TY  - JOUR
A1  - Altintas, Zeynep
A1  - Takiden, Aref
A1  - Utesch, Tillmann
A1  - Mroginski, Maria A.
A1  - Schmid, Bianca
A1  - Scheller, Frieder W.
A1  - Süssmuth, Roderich D.
T1  - Integrated approaches toward high-affinity artificial protein binders obtained via computationally simulated epitopes for protein recognition
JF  - Advanced functional materials
N2  - Widely used diagnostic tools make use of antibodies recognizing targeted molecules, but additional techniques are required in order to alleviate the disadvantages of antibodies. Herein, molecular dynamic calculations are performed for the design of high affinity artificial protein binding surfaces for the recognition of neuron specific enolase (NSE), a known cancer biomarker. Computational simulations are employed to identify particularly stabile secondary structure elements. These epitopes are used for the subsequent molecular imprinting, where surface imprinting approach is applied. The molecular imprints generated with the calculated epitopes of greater stability (Cys-Ep1) show better binding properties than those of lower stability (Cys-Ep5). The average binding strength of imprints created with stabile epitopes is found to be around twofold and fourfold higher for the NSE derived peptide and NSE protein, respectively. The recognition of NSE is investigated in a wide concentration range, where high sensitivity (limit of detection (LOD) = 0.5 ng mL(-1)) and affinity (dissociation constant (K-d) = 5.3 x 10(-11)m) are achieved using Cys-Ep1 imprints reflecting the stable structure of the template molecules. This integrated approach employing stability calculations for the identification of stabile epitopes is expected to have a major impact on the future development of high affinity protein capturing binders.
KW  - artificial protein binders
KW  - cancer markers
KW  - computationally simulated epitopes
KW  - molecular imprinting
KW  - protein recognition
Y1  - 2019
U6  - https://doi.org/10.1002/adfm.201807332
SN  - 1616-301X
SN  - 1616-3028
VL  - 29
IS  - 15
PB  - Wiley-VCH
CY  - Weinheim
ER  - 
TY  - JOUR
A1  - Hartmann, Tobias
A1  - Schrapers, Peer
A1  - Utesch, Tillmann
A1  - Nimtz, Manfred
A1  - Rippers, Yvonne
A1  - Dau, Holger
A1  - Mroginski, Maria Andrea
A1  - Haumann, Michael
A1  - Leimkühler, Silke
T1  - The Molybdenum Active Site of Formate Dehydrogenase Is Capable of Catalyzing C-H Bond Cleavage and Oxygen Atom Transfer Reactions
JF  - Biochemistry
N2  - Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pK(a) of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase.
Y1  - 2016
U6  - https://doi.org/10.1021/acs.biochem.6b00002
SN  - 0006-2960
VL  - 55
SP  - 2381
EP  - 2389
PB  - American Chemical Society
CY  - Washington
ER  - 
TY  - JOUR
A1  - Peng, Lei
A1  - Utesch, Tillmann
A1  - Yarman, Aysu
A1  - Jeoung, Jae-Hun
A1  - Steinborn, Silke
A1  - Dobbek, Holger
A1  - Mroginski, Maria Andrea
A1  - Tanne, Johannes
A1  - Wollenberger, Ursula
A1  - Scheller, Frieder W.
T1  - Surface-Tuned Electron Transfer and Electrocatalysis of Hexameric Tyrosine-Coordinated Heme Protein
JF  - Chemistry - a European journal
N2  - Molecular modeling, electrochemical methods, and quartz crystal microbalance were used to characterize immobilized hexameric tyrosine-coordinated heme protein (HTHP) on bare carbon or on gold electrodes modified with positively and negatively charged self-assembled monolayers (SAMs), respectively. HTHP binds to the positively charged surface but no direct electron transfer (DET) is found due to the long distance of the active sites from the electrode surfaces. At carboxyl-terminated surfaces, the neutrally charged bottom of HTHP can bind to the SAM. For this "disc" orientation all six hemes are close to the electrode and their direct electron transfer should be efficient. HTHP on all negatively charged SAMs showed a quasi-reversible redox behavior with rate constant k(s) values between 0.93 and 2.86 s(-1) and apparent formal potentials E-app(0)' between -131.1 and -249.1 mV. On the MUA/MU-modified electrode, the maximum surface concentration corresponds to a complete monolayer of the hexameric HTHP in the disc orientation. HTHP electrostatically immobilized on negatively charged SAMs shows electrocatalysis of peroxide reduction and enzymatic oxidation of NADH.
KW  - electrochemistry
KW  - electron transfer
KW  - heme proteins
KW  - molecular modeling
KW  - monolayers
Y1  - 2015
U6  - https://doi.org/10.1002/chem.201405932
SN  - 0947-6539
SN  - 1521-3765
VL  - 21
IS  - 20
SP  - 7596
EP  - 7602
PB  - Wiley-VCH
CY  - Weinheim
ER  - 
TY  - JOUR
A1  - Sezer, Murat
A1  - Spricigo, Roberto
A1  - Utesch, Tillmann
A1  - Millo, Diego
A1  - Leimkühler, Silke
A1  - Mroginski, Maria A.
A1  - Wollenberger, Ursula
A1  - Hildebrandt, Peter
A1  - Weidinger, Inez M.
T1  - Redox properties and catalytic activity of surface-bound human sulfite oxidase studied by a combined surface enhanced resonance Raman spectroscopic and electrochemical approach
N2  - Human sulfite oxidase (hSO) was immobilised on SAM-coated silver electrodes under preservation of the native heme pocket structure of the cytochrome b5 (Cyt b5) domain and the functionality of the enzyme. The redox properties and catalytic activity of the entire enzyme were studied by surface enhanced resonance Raman (SERR) spectroscopy and cyclic voltammetry (CV) and compared to the isolated heme domain when possible. It is shown that heterogeneous electron transfer and catalytic activity of hSO sensitively depend on the local environment of the enzyme. Increasing the ionic strength of the buffer solution leads to an increase of the heterogeneous electron transfer rate from 17 s(-1) to 440 s(- 1) for hSO as determined by SERR spectroscopy. CV measurements demonstrate an increase of the apparent turnover rate for the immobilised hSO from 0.85 s(-1) in 100 mM buffer to 5.26 s(-1) in 750 mM buffer. We suggest that both effects originate from the increased mobility of the surface-bound enzyme with increasing ionic strength. In agreement with surface potential calculations we propose that at high ionic strength the enzyme is immobilised via the dimerisation domain to the SAM surface. The flexible loop region connecting the Moco and the Cyt b5 domain allows alternating contact with the Moco interaction site and the SAM surface, thereby promoting the sequential intramolecular and heterogeneous electron transfer from Moco via Cyt b5 to the electrode. At lower ionic strength, the contact time of the Cyt b5 domain with the SAM surface is longer, corresponding to a slower overall electron transfer process.
Y1  - 2010
UR  - http://www.rsc.org/Publishing/Journals/CP/index.asp
U6  - https://doi.org/10.1039/B927226g
SN  - 1463-9076
ER  -