DsRed, a tetrameric fluorescent protein cloned from the Discosoma genus of coral, has shown promise as a longer-wavelength substitute for green fluorescent protein (GFP) mutants for in vivo protein labeling. Bulk and single-molecule studies of the recombinant protein revealed that the DsRed chromophore shows high stability against photobleaching as compared to GFP mutants. Stark modulation spectra confirm that the electronic structure of the DsRed chromophore is similar to that of GFP. However, the tetrameric nature of DsRed leads to intersubunit energy transfer, as evidenced by the molecule's unusually low fluorescence anisotropy when immobilized (0.23 ( 0.02). This value is approximately consistent with an estimate of the energy transfer rates based on preliminary crystallographic information. The fluorescence emission bleaches at a rate linear in the applied excitation intensity, implying that the cessation of emission during pumping at 532 nm is light-driven and, consistent with the tetrameric structure, several photobleaching "steps" were observed for individual complexes. Because more photons are emitted before bleaching, this study suggests that DsRed may be superior to some GFP-based labeling technologies as long as tetramerization is not an issue in physiological studies. IntroductionSingle-molecule spectroscopy (SMS) continues to play a unique role in the elucidation of the dynamics of an increasingly broad range of complex systems. 14,25 By removing the averaging inherent in ensemble measurements, SMS yields a measure of the distribution of molecular properties which is of importance in systems that display static or time-dependent heterogeneity. Concurrently, developments in fluorescence microscopy have made the observation of single molecules of biological interest possible under a wide range of experimental conditions. 26 This versatility can be exploited to examine the molecular complexity of many biological systems.One critical challenge to the application of SMS techniques to biomolecules, especially in vivo, is the introduction of a fluorophore that acts as a reporter of activity, local environment, or spatial location. While certain important proteins display useful amounts of visible fluorescence arising from native cofactors, 12 most require the attachment of an extrinsic fluorophore to serve as the probe. The genetic fusion of naturally fluorescent proteins to a protein of interest ensures a consistent 1:1 stoichiometry in the cell, without the need for external chemical reactions. While the green fluorescent protein (GFP) and its mutants have proved suitable for many applications, 22,27 the relatively high quantum yield of photobleaching (∼10× that for a rhodamine dye 16 ) leaves room for improvement in applications that require a large number of emitted photons, such as in single-molecule studies. Also, no mutant of GFP has been demonstrated to be a strong emitter at long (>550 nm) wavelengths, where the interference from endogenous fluorophores is substantially less severe.Considering th...

The bacterium Caulobacter crescentus divides asymmetrically as part of its normal life cycle. This asymmetry is regulated in part by the membrane-bound histidine kinase PleC, which localizes to one pole of the cell at specific times in the cell cycle. Here, we track single copies of PleC labeled with enhanced yellow fluorescent protein (EYFP) in the membrane of live Caulobacter cells over a time scale of seconds. In addition to the expected molecules immobilized at one cell pole, we observed molecules moving throughout the cell membrane. By tracking the positions of these molecules for several seconds, we determined a diffusion coefficient (D) of 12 ؎ 2 ؋ 10 ؊3 m 2 ͞s for the mobile copies of PleC not bound at the cell pole. This D value is maintained across all cell cycle stages. We observe a reduced D at poles containing localized PleC-EYFP; otherwise D is independent of the position of the diffusing molecule within the bacterium. We did not detect any directional bias in the motion of the PleC-EYFP molecules, implying that the molecules are not being actively transported.single molecule ͉ diffusion ͉ PleC ͉ enhanced yellow fluorescent protein T he inner membranes of bacterial cells contain proteins required for a wide variety of functions, including energy generation, solute transport, signaling, proteolysis, polar morphogenesis, chemotaxis, and cell division (1-3). The size of the diffusion coefficient (D) of these proteins in the membrane can affect their interactions with each other and with cytoplasmic proteins. For example, in Escherichia coli, the MinCDE system for locating the division plane is thought to require a difference in D between the membrane-associated and the cytoplasmic forms of the MinD and MinE proteins for its proper function (4-6). The D values of several cytoplasmic proteins have been measured in E. coli (7). Measurements of D for membrane proteins in eukaryotic cells, using fluorescence recovery after photobleaching (FRAP) (8), single gold bead tracking (9-11), and single-molecule tracking techniques (12, 13), have yielded values ranging from 5 ϫ 10 Ϫ3 to 500 ϫ 10 Ϫ3 m 2 ͞s. Each Caulobacter cell division produces a pair of distinct daughter cells (Fig. 1): a motile swarmer (SW) cell with a single flagellum located at a specific pole and a stalked (ST) cell possessing an adhesive holdfast at the end of the stalk, allowing it to attach to a surface (14, 15). The transmembrane histidine kinase PleC regulates polar organelle formation, motility, and asymmetric cell division in Caulobacter (16). PleC is a 90-kDa inner membrane protein, with four predicted transmembrane domains as obtained from TMPRED (www.ch.embnet.org͞ software͞TMPREDform.html). Cells with mutant PleC do not form stalks or pili and have paralyzed flagella (17)(18)(19). These mutant cells undergo symmetric cell division, producing two daughter cells of similar size, each possessing a paralyzed flagellum. By using conventional fluorescence microscopy, molecules of PleC were found to be localized to the flagellar pole of SW...

Although the catalytic (C) subunit of cAMP-dependent protein kinase is N-myristylated, it is a soluble protein, and no physiological role has been identified for its myristyl moiety. To determine whether the interaction of the two regulatory (R) subunit isoforms (R I and R II ) with the N-myristylated C subunit affects its ability to target membranes, the effect of N-myristylation and the R I and R II subunit isoforms on C subunit binding to phosphatidylcholine͞ phosphatidylserine liposomes was examined. Only the combination of N-myristylation and R II subunit interaction produced a dramatic increase in the rate of liposomal binding. To assess whether the R II subunit also increased the conformational flexibility of the C subunit N terminus, the effect of N-myristylation and the R I and R II subunits on the rotational freedom of the C subunit N terminus was measured. Specifically, fluorescein maleimide was conjugated to Cys-16 in the N-terminal domain of a K16C mutant of the C subunit, and the time-resolved emission anisotropy was determined. The interaction of the R II subunit, but not the R I subunit, significantly increased the backbone flexibility around the site of mutation and labeling, strongly suggesting that R II subunit binding to the myristylated C subunit induced a unique conformation of the C subunit that is associated with an increase in both the N-terminal flexibility and the exposure of the N-myristate. R II subunit thus appears to serve as an intermolecular switch that disrupts of the link between the N-terminal and core catalytic domains of the C subunit to expose the N-myristate and poise the holoenzyme for interaction with membranes. C yclic AMP-dependent protein kinase (cAPK) exists in nearly all eukaryotic cells and plays a critical role in the regulation of cellular growth, metabolism, and homeostasis by catalyzing the phosphorylation of a variety of proteins (1-3). The holoenzyme configuration of cAPK comprises two catalytic (C) subunits and a cAMP-binding regulatory (R) subunit homodimer. Three mammalian forms of the C subunit have been identified (C ␣ , C ␤1,2,3 , and C ␥ ) (1-3). With the exception of some of the novel C ␤ splice variants, all the mammalian isoforms are myristylated (4, 5). Two pharmacologically and structurally distinct types of the R subunits, R I and R II , are known, along with ␣ and ␤ isoforms of each (1-3). Tissue-specific membrane targeting of each type has been observed (1-3) and appears to be mediated by specific membrane anchoring proteins (A-kinase anchoring proteins) that bind to the dimerization domain of the R subunit but not to the C subunit (6).Because protein N-myristylation is often part of a membrane targeting signal that permanently or reversibly steers proteins to membranes (7), it is perplexing that the C subunit is thought to lack a membrane targeting signal and that membrane binding of the holoenzyme results solely from A-kinase anchoring protein interaction with the nonmyristylated R subunits. The x-ray structure of the mammalian C ␣ subunit su...

Green fluorescent proteins bearing the Y66H mutation exhibit strongly blue-shifted fluorescence excitation and emission spectra. However, these blue fluorescent proteins (BFPs) have lower quantum yields of fluorescence (Phi(f) approximately 0.20), which is believed to stem from the increased conformational freedom of the smaller chromophore. We demonstrate that suppression of chromophore mobility by increasing hydrostatic pressure or by decreasing temperature can enhance the fluorescence quantum yield of these proteins without significantly affecting their absorption properties or the shape of the fluorescence spectra. Analysis of the fluorescence lifetimes in the picosecond and nanosecond regimes reveals that the enhancement of the fluorescence quantum yield is due to the inhibition of fast quenching processes. Temperature-dependent fluorescence measurements reveal two barriers ( approximately 19 and 3 kJ/mol, respectively) for the transition into nonfluorescing states. These steps are probably linked with dissociation of the hydrogen bond between the chromophore and His148 or an intervening water molecule and to the barrier for chromophore twisting in the excited state, respectively. The chromophore's hydrogen-bond equilibrium at room temperature is dominated by entropic effects, while below approximately 200 K the balance is enthalpy-driven.

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A combination of site-directed labeling and time-resolved fluorescence anisotropy was used to further elucidate the structure and underlying dynamic features of the type I regulatory (R I R ) subunit of the cAMP-dependent protein kinase. Specifically, the consequences of cAMP and the catalytic (C)-subunit binding on the backbone flexibility around seven sites of cysteine substitution and fluorescein maleimide labeling (Thr 6 Cys, Leu 66 Cys, Ser 75 Cys, Ser 81 Cys, Ser 99 Cys, Ser 145 Cys, and Ser 373 Cys) in the R I R subunit were assessed. Focusing on the fast rotational correlation time, the results indicate that most of the interdomain segment connecting the dimerization/docking (D/D) and tandem cAMP-binding domains is probably weakly associated with the latter domain. Also, this segment becomes more tightly bound to the C subunit upon holoenzyme formation. The results also suggest that there is a short 'hinge' segment (around Leu 66 Cys) that could allow the structured interdomain/cAMP-binding and D/D domains to pivot about each other. Finally, cAMP binding dramatically reduces the backbone flexibility around only the two sites of cysteine substitution in the cAMP-binding domains, suggesting a selective structural stabilization caused by cAMP and a "tight" coupling of low-nanosecond fluctuations selectively within the tandem cAMP-binding domains.Although it is a truism that conformational flexibility and dynamics play essential roles in protein activity, our knowledge of the flexibility and dynamics of most proteins is limited. For example, most of what is known about the flexibility and dynamics of the signaling protein cAMPdependent protein kinase (cAPK) 1 centers around its monomeric catalytic (C) subunit. Substrate binding induces a narrowing of the C-subunit active-site cleft that is formed by its small and large lobes (1), type-II cAPK-regulatory (R II ) subunit binding mobilizes the N-terminus of the myristylated C subunit (2), and the heat-stable cAPKinhibitor peptide, PKIR, undergoes a disorder-order transition upon binding to the C subunit (3).With regard to the homodimeric R subunit, cAMP raises the concentration for chaotropic denaturation, indicating that cAMP stabilizes the R subunit. This also suggests that cAMP reduces all or part of its conformational flexibility (4). Smallangle scattering results suggest a hinge movement at each end of the R II subunit dimerization/docking (D/D) domain, which links the two monomeric R subunits and forms the binding site for cAPK anchoring proteins (AKAPs) (5). The size and the inherent difficulty of crystallizing full-length isoforms of the R subunit have limited the utility of both NMR spectroscopy and X-ray crystallography for the analysis of its flexibility and dynamics. Some success has been achieved by studying fragments of the R subunit. The solution structure of the R II R -subunit D/D domain has been determined by multidimensional NMR spectroscopy (6). Similarly, a ∆1-91 deletion mutant C-terminal fragment of the cAMP-bound R I R subunit ...

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