Evidence for the isomerization and decarboxylation in the photoconversion of the red fluorescent protein DsRed

Recently, it has been shown that the red fluorescent protein DsRed undergoes photoconversion on intense irradiation, but the mechanism of the conversion has not yet been elucidated. Upon irradiation with a nanosecond-pulsed laser at 532 nm, the chromophore of DsRed absorbing at 559 nm and emitting at 583 nm (R form) converts into a super red (SR) form absorbing at 574 nm and emitting at 595 nm. This conversion leads to a significant change in the fluorescence quantum yield from 0.7 to 0.01. Here we demonstrate that the photoconversion is the result of structural changes of the chromophore and one amino acid. Absorption, fluorescence, and vibrational spectroscopy as well as mass spectrometry suggest that a cis-to-trans isomerization of the chromophore and decarboxylation of a glutamate (E215) take place upon irradiation to form SR. At the same time, another photoproduct (B) with an absorption maximum at 386 nm appears upon irradiation. This species is assigned as a protonated form of the DsRed chromophore. It might be a mixture of several protonated DsRed forms as there is at least two ways of formation. Furthermore, the photoconversion of DsRed is proven to occur through a consecutive two-photon absorption process. Our results demonstrate the importance of the chromophore conformation in the ground state on the brightness of the protein as well as the importance of the photon flux to control/avoid the photoconversion process.     

Total synthesis and initial structure-function analysis of the potent V-ATPase inhibitors salicylihalamide A and related compounds

Salicylihalamide A is the first member of a growing class of macrocyclic salicylate natural products that induce a variety of interesting phenotypes in cultured mammalian cells. Salicylihalamide A was reported to be a unique and highly differential cytotoxin and a potent inhibitor of the mammalian vacuolar (H(+))-ATPase. The total synthesis of both enantiomers of salicylihalamide A, a revision of the absolute configuration assigned to the natural product, and extensive structure-function studies with synthetic salicylihalamide variants are reported. These studies were possible only due to a highly efficient synthetic strategy that features (1) a remarkably E-selective ring-closing olefin metathesis to construct the 12-membered benzolactone skeleton 29, (2) a mild stereocontrolled elaboration to E-alkenyl isocyanate 41, and (3) addition of carbon, oxygen, and sulfur nucleophiles to isocyanate 41 to obtain salicylihalamide A and congeners. We demonstrate for the first time that salicylihalamide A is a potent inhibitor of fully purified reconstituted V-ATPase from bovine brain, and have identified several similarly potent side chain modified derivatives, including salicylihalamide dimers 43-45. In combination, these studies have laid the foundation for ongoing studies aimed at a comprehensive understanding of salicylihalamide's mode-of-action, of potential relevance to the development of lead compounds for the treatment of osteoporosis and cancer.     

Synthesis of 3,6-diaminophthalimides for ureidophthalimide-based foldamers

[reaction: see text]. Herein, we report an improved methodology for the synthesis of a variety of 3,6-diaminophthalimides in high yields. This enables decoration of the periphery of foldamers with a wide range of functionalities.     

Metal-catalyzed regioselective oxy-functionalization of internal alkynes: an entry into ketones, acetals, and spiroketals

[reaction: see text] Platinum(II) and an unusual cationic gold(I) complex were identified as mild catalysts for the room temperature cycloisomerization or tandem hydroalkoxylation/acetal formation of unactivated internal alkynols. Under the appropriate conditions, 5-endo, 5-exo, 6-endo, and 6-exo cycloisomerization modes are all available.     

Numerical accuracy of real inversion formulas for the Laplace transform

In this article, we investigate and compare a number of real inversion formulas for the Laplace transform. The focus is on the accuracy and applicability of the formulas for numerical inversion. In this contribution, we study the performance of the formulas for measures concentrated on a positive half-line to continue with measures on an arbitrary half-line. As our trial measure concentrated on a positive half-line, we take the broad Gamma probability distribution family.     

Comparing frontier methods for economic–environmental trade-off analysis

This paper uses a mechanistic frontier approach as a reference to evaluate the ability of conventional parametric (SFA) and non-parametric (DEA) frontier approaches for analyzing economic–environmental trade-offs. Conventional frontier approaches are environmentally adjusted through incorporating the materials balance principle. The analysis is worked out for the Flemish pig finishing case, which is both representative and didactic. Results show that, on average, SFA and DEA yield adequate economic–environmental trade-offs. Both methods are good estimators for technical efficiency. Cost allocative and environmental allocative efficiency scores are less robust, due to the well-known methodological advantages and disadvantages of SFA and DEA. For particular firms, SFA, DEA and the mechanistic approach may yield different economic–environmental trade-offs. One has therefore to be careful when using conventional frontier approaches for firm-specific decision support. The mechanistic approach allows for optimizing performances per average present finisher, which is the production unit in pig finishing. Conventional frontier methods do not allow for this optimization since the number of average present finishers varies along the production functions. Since the mechanistic production function is based on underlying growth, feed uptake and mortality functions, additional firm-specific indicators can also be calculated at each point of the production function.     

Characterization and sequence confirmation of unnatural amino acid containing peptide libraries using electrospray ionization mass spectrometry

The content of 24 12-membered heptapeptide libraries was investigated using capillary liquid chromatography coupled with an electrospray ionization quadrupole orthogonal acceleration time-of-flight mass spectrometer. Adjustment of the chromatographic parameters led to the separation of most of the components. Extraction of the [M + 2H]2+ ions allowed us to demonstrate the presence of all expected species in the library and to evaluate their relative abundance in the mixture. Rapid sequence confirmation was achieved by subtraction of product ion spectra, a way to eliminate common ions and to simplify the spectra for interpretation. This technique can also easily be applied to other libraries consisting of components with a common core.     

Synthesis and Conformational Analysis of 2′-Deoxy-2′-(3-methoxybenzamido)adenosine,a rational-designed inhibitor of trypanosomal glyceraldehyde phosphate dehydrogenase (GAPDH)

A series of 2′-benzamido-2′-deoxyadenosine analogues were synthesized in an effort to find new lead structures for the treatment of sleeping sickness. The 2′-deoxy-2′-(3-methoxybenzamido)adenosine ( 1h ) was proved to be a selective inhibitor of the parasite glyceraldehyde 3-phosphate dehydrogenase which confirms the modeling studies. The solution-state conformation of 2′-(thiophene-2-carboxamido) analogue 1d demonstrates a 2′-endo conformation, an orientation of the thiophene ring under the ribose moiety, and the base part occupying a ‘syn’/‘anti’ equilibrium.     

Characterization of modification sites during peptide synthesis using liquid secondary ion/collision-induced dissociation mass spectrometry and a computer program

Side-reactions often occur during peptide synthesis resulting in modified amino acid moieties. To identify these residues, liquid secondary ion/collision-induced dissociation mass spectra were recorded. The main fragments are generated by cleavage of the peptide bond. To facilitate interpretation of the spectra and assignment of the structure, a simple, but flexible and efficient, computer program is presented. The program allows the verification of the correct structure of the synthesized peptides and the deduction of the type of side-products formed, such as alkylation of tryptophan residues.     

Motion Planning in Environments with Low Obstacle Density

We present a simple and efficient paradigm for computing the exact solution of the motion planning problem in environments with a low obstacle density. Such environments frequently occur in practical instances of the motion planning problem. The complexity of the free space for such environments is known to be linear in the number of obstacles. Our paradigm is a new cell decomposition approach to motion planning and exploits properties that follow from the low density of the obstacles in the robot's workspace. These properties allow us to decompose the workspace, subject to some constraints, rather than to decompose the higher-dimensional free configuration space directly. A sequence of uniform steps transforms the workspace decomposition into a free space decomposition of asymptotically the same size. The approach leads to nearly optimal O(n \log n) motion planning algorithms for free-flying robots with any fixed number of degrees of freedom in workspaces with low obstacle density. Received October 17, 1995, and in revised form July 8, 1997, and February 23, 1998.