The ability to genetically incorporate amino acids modified with spectroscopic reporters site-specifically into proteins with high efficiency and fidelity has greatly enhanced the ability to probe local protein structure and dynamics. Here, we have synthesized the unnatural amino acid (UAA), 4-cyano-L-phenylalanine (pCNPhe), containing the nitrile vibrational reporter and three isotopomers ((15)N, (13)C, (13)C(15)N) of this UAA to enhance the ability of pCNPhe to study local protein environments. Each pCNPhe isotopic variant was genetically incorporated in an efficient, site-specific manner into superfolder green fluorescent protein (sfGFP) in response to an amber codon with high fidelity utilizing an engineered, orthogonal aminoacyl-tRNA synthetase. The isotopomers of 4-cyano-L-phenylalanine permitted the nitrile symmetric stretch vibration of these UAAs to be unambiguously assigned utilizing the magnitude and direction of the isotopic shift of this vibration. The sensitivity of the nitrile symmetric stretching frequency of each isotopic variant to the local environment was measured by individually incorporating the probes into two distinct local environments of sfGFP. The UAAs were also utilized in concert to probe multiple local environments in sfGFP simultaneously to increase the utility of 4-cyano-L-phenylalanine.

The genetic incorporation of unnatural amino acids (UAAs) with high efficiency and fidelity is a powerful tool for the study of protein structure and dynamics with site-specificity in a relatively nonintrusive manner. Here, we illustrate the ability of L-4-nitrophenylalanine to serve as a sensitive IR probe of local protein environments in the 247 residue superfolder green fluorescent protein (sfGFP). Specifically, the nitro symmetric stretching frequency of L-4-nitrophenylalanine was shown to be sensitive to both solvents that mimic different protein environments and (15)N isotopic labeling of the three-atom nitro group of this UAA. (14)NO(2) and (15)NO(2) variants of this UAA were incorporated utilizing an engineered orthogonal aminoacyl-tRNA synthetase/tRNA pair into a solvent exposed and a partially buried position in sfGFP with high efficiency and fidelity. The combination of isotopic labeling and difference FTIR spectroscopy permitted the nitro symmetric stretching frequency of L-4-nitrophenylalanine to be experimentally measured at either site in sfGFP. The (14)NO(2) symmetric stretching frequency red-shifted 7.7 cm(-1) between the solvent exposed and partially buried position, thus illustrating the ability of this UAA to serve as an effective IR probe of local protein environments.

CALI and the labelling specificity that fluorescent proteins provide is very useful to avoid uncontrolled photodamage. Indeed, fluorescent proteins have been successfully used in CALI, although of the inactivation mechanisms by ROS are dependent on the fluorescent protein used and are not fully understood [2,3]. Here, we present a quantitative study of the ability of TagRFP to produce ROS, in particular singlet oxygen. TagRFP is able to photosensitize singlet oxygen with an estimated quantum yield of 0.004 [4]. This is the first estimation of a quantum yield of singlet oxygen production value for a GFP-like protein. We also find that TagRFP has a short triplet lifetime, which reflects relatively high oxygen accessibility to the chromophore compared to EGFP. Our results provide photophysical insight that allows the understanding of the mechanism behind CALI. Moreover, it has implications in improving photobleaching in fluorescent proteins.[1] K.

The steric configurations of (-)-galbelgin and galgravin (isomeric forms 111) have been investigated by fission of the tetrahydrofuran ring with sodium and ethanol in liquid ammonia, and examination of the products. Reduction of ( f)-1 : 2-(VII) and ( f)-3 : 4-dehydrogalbulin (VIII) by lithium and ammonia leads predominantly to ( -J-) -galbulin which accordingly has the configuration (I1 + its enantiomer) . The action of perchloric acid in acetic acid on galgravin and galbelgin has been investigated. We conclude that galgravin is probably (XII) and galbelgin probably (XIV) .THE lignans (-)-galbulin (II), (-)-galbacin (111; but with R = 3 : 4-methylenedioxyphenyl) , (-)-galcatin (I), galgravin, and (-)-galbelgin, the last two being stereoisomeric forms of (111), were isolated from Himantandra barks1 They offer opportunities for stereochemical studies since they carry only methyl groups instead of the oxygenated side-chains hitherto usually encountered in this series. While the present work was in progress, Schrecker and Hartwell also carried out investigations in this field and arrived at some similar conclusions although by different routes.(-)-Galcatin (I) has the same optical configuration as (-)-galbulin (11) since it can be converted into the latter by alkaline demethylenation and methylation. Schrecker and Hartwell concluded on the basis of optical rotations that this relation was highly probable.(-)-Galcatin (I) * has been converted into (-)-galbulin (11). CHR y o m : e tleO@Me 0 / \ -'FH , CHMe Me (Ill) * This and other formulac represent absolute configurations, except that for (&)-forms only one form is shown.

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