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Time-resolved fluorescence spectroscopy David P Millar


Time-resolved fluorescence spectroscopy is used to monitor molecular interactions and motions that occur in the picosecond-nanosecond time range, and is especially useful in the analysis of biomolecular structure and dynamics. Recent advances in the application of time-resolved fluorescence spectroscopy to biological systems have led to a better understanding of the origin of nonexponential fluorescence decay in proteins, the use of tryptophan analogs as unique spectroscopic probes of protein-protein interactions, the detailed characterization of protein-folding processes and intermediates, and the development of new approaches to the study of DNA-protein interactions.

fluorescence spectroscopy to studies of proteins, nucleic acids and protein-nucleic acid complexes. There are two types of measurements that can be performed using time-resolved fluorescence spectroscopic techniques. T h e first is the measurement of the decay of the total fluorescence intensity following pulsed (or modulated) excitation. This is used to determine the fluorescence lifetime of a fluorophore, which reflects the average time that a molecule remains in the singlet excited state. T h e utility of fluorescence lifetime measurements stems from the fact that many dynamic events can deactivate the excited state and hence influence the lifetime; these events include solvent relaxation, fluctuations in macromolecular conformation, rotations of side chains, interactions with neighboring residues, and quenching by exogenous agents. Accordingly, the fluorescence lifetime of a fluorophore in a protein or nucleic acid is highly dependent upon its local environment and can vary from a few picoseconds to tens of nanoseconds. Modern instrumentation for time-resolved fluorescence measurements can readily quantify lifetimes across this entire range. In reality, many biological macromolecules exhibit complex intensity decay patterns characterized by two or more lifetimes. As discussed below, the origin of nonexponential fluorescence decay in proteins is beginning to be understood in structural terms. Complex intensity decays can also be caused by nonradiative energy transfer to an acceptor probe, especially when there is significant dispersion in the donor-acceptor distance. Analysis of such decay profiles can be used to recover the distribution of interprobe distances, which is useful for characterizing molecular flexiblity and conformational disorder. T h e other type of time-resolved fluorescence experiment involves measurement of the polarization anisotropy decay and is used to characterize molecular motions. T h e anisotropy decay monitors reorientation of the emission dipole during the lifetime of the excited state and can report on local fluorophore motion, segmental mobility or rotation of an entire macromolecule. Anisotropy decay measurements can therefore provide hydrodynamic data that describe the rotational diffusion of a protein or nucleic acid. A more exciting application of the technique is its use as a real-time prebe of the rapid (picosecond) internal dynamics of biological macromolecules. Experiments of this type can be used to corroborate the predictions of molecular dynamics simulations [11. T h e studies reviewed below exemplify recent contributions of time-resolved fluorescence spectroscopy to the characterization of the structure, dynamics and interactions

Addresses The Scripps Research Institute, Department of Molecular Biology, MB-19, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA; e-mail: millar@scripps.edu Current Opinion in Structural Biology 1996, 6:637-642

Current Biology Ltd ISSN 0959-440X


Abbreviations DHFR dihydrofolatereductase FRET fluorescenceresonance energy transfer TBP TATA-box binding protein TCSPC time-correlatedsingle photon counting

Introduction

Time-resolved fluorescence spectroscopy is a very promising technique for investigating the structure and dynamics of biological macromolecules, especially proteins and nucleic acids. T h e technique monitors dynamic events occurring within the environment of a fluorophore and can also be used to characterize segmental and overall motions. Recent advances in instrumentation have extended time-resolved fluorescence measurements into the picosecond region. In addition, the increasing use of molecular biological techniques to produce proteins for spectroscopic characterization has also expanded the scope of time-resolved fluorescence studies. Thus, site-directed mutagenesis techniques can be used to limit the number of intrinsic fluorophores in a protein of interest, thereby simplifying the interpretation of fluorescence data, or to introduce fluorophores into specific segments of a protein. Recently it has become possible to replace intrinsic tryptophan residues in proteins with analogs possessing unique spectroscopic properties. Similarly, advances in oligonucleotide synthesis and site-specific labeling methodologies have led to corresponding improvements in the ability to characterize structural and dynamic features of nucleic acids using time-resolved fluorescence techniques. T h e purpose of this article is to review recent progress made in the application of time-resolved

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of proteins and nucleic acids. It has not been possible to review all the literature in this field; therefore, the studies reviewed here have been chosen to highlight new methodologies and to illustrate the information that is forthcoming from time-resolved fluorescence analyses of protein and nucleic acid systems. Regrettably, the applications of time-resolved fluorescence spectroscopy to studies of membranes and photosynthetic systems, although important, are not reviewed here. Likewise, the use of time-resolved fluorescence techniques in imaging applications is not discussed here.

Instrumentation and data analysis


T h e ability to record fluorescence-intensity decays with picosecond time resolution, using either time-correlated single photon counting (TCSPC) or multifrequency phase/ modulation methods, has become routine in the past few years. This has principally been achieved through technical improvements in both light sources and detection systems. Synchronously pumped dye lasers capable of generating high repetition-rate light pulses with a duration of just a few picoseconds have been in use in many laboratories for a number of years. These lasers, when used in conjunction with nonlinear optical systems for frequency doubling, provide a moderate degree of tunability within selected spectral regions from the ultraviolet to the near infrared. Recently, high repetition-rate mode-locked (pulsed) lasers utilizing a titanium doped sapphire crystal as the active medium have become commercially available. Apart from the convenience of a solid-state gain medium, these lasers generate shorter pulses than dye lasers and also provide a much wider tuning range (when used with suitable frequency doubling and tripling systems). In addition, the high intensity output from Ti:sapphire lasers permits direct excitation of biological chromophores by means of two- and three-photon absorption [2,3]. T h e high excitation intensities produced by these lasers can also lead to other nonlinear optical effects, such as stimulated emission. Stimulated emission depletes the excited-state population and can lead to an apparent shortening of the fluorescence lifetime. When properly accounted for, however, such effects can be exploited in the design of new types of time-resolved fluorescence experiments [4]. Apart from the use of picosecond and subpicosecond laser pulses for excitation, the time resolution of fluorescence decay measurements has also been improved by using microchannel plate photomultipliers to detect the emitted fluorescence. These devices provide extremely fast response to the fluorescence and have high quantum efficiencies throughout the ultraviolet and visible regions of the spectrum. Detailed descriptions of modern instrumentation for time-resolved fluorescence measurements are presented elsewhere [5%6,7]. Analytical methods play an important role in the interpretation of time-resolved fluorescence data, especially in studies of biological macromolecules. The total fluorescence intensity of a simple fluorophore in a homogeneous

environment decays as a monoexponential function of time. T h e intensity decays observed for most biological macromolecules are more complex, however, and different mathematical expressions must be employed for quantitative analysis. A number of different analytical models are used to treat these complex decays and the reader is referred to earlier reviews and publications for details of the various methods [5",8-10]. Conventionally, the fluorescence decay is resolved in terms of a small number of discrete exponential components. An alternative model, based on a continuous distribution of lifetimes, has also been invoked to account for the heterogeneous fluorescence decay of biological macromolecules [11]. Indeed, it is usually possible to fit an observed decay curve using either model. In view of the different physical interpretations implied by discrete lifetime components or a continuous distribution of lifetimes, it is clearly important to have objective criteria that can be used to decide whether a lifetime distribution is actually present. In a recent study, Vix and Lami [12 ] suggest that such criteria can be established by recording a sample of 20 separate decays, each of which is fitted with a multiexponential function. T h e presence of a lifetime distribution is reflected in the pattern of Z2 values obtained for each fit for the 20 decays. Not surprisingly, the results of this study show that it is only possible to discriminate between discrete and distributed lifetime models if the lifetime distribution is sufficiently broad. Significantly, the authors point out that they find no evidence for lifetime distributions among any of several single-tryptophan proteins that have been examined in their laborator3; indicating that the distributions are either too narrow to be detected, or that the fluorescence intensity decays of the various proteins are actually described by discrete lifetime components. Numerical methods used for data analysis can also determine the effective time resolution of a fluorescence decay experiment. Of particular importance are schemes for discretization of the convolution integrals required for the analysis of T C S P C data. Bazjer et al. [13"] have considered the merits of different discretization schemes and have shown that more accurate algorithms can result in better recovery" of picosecond decay times. Interestingly, their results show that a reduction in the width of the instrument response function does not necessarily lead to better time resolution, unless a suitably accurate discretization formula is used.

Time-resolved fluorescence studies of proteins Intrinsic tryptophan fluorescence


The intrinsic fluorescence of proteins is generally dominated by emissions from tryptophan residues. The emission properties and excited-state decay of tryptophan are quite sensitive to the surrounding environment, making it a potentially useful probe of protein structure and dynamics. Moreover, many proteins contain just a

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single tryptophan residue, which considerably simplifies the interpretation of the fluorescence data. In other cases, site-directed mutagenesis techniques have been used to generate single tryptophan mutants in which the tryptophan is incorporated into a specific region of a protein, as noted above. In almost all cases, proteins containing a single tryptophan residue exhibit muhiexponential fluorescence decay kinetics. The origin of this heterogeneous decay has been the subject of intensive study. Recently, significant progress has been made in the interpretation of tryptophan fluorescence in terms of protein structure. T h e most popular current hypothesis is that the heterogeneous intensity decay of tryptophan in proteins is due to the existence of different rotameric conformations of the tryptophan side chain. T h e basic premise underlying this model is that tryptophan can populate a number of low energy conformations, as a result of rotation about the Cot-C[3 and/or the C ~ - C y bonds, with each conformer exhibiting a distinct fluorescence lifetime. T h e rotamer model was initially proposed to explain the biexponential fluorescence decay of pure tryptophan in aqueous solution [14,15] and was subsequently shown to be applicable to small tryptophan-containing peptides [16] on the basis of studies which demonstrate that the pre-exponential factors in the fluorescence decay correspond to the individual rotamer populations determined by N M R spectroscopy [17]. Recent studies by Dahms eta/. [18 ] on the time-resolved fluorescence of erabutoxin-b crystals, have now extended the rotamer model to tUptophan residues in proteins. T h e s e investigators showed that the intensity decay of the single tryptophan of erabutoxin-b is characterized by three decay times and that the relative contributions of each component to the overall fluorescence decay vary as the crystal is rotated with respect to the polarization direction of the excitation beam. This angular dependence indicates that each species is oriented differently with respect to the crystal axes, just as expected for tryptophan rotamers within the ordered environment of the protein crystal. T h e results of this study are especially interesting, given that the tryptophan residue is modeled as a single conformer in the crystal structure of erabutoxin b. Thus, time-resolved fluorescence studies of proteins may reveal minor populations of alternate tryptophan side-chain rotamers that arc not resolved by X-ray cry.stallographic techniques. Why do the different tryptophan rotamers exhibit distinct decay times? This presumably reflects interactions between the excited indole moiety and the surrounding amino acid residues and peptide groups that arc unique to each conformational state. Thus, in principle, the time-resolved fluorescence of an intrinsic tryptophan residue contains information on protein structure. It is not yet possible to interpret fluorescence decay times in terms of specific structures, however, because of an incomplete understanding of the quenching mechanisms

involved. Nevertheless, this remains a long term goal of research in this field. In the meantime, some structural details can be gleaned from the pre-exponential factors in the intensity decay, as these represent the relative populations of the different rotamers. Indeed, Szabo and colleagues [19,20 ] have proposed that the pre-exponential factors are related to the secondary structure in the region of the tryptophan residue, via the effect of the main chain on the rotamer populations. Support for this hypothesis comes from studies in which the rotamer populations deduced from the fluorescence decay of a single tryptophan residue in a series of model peptides were correlated with the amount of o~-helical or [3-sheet secondary structure [19,20]. These studies provide the basis for using tryptophan as a site-specific structural probe, and significantly improve the ability to interpret tryptophan fluorescence in terms of protein structure.
Tryptophan analogs

While tryptophan is useful as a probe of protein structure and dynamics, its utility in studies of protein-protein interactions is hampered by difficulties in distinguishing the fluorescence signals from a mixture of two or more proteins. To circumvent this problem, a number of groups have used 5-hydroxytryptophan (5-OH-Trp) [21,22] and 7-azatryptophan (7-aza-Trp) [23,24] as alternative intrinsic fluorophores in proteins. T h e utility of these analogs stems from their distinctive spectral features, which permit selective excitation and observation of their fluorescence in the presence of other proteins containing tUptophan. With the ability to incorporate these analogs in a given peptidc or protein of interest by using biosynthetic or chemical methods, exciting new opportunities arise for probing local structural and dynamic features in a variety of macromolect, lar complexes. Potential applications include the study of peptide binding to proteins, protein-protein and hormone-receptor interactions, and nucleic acid-protein complexation. Petrich, Schwabacher and colleagues [25 ] have recently demonstrated the potential of using 7-aza-Trp as a probe of small-molecule-protein interactions. This group has also reported that the Nl-methylated derivative of 7-aza-Trp affords even greater spectroscopic distinguishability with respect to tryptophan [26]. In another recent study, 5-OH-Trp was incorporated into insulin, in place of a tyrosine residue, using chemical synthetic procedures [27]. T h e resultii,g insulin analog was shown to be biologically active and the fluorescence data indicated that the 5-OH-Trp group is located on the surface of the insulin molecule. This spectrally-enhanced insulin analog should be useful for studies of hormone-receptor interactions. Shen et al. [28"] have utilized tryptophan analogs in a study of the protein-protein interactions involved in transcription initiation and activation. These authors introduced 7-aza-Trp or 5-OH-Trp into the activation domain of the herpes virus VP16 protein, and used the fluorescence of these analogs to monitor conformational changes of the activation domain upon interaction with

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basal transcription factors. Their results illustrate the potential of studies of this type to yield molecular details of complex multiprotein assemblies under solution conditions.

Protein dynamics
As noted above, the fluorescence anisotropy decay technique is of great importance as a real-time method for the detailed investigation of picosecond internal motions of proteins. The state of the art in this area is illustrated by two recent reports. Broos etal. [29 ] have used fluorescence anisotropy decay to examine the internal dynamics of enzymes suspended in organic solvents. Enzymes exhibit interesting properties in anhydrous organic media, including enhanced thermal stability and altered substrate specificity and enantioselectivity [30]. The results of Broos et a/. show that the enzymes ct chymotrypsin and subtilisin carlsberg have markedly reduced flexibility under these conditions, an effect that the authors correlate with the diminished activity and altered enantioselectivity of the enzymes. Silva and Prendergast [31 ] have used similar methods to probe the internal dynamics of the single tryptophan residue of FKBP12, a protein that is important in the immunosuppressant action of FK506 and rapamycin. The results provide evidence for the occurence of an extremely rapid motion of the tryptophan side chain in the uncomplexed FKBP12 protein, whereas the side chain appears to be immobilized in the complexes of FKBP12 with FK506 or rapamycin. On the basis of these findings, the authors suggest that the dynamics of the tryptophan side chain play a role in drug binding to FKBP12. In addition, the authors correlated the experimental data with simulations of the tryptophan side chain dynamics generated by the minimum perturbation mapping technique. These simulations also showed that binding of the immunosuppressants to FKBP12 restricts the internal motion of the tryptophan side chain.

Time-resolved fluorescence methods also provide new ways of probing the fluctuating structure of unfolded proteins and of characterizing molten globule states. Such states exist as a conformational ensemble and are difficult to characterize by conventional structural methods. T h e distribution of distances between two points along the polypeptide chain, measured by time-resolved fluorescence resonance energy transfer ( F R E T ) between donor and acceptor probes, can provide direct information on the ensemble of different conformations present under various conditions. T h e ability of time-resolved F R E T methods to provide unique information on the dynamic conformations of unfolded and partially folded proteins is amply demonstrated in recent studies by Haas and coworkers on reduced bovine trypsin inhibitor [33] and ribonuclease A, in native and denatured states [34]. Similar methods were used by Rischel et al. [35] to characterize the structure of the molten globule state of apomyoglobin and by Wu and Brand [36] to probe the conformational distribution of a staphylococcal nuclease mutant under various conditions.

Time-resolved fluorescence studies of DNA and DNA-protein complexes Fluorescence spectroscopy is becoming increasingly important as a technique for studying nucleic acids [37]. Since the intrinsic fluorescence of DNA is generally very weak, investigators have, in the past, used intercalating dyes as extrinsic labels. Recently, new types of DNA ligands have been used as fluorescent probes. In addition, it is now possible to synthesize oligonucleotides containing fluorescent nucleotides or other labels. Synthetic methods provide a convenient means of controlling the position of the probe within the DNA sequence. DNA dynamics Fluorescence anisotropy decay of intercalating dyes has been used extensively in investigations of the torsional dynamics of DNA. Recently, two groups have examined the utility of other classes of DNA-binding ligands in fluorescence anisotropy decay studies. Barcellona and Gratton [38 ] have used the minor groove binder, DAPI, as a probe of DNA torsional motions. Lakowicz et al. [39] have reported time-dependent anisotropy data for ruthenium metal-ligand complexes bound to DNA. These metal complexes have long emission lifetimes and are potentially useful as probes of the slow bending motions of DNA.
While extrinsic probes have been useful in revealing the overall dynamics of DNA, they do not report directly on the rapid motions of the DNA bases themselves. Georghiou et al. [40] have reported anisotropy decay measurements of double-stranded oligonucleotides and polynucleotides, using the intrinsic fluorescence of thymine bases excited at 293 nm. Their results provide evidence for large-amplitude base motions in DNA with subnanosecond correlation times. The conclusions are

Protein folding Fluorescence spectroscopy is an important tool in studies of protein folding. The recent introduction of fluorescence decay techniques promises to take fluorescence-based studies of protein folding to a new high. Jones et al. [32 ] have recently carried out fluorescence decay measurements during the kinetic refolding of Esherichia. coli dihydrofolate reductase (DHFR). By combining stoppedflow mixing methods with very rapid acquisition of TCSPC data, the authors were able to measure the emission lifetimes and anisotropy decay times of the intrinsic tryptophans of D H F R and of the extrinsic dye 1-anilinonaphthalene-8-sulfonate (ANS) throughout the course of the folding reaction. The results yielded much more detailed information on the nature of the folding intermediates than that obtained in earlier studies based on real-time measurements of the total fluorescence intensity.

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consistent with earlier anisotropy decay studies utilizing the fluorescent base analog, 2-aminopurine [41,42]. Time-resolved fluorescence techniques are also extremely useful for characterizing the dynamics of more complex nucleic acid structures. Yang and I [43] have characterized the overall structure and conformational flexibility of three-way DNA junctions, with and without unpaired bases at the branch point, using time-resolved F R E T between dyes attached pairwise to the junction arms. Our results revealed that base bulges have a significant impact on the static and dynamic structure of the three-way junction.

Acknowledgements
This work was supported by grants from the National Institutes of Health (GM44060) and National Science Foundation (MCB-9019250).

References and recommended reading


Papers of particular interest, published within the annual period of review, have been highlighted as: * 1. of special interest of outstanding interest Axelsen PH, Gratton E, Prendergast FG: Experimentally verifying molecular dynamics simulations through fluorescence anisotropy measurements. Biochemistry 1991,30:1173-1179. Kierdaszuk B, Gryczynski I, Modrakwojcik A, Bzowska A, Shugar D, Lakowicz JR : Fluorescence of tyrosine and tryptophan in proteins using one- and two-photon excitation. Photochem Photobiol 1995, 61:319-324. Gryczynski I, Malak H, Lakowicz JR: Three-photon excitation of a tryptophan derivative using a fs-Ti-sapphire laser. Biospectroscopy 1996, 2:9-15. Kusba J, Bogdanov V, Gryczynski I, Lakowicz JR: Theory of light quenching: effects on fluorescence polarization, intensify, and anisotropy decays. Biophys J 1994, 67:2024-2040. Holzwarth AR: Time-resolved fluorescence spectroscopy.

2.

DNA-protein interactions
A promising new development in the biological application of time-resolved fluorescence spectroscopy is in the study of DNA-protein interactions. Complexation of DNAbinding proteins with DNA may be detected through changes in the protein's intrinsic tryptophan emission, or through changes in the fluorescence lifetime and rotational properties of probes attached to the DNA. Both approaches are illustrated by recent studies. Perez-Howard et al. [44 ] have analyzed the fluorescence anisotropy decay of the single tryptophan residue of the TATA-box binding protein (TBP) from yeast. The data revealed that TBP exists in solution as a muhimer, which, upon binding to DNA, dissociates into a monomeric complex. Bailey et al. [45 ] have reported studies in which a fluorescein probe was attached to DNA at various positions within and adjacent to the recognition sequence of the TyrR regulatory protein. Measurements of the fluorescence lifetime and anisotropy decay of the probe at each position, in both the absence and presence of TyrR, were used to define the points of contact between the DNA and protein. Indeed, the strong dependence of the fluorescence parameters on the probe position observed in this study, and also in a similar study which used a different DNA-binding protein [46], suggests that time-resolved fluorescence measurements may be useful for elucidating structural features of a variety of DNA-protein complexes.

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A recent review of time-resolved fluorescence spectroscopy as applied to studies of biochemical systems, with a particular emphasis on instrumentation and measurement techniques. 6. Small EW: Laser sources and microchannel plate detectors for pulse fluorometry. In Topics in F/uorescence Spectroscopy, vol 1. Edited by Lakowicz JR. New York: Plenum; 1991:97-182. Lakowicz JR, Gryczynski I: Frequency-domain fluorescence spectroscopy. In Topics in F/uorescence Spectroscopy, vol 1. Edited by Lakowicz JR. New York: Plenum; 1991:293-335. Prendergast FG: Time-resolved fluorescence techniques: methods and applications in biology. Curr Opin Struct Bio/ 1991, 1:1054-1059. Livesy AK, Brochon, JC: Analyzing the distribution of decay constants in pulse fluorometry using the maximum entropy method. Biophys J 1987, 52:693-706. Bazjer ~', Sharp JC, Sedarous SS, Prendergast FG: Pade-Laplace method for the analysis of time-resolved fluorescence decay curves. Eur .J Biophys 1980, 18:101-115. Alcala JR, Gratton E, Prendergast FG: Fluorescence lifetime distributions in proteins. Biophys J 1987, 51:597-604.

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Vix A, Lami H: Protein fluorescence decay: discrete components or distribution of lifetimes? Really no way out of the dilemma? Biophys J 1995, 68:1145-1151. Specific criteria are given for distinguishing between discrete and distributed lifetime models in the analysis of fluorescence decay data. Bazjer ~', Zelic A, Prendergast FG: Analytical approach to the recovery of short fluorescence lifetimes from fluorescence decay curves. Biophys J 1995, 69:1148-1161. New methods are presented for the numerical calculation of convolution integrals in TCSPC, leading to improved recovery of short fluorescence lifetimes. 14. Szabo AG, Rayner DM: Fluorescence decay of tryptophan conformers in aqueous solution. J Am Chem Soc 1980, 102:554-563. Petrich JW, Chang MC, McDonald DB, Fleming GR: On the origin of nonexponential fluorescence decay in tryptophan and its derivatives. J Am Chem Soc 1983, 105:3824-3832. Chen RF, Knutson JR, Ziffer H, Porter D: Fluorescence of tryptophan dipeptides: correlations with the rotamer model. Biochemistry 1991, 30:5184-5195. Ross JBA, Wyssbrod HR, Porter IRA, Schwartz GP, Michaels CA, Laws WR: Correlation of tryptophan fluorescence intensity decay parameters with 1H NMR-determined rotamer conformations: [tryptophan2]oxytocin. Biochemistry 1992, 31:1585-1594. Dahms TES, Willis KJ, Szabo AG: Conformational heterogeneity of tryptophan in a protein crystal. J Am Chem Soc 1995, 117:2391-2326. 13.

Conclusions
T h e development of instrumentation for picosecond time-resolved measurements, coupled with improved techniques for the manipulation of proteins and nucleic acids, has resulted in significant advances in the application of fluorescence spectroscopic techniques to biologically important systems. The studies reviewed here illustrate the increasing ability to interpret time-resolved fluorescence parameters in terms of biomolecular structure and dynamics. I anticipate that the techniques described in this review will be used more and more to characterize structural and dynamic features of complex biological assemblies, including protein-protein and protein-nucleic acid complexes.

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The existence of three ground-state tryptophan rotamers in crystalline erabutoxin b is established by time-resolved fluorescence measurements obtained as a function of crystal orientation. 19. Willis K.I, Neugebauer W, Sikorska M, Szabo AG: Probing czhelical secondary structure at a specific site in model peptides via restriction of tryptophan side-chain retainer conformation. Biophys J 1994, 66:1623-1630.

An impressive study demonstrating that time-resolved fluorescence data can be collected during the course of a rapid protein-folding reaction. 33. Ittah V, Haas E: Nonlocal interactions stabilize long range loops in the initial folding intermediates of reduced bovine pancreatic trypsin inhibitor. Biochemistry 1995, 34:4493-4506.

Dahms TES, Szabo AG: Probing local secondary structure by fluorescence: time-resolved and circular dichroism studies of highly purified neurotoxins. Biophys J 1995, 69:569-576, This paper demonstrates that the relative rotamer proportions, determined from the time-resolved fluorescence of an intrinsic tryptophan residue, is indicative of local ~-sheet secondary structure. 21. Hogue CW, Rasquinha I, Szabo AG, MacManus JP: A new intrinsic fluorescent probe for proteins. Biosynthetic incorporation of 5-hydroxytryptophan into oncomodulin. FEBS Lett 1892, 310:269-272. Ross JBA, Senear DF, Waxman E, Kombo BB, Rusinova E, Huang YT, Laws WR, Hasselbacher CA: Spectral enhancement of proteins: biological incorporation and fluorescence characterization of 5-hydroxytryptophan in bacteriophage ;L cl represser. Proc Nat/Acad Sci USA 1992, 89:12023-12027. Negrerie MJ, Gai F, Petrich JW: Photophysics of a novel optical probe: 7-azaindole. J Phys Chem 1991, 95:8663-8670. Hogue CWV, Szabo AG: Characterization of aminoacyladenylates in B. subtilis tryptophanyl-tRNA synthetase, by the fluorescence of tryptophan analogs 5-hydroxytryptophan and 7-azatryptophan. Biophys Chem 1993, 48:159-169.

20.

Buckler DR, Haas E, Scheraga HA: Analysis of the structure of ribonuclease A in native and partially denatured states by time-resolved nonradiative dynamic excitation energy transfer between site-specific extrinsic probes. Biochemistry 1995, 34:15965-15978. An impressive combination of protein chemistry, time-resolved fluorescence and sophisticated data analysis is used to characterize the conformation and dynamic flexibility of ribonuclease A under native and denaturing conditions. 35. Rischel C, Thyberg P, Rigler R, Poulsen FM: Time-resolved fluorescence studies of the molten globule state of apomyoglobin. J Mo/ Bio/1996, 257:877-885. Wu P, Brand L: Conformational flexibility in a staphylococcal nuclease mutant K45C from time-resolved resonance energy transfer measurements. Biochemistry 1994, 33:10457-10462.

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37. Millar DP: Fluorescence studies of DNA and RNA structure and dynamics. Curr Opin Struct Biol 1996, 6:322-326. Applications of steady-state and time-resolved fluorescence techniques used to study DNA and RNA are reviewed, with an emphasis on studies that provide information on local and global aspects of nucleic acid structure. 38. Barcellona ML, Gratton E: Torsional dynamics and orientation of DNA-DAPI complexes. Biochemistry 1996, 35:321-333. A combined analysis of DNA torsional dynamics and DAPI-DAPI energy transfer is used to delimit the torsion elastic constant recovered from the anisotropy decay of DAPI bound to DNA. 39. Lakowicz JR, Malak H, Gryczynski I, Castellano FN, Meyer G J: DNA dynamics observed with long lifetime metal-ligand complexes. Biospectroscopy 1995, 1:163-168.

Rich RL, Gai F, Lane JW, Petrich JW, Schwabacher AW: Using 7-azatryptophan to probe small molecule-protein interactions on the picosecond time scale: the complex of avidin and biotinylated 7-azatryptophan. J Am Chem Soc 1995, 117:733-739. Reports a time-resolved fluorescence study of bietinylated 7-aza-Trp bound to avidin which demonstrates the optical selectivity of 7-aza-Trp in the presence of several tryptephan residues. 26. Rich RL, Smirnov AV, Schwabacher AW, Petrich JW: Synthesis and photophysics of the optical probe N 1-methyl-7azatryptophan. J Am Chem Soc 1995, 117:11850-11853. Laws WR, Schwartz GP, Rusinova E, Burke GT, Chu Y-C, Katsoyannis PG, Ross JBA: 5-Hydroxytryptophan: an absorption and fluorescence probe which is a conservative replacement for [A14 tyrosine] in insulin. J Protein Chem 1995, 14:225-231.

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Georghiou S, Bradrick TD, Philippetis A, Beechem JM: Largeamplitude picosecond anisotropy decay of the intrinsic fluorescence of double-stranded DNA. Biophys J 1996, 70:1909-1922. Time-resolved anisotropy measurements of the intrinsic fluorescence of thymine residues in synthetic polynucleotides provide evidence for picosecond base motions in DNA. 41. Nordlund TM, Andersson S, Nilsson L, Rigler R, Graslund A, McLaughlin LW: Structure and dynamics of a fluorescent DNA oligomer containing the EcoRI recognition sequence: fluorescence, molecular dynamics, and NMR studies. Biochemistry 1989, 28:9095-91 03. Guest CR, Hochstrasser RA, Sowers LC, Millar DP: Dynamics of mismatched base pairs in DNA. Biochemistry 1991, 30:3271-3279.

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Shen F, Triezenberg S J, Hensley P, Porter D, Knutson JR: Transcriptional activation domain of the herpesvirus protein VP16 becomes conformationally constrained upon interaction with basal transcription factors. J Bio/Chem 1996, 271:4827-4837. An elegant example of the use of tryptophan analogs in studies of protein-protein interactions. The results provide evidence for conformational changes in the transcriptional activator VP16 due to interactions with the basal transcription factors TBP and TFIIB. 29. Breos J, Visser AJWG, Engbersen JF_J, Verboom W, Van Hook A, Reinhoudt DN: Flexibility of enzymes suspended in organic solvents probed by time-resolved fluorescence anisotropy. Evidence that enzyme activity and enantioselectivity are directly related to enzyme flexibility. J Am Chem Soc 1995, 117:12657-12663. Provides direct evidence for restricted molecular flexibility of enzymes suspended in organic solvents and suggests a relationship between enantio-selectivity and enzyme flexibility. 30. 31. Klibanov AM: Enzymatic catalysis in anhydrous organic solvents. Trends Biochem Sci 1989, 14:141-144.

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Yang M, Millar DP: Conformational flexibility of three-way DNA junctions containing unpaired nucleotides. Biochemistry 1996, 35:7959-7967. Measurements of time-resolved FRET are combined with donor-accepter distance distribution analysis to characterize the impact of bulged bases on the overall geometry and flexibility of three-way DNA junctions. Perez-Howard GM, Well PA, Beechem JM: Yeast TATA binding protein interaction with DNA: fluorescence determination of oligomeric state, equilibrium binding, on-rate, and dissociation kinetics. Biochemistry 1995, 34:8005-8017. A comprehensive study of TBP-DNA interactions based on measurements of fluorescence signals derived from both intrinsic tryptophan residues and extrinsic probes attached to DNA. Bailey M, Hagmar P, Millar DP, Davidson BE, Tong G, Haralambidis J, Sawyer WH: Interaction between the Escherichia coil regulatory protein TyrR and DNA: a fluorescence footprinting study. Biochemistry 1995, 34:15802-15812. This paper demonstrates that the time-resolved fluorescence properties of a fluorescein probe attached to DNA can be used to distinguish between the different DNA footprints associated with the dimeric and hexameric forms of the TyrR represser protein. 46. Guest CR, Hochstrasser RA, Dupuy C, Allen D J, Benkovic S J, Millar DP: Interaction of DNA with the Klenow fragment of DNA polymerase I studied by time-resolved fluorescence spectroscopy. Biochemistry 1991, 30:8759-8770. 45. 44.

43.

Silva ND, Prendergast FG: Tryptophan dynamics of the FK506 binding protein: time-resolved fluorescence and simulations. Biophys J 1996, 70:1122-1137. A very thorough study of the internal dynamics of the single tryptophan residue of FKBP12, both free and bound to immunosuppressants, which combines time-resolved anisetropy measurements with theoretical simulations. 32. Jones BE, Beechem JM, Matthews CR: Local and global dynamics during the folding of Escherichia coil dihydrofolate reductase by time-resolved fluorescence spectroscopy. Biochemistry 1995, 34:1868-1877.

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