Synthetic OCP heterodimers are photoactive and recapitulate the fusion of two primitive carotenoproteins in the evolution of cyanobacterial photoprotection

The orange carotenoid protein (OCP) governs photoprotection in the majority of cyanobacteria. It is structurally and functionally modular, comprised of a C‐terminal regulatory domain (CTD), an N‐terminal effector domain (NTD) and a ketocarotenoid; the chromophore spans the two domains in the ground state and translocates fully into the NTD upon illumination. Using both the canonical OCP1 from Fremyella diplosiphon and the presumably more primitive OCP2 paralog from the same organism, we show that an NTD‐CTD heterodimer forms when the domains are expressed as separate polypeptides. The carotenoid is required for the heterodimeric association, assembling an orange complex which is stable in the dark. Both OCP1 and OCP2 heterodimers are photoactive, undergoing light‐driven heterodimer dissociation, but differ in their ability to reassociate in darkness, setting the stage for bioengineering photoprotection in cyanobacteria as well as for developing new photoswitches for biotechnology. Additionally, we reveal that homodimeric CTD can bind carotenoid in the absence of NTD, and name this truncated variant the C‐terminal domain‐like carotenoid protein (CCP). This finding supports the hypothesis that the OCP evolved from an ancient fusion event between genes for two different carotenoid‐binding proteins ancestral to the NTD and CTD. We suggest that the CCP and its homologs constitute a new family of carotenoproteins within the NTF2‐like superfamily found across all kingdoms of life.     

Photo-stimulated leaving group isomerization of acyl donor esters in protease-catalyzed hydrolysis reactions

Abstract

Hydrolysis studies of photo-switchable N-maleyl-amino acid-(4-phenylazophenyl) esters of Ala, Gly, Met, Phe, and Pro were performed using the proteases trypsin and chymotrypsin. It has been found out that the cis-isomers were hydrolyzed faster than the trans-isomers. In dependence of the amino acid in the P1 position the velocity graduation is Phe?>?Met?>?Ala?>?Gly?>?Pro for trypsin and Phe???Met?>?Ala?>?Gly?>?Pro for chymotrypsin for both isomers.     

Switching between low and high affinity for the Syk tandem SH2 domain by irradiation of azobenzene containing ITAM peptidomimetics

Spleen tyrosine kinase (Syk) plays an essential role in IgE receptor signaling (FcεRI), which leads to mast cell degranulation. Divalent binding of the tandem SH2 domain (tSH2) of Syk to the intracellular ITAM motif of FcεRI activates the kinase domain of Syk, and thereby initiates cell degranulation. The inter SH2 domain distance in Syk tSH2 might be important for Syk kinase activation. In this study, photoswitchable ITAM peptidomimetics containing an azobenzene moiety were synthesized. Irradiation of these constructs changes the distance between the two SH2 binding epitopes and therefore, they may be used as photoswitches. The affinity of the cis‐ and trans‐isomer for tSH2 was assayed with SPR. The ITAM peptidomimetic with the smallest linker displayed the largest difference in affinity between the two isomers (at least 100‐fold), and the affinity of the cis‐isomer was comparable to monovalent binding. The ITAM mimics with larger photoswitchable linkers displayed modest differences. These results indicate that Syk tSH2 is able to adapt the inter SH2 domain distance to ligands larger than native ITAM, but not to smaller ones. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.