analogy to photoaffinity labeling [28], the conjugate is linked covalently to the protein in the course of a photoreaction. This avoids dissociation under the conditions of 2-DGE. The new concept has been proven feasible for the labeling of kinases [29].

Kinases play a major role in many regulatory mechanisms of all living cells. Phosphorylation/dephosphorylation processes trigger important metabolic pathways and participate in internal and external adaptation mechanisms. Systematic analysis of completely sequenced genomes provided crucial advances in kinase research. Although progress has been made in understanding detailed functions of plant kinases, it is still very difficult to assign the vast amount of recently identified kinase genes by systematic genomics (more than 196 in A. thaliana) to a particular cell regulation process. Ser/Thr kinases, of particular interest in plants, make up more than 12 groups because of sequence relationships. Thylakoid membranes contain Ser/Thr kinases functionally involved in adaptation mechanisms as biological responses to changes in environmental conditions (e.g., light) [35-37]. Mapping of in vivo protein phosphorylation sites of surface-exposed phosphorylated peptides in photosynthetic membranes of the green alga C. reinhardtii has recently been studied in detail by IMAC in combination with nESI-Q-TOF-MS/MS sequence analysis [38]. A total of 19 in vivo phosphorylation sites were mapped in the proteins corresponding to 15 genes in C. reinhardtii [38], with many of them belonging to light harvesting complex (LHC) proteins and photosystem II (PS II) proteins. Protein phosphorylation of these proteins is highly sensitive to inhibitors of cAMP-dependent kinases [39, 40]. Some of these kinases have been recently identified as thylakoid-associated enzymes in A. thaliana and in C. reinhardtii using forward and reverse genetic approaches [41-44]. Two of them, the LHC kinases STN7 in Arabidopsis and STT7 in Chlamydomonas [41, 42], are sensitive to the isoquinoline sulfonamide-type inhibitor H-9, which interacts with their ATP binding site [43].

A chemical probe for kinases was designed on the basis of H-9, with a photore-active group and a fluorophor being connected to the reversibly binding recognition unit H-9 across a polar linker. This conjugate aims at tagging Ser/Thr kinases by photochemical cross-linking followed by 2-DGE. The attachment site of the linker was selected on the basis of the crystal structure of a complex between a H-9 derivative and a kinase [45]. 4-Bpa was employed as the photoreactive group, because it is stable to common protic solvents and brings about efficient single-site modification [28] upon photochemical triplet insertion into CH bonds after irradiation at 350 nm.

A series of commercially available kinases was subjected to an in vitro assay in order to examine the specificity of photoaffinity labeling with the photoreactive and fluorescent H-9 conjugate. Three kinases (Figure 4.4, lanes 1, 2, and 4) were fluores-cently tagged to a high extent, whereas the labeling efficiency of a fourth one (lane 3) was quite poor. The specificity of the photoaffinity labeling process is emphasized by the fact that, for example, only the catalytic subunit of cAMP-dependent kinase was tagged, while the regulatory units remained unlabeled. The qualitative data were supported in real-time binding studies using SPR [29]. Potential radiationless fluorescence resonance energy transfer (FRET) within the conjugate—for example, between the benzophenone triplet state and the neighboring fluorescein—could be ruled out according to excitation-emission spectroscopy (EES) [29]. Energy transfer between the silver stain fluorescence image silver stain fluorescence image

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