that they inflict to the host cell is mainly localized in membrane proteins. This photosensitized damage must occur in competition with the rapid diffusion of singlet oxygen through the lipid phase and its escape into the aqueous phase. In this article we show that the extent of damage can be modulated by employing modified hemato- and protoporphyrins, which have alkyl spacers of varying lengths between the tetrapyrrole ring and the carboxylate groups that are anchored at the lipid/water interface. The chromophore part of the molecule, and the point of generation of singlet oxygen, is thus located at a deeper position in the bilayer. The photosensitization efficiency was measured with 9,10-dimethylanthracene, a fluorescent chemical target for singlet oxygen. The vertical insertion of the sensitizers was assessed by two fluorescence-quenching techniques: by iodide ions that come from the aqueous phase; and by spin-probe-labeled phospholipids, that are incorporated into the bilayer, using the parallax method. These methods also show that temperature has a small effect on the depth when the membrane is in the liquid phase. However, when the bilayer undergoes a phase transition to the solid gel phase, the porphyrins are extruded toward the water interface as the temperature is lowered. These results, together with a previous publication in this journal, represent a unique and precedental case where the vertical location of a small molecule in a membrane has an effect on its membranal activity. Abstract | Full Text | PDF (155 kb)

• …more

Copyright © 2005 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 89, Issue 5, L37-L39, 1 November 2005

doi:10.1529/biophysj.105.071027

Biophysical Letters

Fluorescence Quenching by TEMPO: A Sub-30 Å Single-Molecule Ruler

Peizhi Zhu¹, Jean-Pierre Clamme¹ and Ashok A. Deniz^,  

Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

Address reprints and inquiries to Ashok A. Deniz, Tel.: 858-784-9192.

¹ Peizhi Zhu and Jean-Pierre Clamme contributed equally to this work.

The study of conformational distributions and dynamics of biomolecules has been revolutionized in recent years by single-molecule fluorescence resonance energy transfer (FRET), which is an efficient tool for measuring distances between 30 and 80Å. In contrast, fewer examples of single-molecule studies have been reported for monitoring shorter distances in the sub-30Å range. A general methodology with such a capability would be very useful for studying detailed molecular structures, such as those of protein and RNA secondary structures during folding, binding, and assembly. At the single-molecule level, fluorescence self-quenching has been used for protein folding studies 1, and fluorescence quenching of organic dyes by tryptophan or guanosine via photoinduced electron transfer has also been reported to measure short distances and fluctuations in peptides, proteins, and DNA 2,3,4,5. However, there has been no description so far of a method analogous to FRET, where dye and quencher are independently attached to points of interest on a biomolecule, allowing the distance between these points to be monitored.

TEMPO is a small organic nitroxide radical. Several studies have used its fluorescence quenching and electron paramagnetic resonance characteristics to obtain structural information for proteins and RNA, and have shown that its conjugation is well tolerated in these biomolecules 6,7. Furthermore, the distance dependence of the fluorescence quenching rate for a related nitroxide radical PROXYL has been studied by ensemble fluorescence spectroscopy, and shows an exponential distance dependence 8, providing additional support for TEMPO quenching as an attractive candidate for a short-range single-molecule ruler. Based on these features, in this work, we have used fluorescence correlation spectroscopy (FCS) to demonstrate intramolecular quenching by TEMPO of 5-carboxytetramethylrhodamine (5-TAMRA, a commonly used dye for single-molecule studies) as a sub-30Å distance ruler for small ensemble/single-molecule measurements.

To this end, we labeled 29-mer DNA molecules with 5-TAMRA and TEMPO at different positions, producing a DNA series with different distances between the dye and quencher (sequence and constructs are shown in Fig. 1). To calculate theoretical distances between dye and quencher, we built a double-stranded DNA model (corresponding to the first 10 basepairs of the 29-mer sequence) using Hyperchem 7.51. We then used this model to optimize structures corresponding to each of our doubly labeled constructs using the BIO+(CHARMM) molecular mechanics force field, and calculated dye-quencher distances. Double labeling and distance calculation protocols are detailed in the Supplementary Material .

Display large version of this figure

Figure 1

Sequences and dye/quencher labeling positions for the DNA constructs. Theoretical dye-quencher distances from molecular mechanics (see text and Supplementary Material ) are also shown above each construct.

In our experiments, we first performed steady-state ensemble fluorescence measurements for each construct with and without the quencher. The quenching efficiencies (Q_ens) were calculated using the average fluorescence intensities for the DNA constructs with and without quencher (Supplementary Material ). Q_ens varied from >50% at 10.5Å, to close to zero at 47Å. The quenching rate constants were calculated from Q_ens (Supplementary Material ) and are plotted as a function of the theoretical quencher-dye distance (R) in Fig. 2, showing an exponential decrease with increasing distance (solid line, Fig. 2) in the 10–30Å range, consistent with previous work 8,9,10.

Display large version of this figure

Figure 2

Distance dependence of TEMPO quenching rate constant (k_TEMPO (s⁻¹)) measured by ensemble fluorescence (black solid circle) (solid line is single exponential decay fit) and FCS (red solid square). Dashed line is the quenching component expected due to FRET between TAMRA and TEMPO with R₀=8Å. Inset shows FCS curves of DNA-TAMRA (black), DNA-TAMRA-TEMPO (red), and DNA-TAMRA in the presence of 10mM iodide (green). Error bars are ±1 standard deviation.

We next used FCS to investigate the properties of the fluorescence quenching of 5-TAMRA by TEMPO at a small ensemble/single-molecule level. FCS is based on the time correlation of fluorescence fluctuations observed in a subfemtoliter volume of a sample solution. In this study, FCS data were recorded on a homebuilt setup (see Supplementary Material ), averaged (10 files of 60s each), and the correlation functions G(τ) analyzed to extract N, the average number of molecules in the excitation volume, and τ_D, the characteristic diffusion time of the particle 11,12. The average diffusion coefficient without (D_T=8.1 (±0.6)×10⁻¹¹m²s⁻¹) and with the quencher (D_TQ=7.8 (±0.5)×10⁻¹¹m²s⁻¹) are similar, showing that the addition of the quencher does not significantly alter the diffusional properties of the DNA. Our results also show that the number of molecules N (proportional to concentration) with the quencher is not significantly different from that without quencher (data not shown), thus excluding a quenching mechanism involving a long-lived (greater than the ∼200μs diffusion time) dark state, which would lower the number of fluorescent molecules detected in the focal volume. Moreover, no significant increase in the triplet state fraction was observed in the presence of the quencher (Fig. 2, inset, black/red curves with/without quencher, respectively). By comparison, quenching by iodide, known to occur by intersystem crossing to the triplet state, produces an FCS curve (Fig. 2, inset, green) displaying an additional fast decay. These results suggest that TAMRA quenching by TEMPO does not occur due to enhanced intersystem crossing of TAMRA to a dark triplet state.

To evaluate the quenching observed by FCS, the average count rate (I) for each construct was recorded and used to calculate the brightness or count rate per molecule (η) defined as η=I/N. The quenching rate constants were then derived from the quenching efficiencies (derived from η for constructs with and without quencher; see Supplementary Material ). This quenching rate constant is plotted as a function of the dye-quencher distance in Fig. 2, also showing an exponential decay in good agreement with the ensemble data. Finally, Fig. 2 also shows the expected quenching based on a FRET mechanism and an R₀ of 8Å (Supplementary Material ), observed to drop below 5% above 13Å. A comparison with the experimental data shows that a FRET mechanism of quenching cannot be making a significant contribution to the observed quenching above ∼13Å.

Our FCS distance dependence results show that TAMRA fluorescence quenching by TEMPO can be used to measure sub-30Å distance changes with ∼5Å resolution in biomolecules at single-molecule resolution. The results also rule out a predominant role of FRET or intersystem crossing in the mechanism of the quenching process. Further technical refinements will include improving accuracy by using single-molecule fluorescence lifetime measurements as well as monitoring long time trajectories on surface immobilized molecules. We believe that this single-molecule distance measurement technique will complement FRET for measuring localized conformational changes for biomolecules during their folding, assembly, and function, including monitoring conformational fluctuations using FCS measurements 5.