The development of a method is described for the chemical labeling of proteins which occurs with high target specificity, proceeds within seconds to minutes, and offers a free choice of the reporter group. The method relies upon the use of peptide templates, which align a thioester and an Nterminal cysteinyl residue such that an acyl transfer reaction is facilitated at nanomolar concentrations. The protein of interest is N-terminally tagged with a 22 aa long Cys-E3 peptide (acceptor), which is capable of forming a coiled-coil with a reporter-armed K3 peptide (donor). This triggers the transfer of the reporter to the acceptor on the target protein. Because ligation of the two interacting peptides is avoided, the mass increase at the protein of interest is minimal. The method is exemplified by the rapid fluorescent labeling and fluorescence microscopic imaging of the human Y 2 receptor on living cells.

Biological nanopores are emerging as powerful and low-cost sensors for real-time analysis of biological samples. Proteins can be incorporated inside the nanopore, and ligand binding to the protein adaptor yields changes in nanopore conductance. In order to understand the origin of these conductance changes and develop sensors for detecting metabolites, we tested the signal originating from 13 different protein adaptors. We found that the quality of the protein signal depended on both the size and charge of the protein. The engineering of a dipole within the surface of the adaptor reduced the current noise by slowing the protein dynamics within the nanopore. Further, the charge of the ligand and the induced conformational changes of the adaptor defined the conductance changes upon metabolite binding, suggesting that the protein resides in an electrokinetic minimum within the nanopore, the position of which is altered by the ligand. These results represent an important step toward understanding the dynamics of the electrophoretic trapping of proteins inside nanopores and will allow developing next-generation sensors for metabolome analysis.

Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is an interesting target for biotechnological applications, because of its vast substrate variety combined with high P450 monooxygenase activity. The low stability in vitro could be overcome by immobilization on surfaces. Here we describe a novel method for immobilization on metal surfaces by using selectively binding peptides. A P450 BM3 triple mutant (3M-P450BM3: A74G, F87V, L188Q) was purified as protein thioester and ligated to indium tin oxide or gold binding peptides (BP) named HighSP-BP and Cys-BP, respectively. The ligation products were characterized by Western Blot and tryptic digestion combined with mass spectrometry, and displayed high affinity binding on the depicted surfaces. Next, we could demonstrate by benzyloxyresorufin O-dealkylation assay (BROD assay) that the activity of immobilized ligation products is higher than for the soluble form. The study provides a new tool for selective modification and immobilization of P450 variants.

Industrial biotechnology aims to exploit cytochrome P450 enzymes to access their sophisticated catalytic activity for challenging chemical reactions on inert C−H bonds. Limited by the need for NADPH, approaches to bind P450 enzymes to electrode surfaces for an artificial electron supply are promising. Here, we demonstrate that a recombinant fusion of an indium tin oxide binding peptide and the multi‐domain class VIII cytochrome P450 BM3 can be used in electrically driven catalysis. Bioelectrocatalytic activity is analyzed by direct product quantification resulting in superior activity of the specifically immobilized P450 BM3 in contrast to unspecifically adsorbed enzyme. Spacer and anchor point studies imply that enzyme flexibility and alignment are crucial factors to achieve high activity on the electrode. Furthermore, we demonstrate that our approach is also feasible for pharmaceutical application using naringenin as substrate.

Eine neue Methode zur chemischen Proteinmarkierung läuft mit hoher Zielspezifität innerhalb weniger Minuten ab und ermçglicht eine freie Wahl des Reportermoleküls. Sie beruht auf Peptidtemplaten, die einen Thioester und ein Nterminales Cystein so ausrichten, dass eine Acyltransferreaktion bei nanomolarer Konzentration mçglich wird. Das Zielprotein wird N-terminal mit einem 22 Aminosäure langen Cys-E3-Peptid (Akzeptor) ausgestattet, das mit einem Reportertragenden K3-Peptid (Donor) ein Coiled-Coil bilden kann. Dadurch wird die Übertragung des Reportermoleküls auf den Akzeptor am Zielprotein ausgelçst. Weil die Ligierung der wechselwirkenden Peptide vermieden wird, ist der Massenzuwachs am Zielprotein minimal. Zur Veranschaulichung der Methode werden die rasche Fluoreszenzmarkierung und die fluoreszenzmikroskopische Untersuchung des humanen Y2-Rezeptors an lebenden Zellen gezeigt.