Zeitauflösende Schwingungsspektroskopie an Bacteriorhodopsin und Halorhodopsin

The Proteins Bacteriorhodopsin (BR) and Halorhodopsin (HR) can be found in the cell membrane of the halophilic archaebacterium Halobacterium salinarum. Under extreme circumstances, BR as a light-driven proton pump provides an alternative to the respiratory chain in order to establish a proton gradient. HR as a light-driven chloride pump is used to maintain the Chloride concentration in the cytoplasm under varying conditions. The previously published ground state x-ray structures of both Proteins-in the case of BR additional structures of photocycle intermediates are known-form an important basis for the understanding of the functional mechanism, although they fail to provide a dynamic picture of the process. However, this picture is provided by time-resolving vibrational spectroscopy, which is able to detect transient changes in the structure of the Proteins. Fourier-Transform Infrared (FTIR)-spectroscopy on BR with polarized measuring light presents an additional parameter, the linear dichroism, to tracé the structural changes during the photocycle. These measurements are used not only for improved separation of the photocycle intermediates, but also to calculate orientations of transition dipole moments and thereby of structural elements . In addition, it is possible to resolve transient protonations of amino acid side chains, an important part of the mechanism of a proton pump and also invisible with the x-ray technique . In contrast to BR not much is known about the photocycle of the Chloride pump HR. In this work, the influence of the Chloride concentration on the kinetics is investigated with time-resolving FTIR-spectroscopy. The use of the attenuated total reflection ATR-technique, here with a new diamond ATR cell, allows reproducible adjustment of the sait concentration in infrared experiments in the range between 0 to 5 mol/L. An alternative photocycle model has been formulated based on the respective influence on the formation and decay of the intermediates . Resonance Raman measurements on both proteins give additional information on the assignment of vibrational modes and demonstrate the potential of the spectroscopic setup as a micro-Raman-spectrometer for the investigation of protein crystals

Time-resolved methods in biophysics. 10. Time-resolved FT-IR difference spectroscopy and the application to membrane proteins

Radu I, Schleeger M, Bolwien C, Heberle J. Time-resolved methods in biophysics. 10. Time-resolved FT-IR difference spectroscopy and the application to membrane proteins. PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES. 2009;8(11):1517-1528.The introduction of time-resolved Fourier transform infrared (FT-IR) spectroscopy to biochemistry opened the possibility of monitoring the catalytic mechanism of proteins along their reaction pathways. The infrared approach is very fruitful, particularly in the application to membrane proteins where NMR and X-ray crystallography are challenged by the size and protein hydrophobicity, as well as by their limited time-resolution. Here, we summarize the principles and experimental realizations of time-resolved FT-IR spectroscopy developed in our group and compare with aspects emerging from other laboratories. Examples of applications to retinal proteins and energy transduction complexes are reviewed, which emphasize the impact of time-resolved FT-IR spectroscopy on the understanding of protein reactions on the level of single bonds

Transient binding of CO to Cu(B) in cytochrome c oxidase is dynamically linked to structural changes around a carboxyl group: a time-resolved step-scan Fourier transform infrared investigation.

The redox-driven proton pump cytochrome c oxidase is that enzymatic machinery of the respiratory chain that transfers electrons from cytochrome c to molecular oxygen and thereby splits molecular oxygen to form water. To investigate the reaction mechanism of cytochrome c oxidase on the single vibrational level, we used time-resolved step-scan Fourier transform infrared spectroscopy and studied the dynamics of the reduced enzyme after photodissociation of bound carbon monoxide across the mid-infrared range (2300-950 cm(-1)). Difference spectra of the bovine complex were obtained at -20 degrees C with 5 micros time resolution. The data demonstrate a dynamic link between the transient binding of CO to Cu(B) and changes in hydrogen bonding at the functionally important residue E(I-286). Variation of the pH revealed that the pK(a) of E(I-286) is >9.3 in the fully reduced CO-bound oxidase. Difference spectra of cytochrome c oxidase from beef heart are compared with those of the oxidase isolated from Rhodobacter sphaeroides. The bacterial enzyme does not show the environmental change in the vicinity of E(I-286) upon CO dissociation. The characteristic band shape appears, however, in redox-induced difference spectra of the bacterial enzyme but is absent in redox-induced difference spectra of mammalian enzyme. In conclusion, it is demonstrated that the dynamics of a large protein complex such as cytochrome c oxidase can be resolved on the single vibrational level with microsecond Fourier transform infrared spectroscopy. The applied methodology provides the basis for future investigations of the physiological reaction steps of this important enzyme

Diamond-Coated Silicon ATR Elements for Process Analytics

Infrared attenuated total reflection (ATR) spectroscopy is a common laboratory technique for the analysis of highly absorbing liquids or solid samples. However, ATR spectroscopy is rarely found in industrial processes, where inline measurement, continuous operation, and minimal maintenance are important issues. Most materials for mid-infrared (MIR) spectroscopy and specifically for ATR elements do not have either high enough infrared transmission or sufficient mechanical and chemical stability to be exposed to process fluids, abrasive components, and aggressive cleaning agents. Sapphire is the usual choice for infrared wavelengths below 5 µm, and beyond that, only diamond is an established material. The use of diamond coatings on other ATR materials such as silicon will increase the stability of the sensor and will enable the use of larger ATR elements with increased sensitivity at lower cost for wavelengths above 5 µm. Theoretical and experimental investigations of the dependence of ATR absorbances on the incidence angle and thickness of nanocrystalline diamond (NCD) coatings on silicon were performed. By optimizing the coating thickness, a substantial amplification of the ATR absorbance can be achieved compared to an uncoated silicon element. Using a compact FTIR instrument, ATR spectra of water, acetonitrile, and propylene carbonate were measured with planar ATR elements made of coated and uncoated silicon. Compared to sapphire, the long wavelength extreme of the spectral range is extended to approximately 8 μm. With effectively nine ATR reflections, the sensitivity is expected to exceed the performance of typical diamond tip probes

Neue Methoden der laserbasierten Gasanalytik

Modern technology without lasers is hardly to be imagined. Due to technological progresses in manufacturing, quality, and width of applications they have successfully entered the field of optical online and inline process analytical instrumentation. Opportunities by application of laser-based methods for gas analysis will be demonstrated in the field of infrared spectroscopy and Raman spectroscopy (vibrational spectroscopy). Using Raman spectroscopy in hollow-core photonic crystal fibers low gas concentrations can be detected with measurement times of a few seconds. Using quantum cascade lasers trace methane emissions indicating a potential leakage can be detected from a distance and by applying lasers and nonlinear optical components, highly sensitive and fast infrared detectors operating at room temperature for gas analysis can be realized

Monitoring the Wobbe Index of Natural Gas Using Fiber-Enhanced Raman Spectroscopy

The fast and reliable analysis of the natural gas composition requires the simultaneous quantification of numerous gaseous components. To this end, fiber-enhanced Raman spectroscopy is a powerful tool to detect most components in a single measurement using a single laser source. However, practical issues such as detection limit, gas exchange time and background Raman signals from the fiber material still pose obstacles to utilizing the scheme in real-world settings. This paper compares the performance of two types of hollow-core photonic crystal fiber (PCF), namely photonic bandgap PCF and kagomé-style PCF, and assesses their potential for online determination of the Wobbe index. In contrast to bandgap PCF, kagomé-PCF allows for reliable detection of Raman-scattered photons even below 1200 cm−1, which in turn enables fast and comprehensive assessment of the natural gas quality of arbitrary mixtures.Published versio

Perfusion-induced redox differences in cytochrome c oxidase: ATR/FT-IR spectroscopy

AbstractAttenuated total reflection (ATR) spectroscopy brings an added dimension to studies of structural changes of cytochrome c oxidase (CcO) because it enables the recording of reaction-induced infrared difference spectra under a wide variety of controlled conditions (e.g. pH and chemical composition), without relying on light or potentiometric changes to trigger the reaction. We have used the ATR method to record vibrational difference spectra of CcO with reduction induced by flow-exchange of the aqueous buffer. Films of CcO prepared from Rhodobacter sphaeroides and beef heart mitochondria by reconstitution with lipid were adhered to the internal reflection element of the ATR device and retained their full functionality as evidenced by visible spectroscopy and time-resolved vibrational spectroscopy. These results demonstrate that the technique of perfusion-induced Fourier-transform infrared difference spectroscopy can be successfully applied to a large, complex enzyme, such as CcO, with sufficient signal/noise to probe vibrational changes in individual residues of the enzyme under various conditions