Probing Local Protein Environments with the Infrared Probe: L-4-Nitrophenylalanine

Abstract

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.

Spectroscopically labeled peptaibiotic analogs: the 4‐nitrophenylalanine infrared absorption probe inserted at different positions into trichogin GA IV

“…Moreover, the main motivation of the present work, namely confirmation of the recently published, interesting application of the Phe(NO 2 ) residue as a potential reporter group of ambient polarity in peptide‐based molecules, was only partially successful because in all environments tested the observed range of the wavenumber of the absorption maximum of the symmetric –NO 2 stretching mode for the membrane‐active trichogin GA IV analogs (1346–1348 cm −1 ) is rather narrow. We tend to attribute this rather modest sensitivity to the absence of water in our environments (in contrast to the measurements performed in the original paper ). Indeed, effective hydrogen‐bonding interactions between the hydrophilic nitro group and the solvent water molecules is a well‐established phenomenon, which apparently is largely responsible for the published blue shift near 1350 cm −1 in the IR absorption spectrum of the Phe(NO 2 ) label from tetrahydrofuran to aqueous solutions .…”

“…We tend to attribute this rather modest sensitivity to the absence of water in our environments (in contrast to the measurements performed in the original paper ). Indeed, effective hydrogen‐bonding interactions between the hydrophilic nitro group and the solvent water molecules is a well‐established phenomenon, which apparently is largely responsible for the published blue shift near 1350 cm −1 in the IR absorption spectrum of the Phe(NO 2 ) label from tetrahydrofuran to aqueous solutions .…”

“…To further expand this ongoing project, in this work, we synthesized, characterized, and investigated the 3D structural, biological, and spectroscopic properties of three Phe(NO 2 ) (4‐nitrophenylalanine)‐containing analogs of trichogin GA IV, each labeled with a side‐chain nitrobenzyl IR absorption probe . These chromophoric residues were incorporated at position 3, 7, or 10, i.e.…”

Biochemical analysis with the expanded genetic lexicon

“…2 (29)) and 3-nitrotyrosine (Fig. 2 (30)) has been used in IR and Raman spectrometry [108][109][110].…”

At the Interface of Chemical and Biological Synthesis: An Expanded Genetic Code

“…1B16), and 3-nitrotyrosine (3-NO 2 Y, Fig. 1B19) have been efficiently incorporated into proteins Taskent-Sezgin et al 2009;Ye et al 2009;Smith et al 2011). To study protein dynamics and microenvironments, photocaged 2 D-labeled o-nitrobenzyl-tyrosine (oNBTyr, Fig.…”