We have employed a rapid fluorescence-based screen to assess the polyspecificity of several aaRSs against an array of unnatural amino acids. We discovered that a p-cyanophenylalanine specific aminoacyl-tRNA synthetase (pCNF-RS) has high substrate permissivity for unnatural amino acids, while maintaining its ability to discriminate against the canonical twenty amino acids. This orthogonal pCNF-RS, together with its cognate amber nonsense suppressor tRNA is able to selectively incorporate 18 unnatural amino acids into proteins, including trifluoroketone, alkynyl, and hydrazino substituted amino acids. In an attempt to better understand this polyspecificity, the x-ray crystal structure of the aaRS/p-cyanophenylalanine complex was determined. A comparison of this structure with those of other mutant aaRSs showed that both binding site size and other more subtle features control substrate polyspecificitiy.
Green fluorescent protein (GFP) from jellyfish Aequorea victoria, the powerful genetically encoded tag presently available in a variety of mutants featuring blue to yellow emission, has found a red-emitting counterpart. The recently cloned red fluorescent protein DsRed, isolated from Discosoma corals (), with its emission maximum at 583 nm, appears to be the long awaited tool for multi-color applications in fluorescence-based biological research. Studying the emission dynamics of DsRed by fluorescence correlation spectroscopy (FCS), it can be verified that this protein exhibits strong light-dependent flickering similar to what is observed in several yellow-shifted mutants of GFP. FCS data recorded at different intensities and excitation wavelengths suggest that DsRed appears under equilibrated conditions in at minimum three interconvertible states, apparently fluorescent with different excitation and emission properties. Light absorption induces transitions and/or cycling between these states on time scales of several tens to several hundreds of microseconds, dependent on excitation intensity. With increasing intensity, the emission maximum of the static fluorescence continuously shifts to the red, implying that at least one state emitting at longer wavelength is preferably populated at higher light levels. In close resemblance to GFP, this light-induced dynamic behavior implies that the chromophore is subject to conformational rearrangements upon population of the excited state.
Abstract:Tetanus toxin and the seven serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G) abrogate synaptic transmission at nerve endings through the action of their light chains (L chains), which proteolytically cleave VAMP (vesicle-associated membrane protein)/ synaptobrevin, SNAP-25 (synaptosome-associated protein of 25 kDa), or syntaxin. BoNT/C was reported to proteolyze both syntaxin and SNAP-25. Clostridia produce several powerful neurotoxins; tetanus toxin and botulinal neurotoxins BoNT/A to BoNT/G cause the clinical manifestations of tetanus and botulism in a large variety of animal species and humans. The toxins are synthesized as single-chain polypeptides with molar masses of ϳ150 kDa. On lysis of the bacteria and activation by proteolytic cleavage, the light chains (L chains; 50 kDa) remain disulfide bound to the heavy chains (H chains; 100 kDa). The extreme neurotoxicity is largely ascribed to the H chains, which bind to neuronal receptors that internalize the holotoxins, and translocate the L chains into the cytosol. Here, the L chains block fusion of synaptic vesicles with the presynaptic membrane (Simpson, 1989).The genes of the eight known clostridial neurotoxins have been cloned and characterized (for review, see Niemann et al., 1994). The L chains contain a Zn 2ϩ -binding motif, His-Glu-X-X-His, also found in an increasing number of zinc-dependent metalloproteases (Jongeneel et al., 1989). Soon after demonstration of cleavage of VAMP (vesicle-associated membrane protein)/synaptobrevin (Trimble et al., 1988) by tetanus toxin and BoNT/B at the same peptide bond (Link et al., 1992;Schiavo et al., 1992), substrates and scissile bonds were identified for all other botulinum serotypes. These studies revealed that BoNT/D, BoNT/F, and BoNT/G also hydrolyze synaptobrevin, although each at a different peptide bond. BoNT/A and BoNT/E cleave SNAP-25 (synaptosome-associated protein of 25 kDa), again at distinct sites close to the C-terminus (for review, see Received March 20, 1998; revised manuscript received July 20, 1998; accepted July 23, 1998. Address correspondence and reprint requests to Dr. T. Binz at Department of Biochemistry, Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, D-30623, Hannover, Germany.The present address of Dr. V. V. Vaidyanathan is Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, U.S.A.Abbreviations used: BoNT, botulinal neurotoxin; GST, glutathione S-transferase; GT, glutathione; L chain, light chain; NSF, Nethylmaleimide sensitive fusion protein; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; SNAP, soluble NSF attachment protein; SNAP-23, 23-kDa isoform of SNAP-25; SNAP-25, synaptosome-associated protein of 25 kDa; hSNAP-23 and mSNAP-23, human and murine SNAP-23, respectively; SNARE, soluble NSF attachment protein receptor; VAMP, vesicle-associated membrane protein.
Confocal fluorescence spectroscopy is a versatile method for studying dynamics and interactions of biomolecules in their native environment with minimal interference with the observed system. Analyzing coincident fluctuations induced by single molecule movement in spectrally distinct detection channels, dual-color fluorescence cross-correlation, and coincidence analysis have proven most powerful for probing the formation or cleavage of molecular bonds in real time. The similarity of the optical setup with those used for laser scanning microscopy, as well as the non-invasiveness of the methods, make them easily adaptive for intracellular measurements, to observe the association and dissociation of biomolecules in situ. However, in contrast to standard fluorescence microscopy, where multiple fluorophores can be spectrally resolved, single molecule detection has so far been limited to dual-color detection systems due to the harsh requirements on detection sensitivity. In this study, we show that under certain experimental conditions, employing simultaneous two-photon excitation of three distinct dye species, their successful discrimination indeed becomes possible even on a single molecule level. This enables the direct observation of higher order molecular complex formation in the confocal volume. The theoretical concept of triple-color coincidence analysis is outlined in detail, along with an experimental demonstration of its principles utilizing a simple nucleic acid reaction system.
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