86-01-1Relevant articles and documents
Microsecond-Resolved Infrared Spectroscopy on Nonrepetitive Protein Reactions by Applying Caged Compounds and Quantum Cascade Laser Frequency Combs
Norahan, Mohamad Javad,Horvath, Raphael,Woitzik, Nathalie,Jouy, Pierre,Eigenmann, Florian,Gerwert, Klaus,K?tting, Carsten
, p. 6779 - 6783 (2021/05/31)
Infrared spectroscopy is ideally suited for the investigation of protein reactions at the atomic level. Many systems were investigated successfully by applying Fourier transform infrared (FTIR) spectroscopy. While rapid-scan FTIR spectroscopy is limited by time resolution (about 10 ms with 16 cm-1 resolution), step-scan FTIR spectroscopy reaches a time resolution of about 10 ns but is limited to cyclic reactions that can be repeated hundreds of times under identical conditions. Consequently, FTIR with high time resolution was only possible with photoactivable proteins that undergo a photocycle. The huge number of nonrepetitive reactions, e.g., induced by caged compounds, were limited to the millisecond time domain. The advent of dual-comb quantum cascade laser now allows for a rapid reaction monitoring in the microsecond time domain. Here, we investigate the potential to apply such an instrument to the huge class of G-proteins. We compare caged-compound-induced reactions monitored by FTIR and dual-comb spectroscopy by applying the new technique to the α subunit of the inhibiting Gi protein and to the larger protein-protein complex of Gαi with its cognate regulator of G-protein signaling (RGS). We observe good data quality with a 4 μs time resolution with a wavelength resolution comparable to FTIR. This is more than three orders of magnitude faster than any FTIR measurement on G-proteins in the literature. This study paves the way for infrared spectroscopic studies in the so far unresolvable microsecond time regime for nonrepetitive biological systems including all GTPases and ATPases.
Photo-electrochemical Bioanalysis of Guanosine Monophosphate Using Coupled Enzymatic Reactions at a CdS/ZnS Quantum Dot Electrode
Sabir, Nadeem,Khan, Nazimuddin,V?lkner, Johannes,Widdascheck, Felix,Del Pino, Pablo,Witte, Gregor,Riedel, Marc,Lisdat, Fred,Konrad, Manfred,Parak, Wolfgang J.
, p. 5844 - 5850 (2016/01/25)
A photo-electrochemical sensor for the specific detection of guanosine monophosphate (GMP) is demonstrated, based on three enzymes combined in a coupled reaction assay. The first reaction involves the adenosine triphosphate (ATP)-dependent conversion of GMP to guanosine diphosphate (GDP) by guanylate kinase, which warrants substrate specificity. The reaction products ADP and GDPare co-substrates for the enzymatic conversion of phosphoenolpyruvate to pyruvate in a second reaction mediated by pyruvate kinase. Pyruvate in turn is the co-substrate for lactate dehydrogenase that generates lactate via oxidation of nicotinamide adenine dinucleotide (reduced form) NADH to NAD+. This third enzymatic reaction is electrochemically detected. For this purpose a CdS/ZnS quantum dot (QD) electrode is illuminated and the photocurrent response under fixed potential conditions is evaluated. The sequential enzyme reactions are first evaluated in solution. Subsequently, a sensor for GMP is constructed using polyelectrolytes for enzyme immobilization.
Enzymatic and molecular characterization of arabidopsis ppGpp pyrophosphohydrolase, AtNUDX26
Ito, Daisuke,Kato, Takahiro,Maruta, Takanori,Tamoi, Masahiro,Yoshimura, Kazuya,Shigeoka, Shigeru
, p. 2236 - 2241 (2013/02/25)
Not only in bacteria but also in plant cells, guanosine- 3',5'-tetraphosphate (ppGpp) is an important signaling molecule, that affects various cellular processes. In this study, we identified nucleoside diphosphates linked to some moiety X (Nudix) hydrola
