Single-molecule techniques offer a unique tool for studying the dynamical behavior of individual molecules and provide the possibility to construct distributions from individual events rather than from a signal stemming from an ensemble of molecules. In biological systems, known for their complexity, these techniques make it possible to gain insights into the detailed spectrum of molecular conformational changes and activities. Here, we report on the direct observation of a single lipase-catalyzed reaction for extended periods of time (hours), by using confocal fluorescence microscopy. When adding a profluorescent substrate, the monitored enzymatic activity appears as a trajectory of ''on-state'' and ''off-state'' events. The waiting time probability density function of the off state and the state-correlation function fit stretched exponentials, independent of the substrate concentration in a certain range. The data analysis unravels oscillations in the logarithmic derivative of the off-state waiting time probability density function and correlations between off-state events. These findings imply that the fluctuating enzyme model, which involves a spectrum of enzymatic conformations that interconvert on the time scale of the catalytic activity, best describes the observed enzymatic activity. Based on this model, values for the coupling and reaction rates are extracted.single enzyme activity ͉ two-state trajectories D ynamics of chemical reactions are conventionally investigated by ensemble measurements. Recent advances in single-molecule spectroscopy have enabled the real-time study of biophysical processes (1-10) and conformational changes (11, 12) of single biomolecules. These studies have demonstrated that new information about such processes can be extracted from single-molecule measurements. In particular, deviations from the standard Michaelis-Menten behavior (13,14), which is expected for bulk enzymatic activity, have been observed (6 -8, 12).Motivated by these findings, we examined the enzymatic activity of individual molecules of the 33-kDa lipase B from Candida antarctica molecules (15, 16) by using confocal fluorescence microscopy. This lipase catalyzes the hydrolysis of esters in aqueous solution following the same reaction mechanism as that of a serine protease (17). To study the catalysis by single lipase, we used a fluorogenic substrate, namely the nonf luorescent ester 2Ј,7Ј-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester, which upon hydrolysis forms a highly fluorescent carboxylic acid product (18,19). This method enabled us to probe the enzymatic activity by monitoring the fluorescence emission from single enzymes. The fluorescence emission displayed blinking of ''on'' and ''off'' events depending on the presence (or absence) of the fluorescent product in the confocal focus (20). By using this approach, we have been able to obtain long trajectories (for time periods of hours) suitable for reliable statistical analysis while varying the concentration of the substrate, thus ...

Insight into the dynamic behavior of chemical processes is typically derived from ensemble measurements. Direct experimental information about the dynamics at the singlemolecular level, however, is sparse and has until recently been primarily deduced from molecular-dynamics simulations. Current advances in single-molecule spectroscopy have paved the way for exploring the behavior of individual molecules in the course of a chemical reaction. Thus far it has proven possible to monitor in real time the dynamic behavior of single-biomolecular processes and observe the enzymatic turnovers of a few motor proteins, [1][2][3][4][5] an oxidase, [6] horseradish peroxidase, [7] and a nuclease. [8] More recently, structural fluctuations of a single flavin reductase [9] and of T4 lysozyme during the course of a reaction [10] have been detected. These few examples clearly demonstrate the tremendous potential of studying an enzymatic process at the single-molecular level.Herein we report the direct observation of the real-time catalysis and substrate kinetics of a single-enzyme-catalyzed

Energy transfer in antenna systems, ordered arrays of chromophores, is one of the key steps in the photosynthetic process. The photophysical processes taking place in such multichromophoric systems, even at the single molecule level, are complicated and not yet fully understood. Instead of directly studying individual antenna systems, we have chosen to focus first on systems for which the amount of chromophores and the interactions among the chromophores can be varied in a systematic way. Dendrimers with a controlled number of chromophores at the rim fulfill those requirements perfectly. A detailed photophysical study of a second-generation dendrimer, containing eight peryleneimide chromophores at the rim, was performed 'J. Am. Chem. Soc., 122 (2000) 9278'. One of the most intriguing findings was the presence of collective on/off jumps in the fluorescence intensity traces of the dendrimers. This phenomenon can be explained by assuming a simultaneous presence of both a radiative trap (energetically lowest chromophoric site) and a non-radiative trap (triplet state of one chromophore) within one individual dendrimer. It was shown that an analogue scheme could explain the collective on/off jumps in the fluorescence intensity traces of the photosynthetic pigment B-phycoerythrin (B-PE) (Porphyridium cruentum). The different values of the triplet lifetime that could be recovered for a fluorescence intensity trace of B-PE were correlated with different intensity levels in the trace, suggesting different chromophores acting as a trap as function of time.

Proteins from the family of the green fluorescent protein (GFP) are presently extensively used in molecular and cellular biology. Recent studies suggest that isomerization of the chromophore occurs upon excitation and is involved in nonradiative deactivation. Using Raman spectroscopy, we report on photoinduced cis-trans isomerization in the red fluorescent protein eqFP611 from the sea anemone Entacmaea quadricolor. The crystal structure of eqFP611 shows that the chemical structure of the chromophore, p-hydroxybenzylidene-imidazolinone with an extended -conjugated system, is nearly identical to the chromophore of other red fluorescent proteins such as DsRed and HcRed. However, the chromophore of eqFP611 has a trans configuration whereas the chromophore of DsRed has a cis configuration. Upon irradiation with 532-nm light, the absorption of eqFP611 peaking at 559 nm diminished, and concomitantly a drastic decrease in the quantum yield of fluorescence as well as more complex decay kinetics was observed. Upon irradiation, changes in the Raman spectrum of eqFP611 were observed, and the relative intensities and peak positions of the irradiated eqFP611 showed striking similarity with the peaks in the Raman spectrum of DsRed. These observations are tentatively interpreted as trans-to-cis isomerization of the chromophore taking place upon irradiation together with the opening of new, nonradiative pathways.

We report on single-molecule fluorescence measurements performed on the phycobiliprotein allophycocyanin (APC). Our data support the presence of a unidirectional Förster-type energy transfer process involving spectrally different chromophores, alpha84 (donor) and beta84 (acceptor), as well as of energy hopping amongst beta84 chromophores. Single-molecule fluorescence spectra recorded from individual immobilized APC proteins indicate the presence of a red-emitting chromophore with emission peaking at 660 nm, which we connect with beta84, and a species with the emission peak blue shifted at 630 nm, which we attribute to alpha84. Polarization data from single APC trimers point to the presence of three consecutive red emitters, suggesting energy hopping amongst beta84 chromophores. Based on the single-molecule fluorescence spectra and assuming that emission at the ensemble level in solution comes mainly from the acceptor chromophore, we were able to resolve the individual absorption and emission spectra of the alpha84 and beta84 chromophores in APC.