Site-specific Fluorescent Labeling Research Articles

Editorial: Fluorescence spectroscopy and nucleic acids

Fluorescence spectroscopy is an established tool for investigating the structure, folding, and conformational dynamics of proteins in solution. The widespread use of fluorescence techniques for the physicochemical characterization of proteins stems from the occurrence of the naturally fluorescent amino acids tryptophan and tyrosine in many proteins and the great sensitivity of the emission properties of these amino acids to perturbations in protein structure. Historically, the application of fluorescence spectroscopy to nucleic acids has been hindered by the lack of suitable intrinsic fluorophores (the exception are certain tRNAs that contain unusual fluorescent bases). However, with the advent of rapid and efficient methods for the solid phase synthesis and site-specific fluorescent labeling of DNA and RNA oligonucleotides, the power of fluorescence spectroscopy can now be brought to bear on nucleic acids. Indeed, the past decade has witnessed a tremendous growth in the number of studies in which fluorescence spectroscopic techniques have been used to characterize the structural properties and fundamental biophysical behaviors of nucleic acids and nucleic acid–protein complexes. Particular advantages of fluorescence-based methods include high detection sensitivity, fast time resolution, and ready adaptability to a wide range of solution conditions. Of the many spectroscopic phenomena that can be exploited to examine nucleic acids, fluorescence resonance energy transfer (FRET) lends itself to the broadest range of applications. This issue of Nucleic Acid Sciences outlines some of the methodologies employed and illustrates the rich variety of information that is forthcoming from FRET-based analyses of nucleic acids, highlighting studies that focus on structural characterization, thermodynamic properties, and kinetic processes. Since the efficiency of resonance energy transfer from a donor fluorophore to an acceptor reports on distances ranging from 10 to 100 Å, measurements of FRET can be used to define the global structure of nucleic acid complexes. This approach is especially powerful in the case of large or disordered complexes that are not amenable to structural analysis by x-ray crystallographic or NMR spectroscopic methods. Showing the broad applicability of this technique to structural questions, FRET has been used to elucidate the helical geometry of multistranded nucleic acid complexes (Singleton and Xiao), to explore the dynamic solution structure of branched DNA molecules (Klostermeier and Millar), to characterize the conformation of bent DNA (Parkhurst et al.), and to probe the global architecture of large macromolecule complexes (Heyduk and Niedziela-Majka). In addition to its utility in probing structural questions, FRET may be used to analyze nucleic acid complex stability and conformational transitions. Precise thermodynamic characterization of the impact of base lesions on DNA duplex stability is possible using sensitive FRET-based methods, giving insight into the biological consequences of DNA damage and the molecular basis of mutagenesis (Plum and Breslauer). As another example, the time-resolved FRET technique can be used to identify and quantify different tertiary structure conformers of RNA, which often play a role in regulating biological activity. This approach has been used to investigate the molecular forces that drive conformational transitions in the hairpin ribozyme (Klostermeier and Millar). Finally, FRET methods can also be harnessed to study the kinetics of nucleic acid conformational transitions, underscoring the great versatility of fluorescence techniques for biophysical characterization of nucleic acids. FRET and rapid kinetic methods have been used to study the dynamics of DNA bending by the transcription factor TBP in real time and to establish a detailed kinetic mechanism for the interaction of TBP with DNA (Parkhurst et al.). Similar methods have been used to monitor large-scale conformational changes during the catalytic cycle of the hairpin and hepatitis delta virus ribozymes (Walter et al.). The studies reviewed in this issue of Nucleic Acid Sciences illustrate the ability of fluorescence techniques to provide structural, thermodynamic, and kinetic information on a variety of biologically important DNA and RNA molecules. It is hoped that these examples will stimulate an even wider application of fluorescence techniques in the study of nucleic acids. While most of the examples presented here involve relatively short oligonucleotides, it is anticipated that the different fluorescent labeling strategies discussed herein will be combined to construct much larger DNA or RNA molecules. Thus, the fluorescence spectroscopic techniques described in this issue can be applied to a variety of dynamic macromolecular complexes, including the replisome, transcription complexes, and the ribosome. In addition, the fluorescence techniques can be extended to the single-molecule level, which should provide new insights into the conformational dynamics and biological function of nucleic acids (single-molecule fluorescence studies have been reviewed recently; T. J. Ha, Current Opinion in Structural Biology, 2001 Vol. 11, pp. 287–292).

Streptokinase Triggers Conformational Activation of Plasminogen through Specific Interactions of the Amino-terminal Sequence and Stabilizes the Active Zymogen Conformation

Cleavage of Arg(561)-Val(562) in plasminogen (Pg) generates plasmin (Pm) through a classical activation mechanism triggered by an insertion of the new amino terminus into a binding pocket in the Pg catalytic domain. Streptokinase (SK) circumvents this process and activates Pg through a unique nonproteolytic mechanism postulated to be initiated by the intrusion of Ile(1) of SK in place of Val(562). This hypothesis was evaluated in equilibrium binding and kinetic studies of Pg activation with an SK mutant lacking Ile(1) (SK(2--414)). SK(2--414) retained the affinity of native SK for fluorescein-labeled [Lys]Pg and [Lys]Pm but induced no detectable conformational activation of Pg. The activity of SK(2--414) was partially restored by the peptides SK(1--2), SK(1--5), SK(1--10), and SK(1--15), whereas Pg(562--569) peptides were much less effective. Active site-specific fluorescence labeling demonstrated directly that the active catalytic site was formed on the Pg zymogen by the combination of SK(1--10) and SK(2--414), whereas sequence-scrambled SK(1-10) was inactive. The characterization of SK(1--10) containing single Ala substitutions demonstrated the sequence specificity of the interaction. SK(1--10) did not restore activity to the further truncated mutant SK(55-414), which was correlated with the loss of binding affinity of SK(55--414) for labeled [Lys]Pm but not for [Lys]Pg. The studies support a mechanism for conformational activation in which the insertion of Ile(1) of SK into the Pg amino-terminal binding cleft occurs through sequence-specific interactions of the first 10 SK residues. This event and the preferentially higher affinity of SK(2--414) for the activated proteinase domain of Pm are thought to function cooperatively to trigger the conformational change and stabilize the active zymogen conformation.

Highly fluorescent protein labeling using dendritic peptide derivatives.

Non-specific fluorescent dyes and photosensitizers are routinely used in clinical practice for the photodetection and photoablation of superficial lesions. Future applications in photomedicine are likely to rely on the selective delivery of photoactive compounds to diseased areas, using specific targeting agents such as antibodies. This fact underlines the need for methods that allow the chemically defined conjugation of several photoactive molecules to a single protein 'vehicle', with full retention of binding affinity. Here, we present methods for the site-specific fluorescent labeling of proteins using dendritic peptides, which had been chemically modified with multiple molecules of fluorescein. Branched peptide derivatives can be stably conjugated to proteins either by reaction with suitable free reactive groups or by using the high-affinity non-covalent interaction between calmodulin and a specific binding peptide. Chemical modification of proteins with one, two or four molecules of fluorescein resulted in a proportional increase in protein fluorescence.

Time-resolved fluorescence methods for analysis of DNA-protein interactions

This chapter describes fluorescence spectroscopy as an established technique for the analysis of macromolecular structure and dynamics and, in more recent studies, is emerging as a promising method for the study of DNA-protein interactions. The chief advantage of fluorescence-based approaches over the gel electrophoretic and filter-binding methods commonly used to characterize DNA protein interactions is the ability to monitor binding events in solution under conditions of true thermodynamic equilibrium. The chapter reviews the application of time-resolved fluorescence spectroscopy to the study of DNA-protein interactions. It provides an overview of general principles, focusing on those time-resolved fluorescence techniques that are most pertinent to the study of DNA-protein interactions. It discusses the instrumentation required for time-resolved fluorescence measurements and the methods employed in site-specific fluorescent labelling of DNA. A number of representative studies are then presented to demonstrate the different experimental strategies that can be used to characterize DNA-protein complexes. The specific examples presented in the chapter describe the ability of time-resolved fluorescence techniques to yield both structural and energetic information on DNA protein interactions.

Site-specific fluorescence labelling of recombinant polyomavirus-like particles.

For the development of gene therapy protocols based on polyomavirus-like particles, we describe a method for fluorescence labelling of virions in order to study virus-cell interactions preceding gene delivery. Site-specific fluorescence labelling of polyomavirus-like particles is achieved via a single cysteine residue and maleimide conjugates of fluorescence dyes (fluorescein, Texas Red). Polyomavirus-like particles can be assembled in vitro from recombinant capsomers produced in E. coli. Since the assembly process is independent of disulfide bond formation, all cysteine residues of the wild-type protein were replaced by serines, and a new unique cysteine residue was introduced for the attachment of the fluorescence marker.

Site-specific confocal fluorescence imaging of biological microstructures in a turbid medium

Normally transparent biological structures in a turbid medium are imaged using a laser confocal microscope and multiwavelength site-specific fluorescence labelling. The spatial filtering capability of the detector pinhole in the confocal microscope limits the number of scattered fluorescent photons that reach the photodetector. Simultaneous application of different fluorescent markers on the same sample site minimizes photobleaching by reducing the excitation time for each marker. A high-contrast grey-level image is also produced by summing confocal images of the same site taken at different fluorescence wavelengths. Monte Carlo simulations are performed to obtain the quantitative behaviour of confocal fluorescence imaging in turbid media. Confocal images of the following samples were also obtained: (i) 15 m diameter fluorescent spheres placed 1.16 mm deep beneath an aqueous suspension of 0.0823 m diameter polystyrene latex spheres, and (ii) hindbrain of a whole-mount mouse embryo (age 10 days) that was stained to fluoresce at 515 nm and 580 nm peak wavelengths. Expression of RNA transcripts of a gene within the embryo hindbrain was detected by a fluorescence-based whole-mount in situ hybridization procedure that we recently tested.

Direct physical measure of conformational rearrangement underlying potassium channel gating.

In response to membrane depolarization, voltage-gated ion channels undergo a structural rearrangement that moves charges or dipoles in the membrane electric field and opens the channel-conducting pathway. By combination of site-specific fluorescent labeling of the Shaker potassium channel protein with voltage clamping, this gating conformational change was measured in real time. During channel activation, a stretch of at least seven amino acids of the putative transmembrane segment S4 moved from a buried position into the extracellular environment. This movement correlated with the displacement of the gating charge, providing physical evidence in support of the hypothesis that S4 is the voltage sensor of voltage-gated ion channels.