Shedding light on reaction mechanisms : structure determination of reactive intermediates and investigation of protein structural dynamics using 2D-IR spectroscopy
Andreas T. Meßmer
- Detailed knowledge of reaction mechanisms is key to understanding chemical, biological, and biophysical processes. For many reasons, it is desirable to comprehend how a reaction proceeds and what influences the reaction rate and its products.
In biophysics, reaction mechanisms provide insight into enzyme and protein function, the reason why they are so efficient, and what determines their reaction rates. They also reveal the relationship between the function of a protein and its structure and dynamics.
In chemistry, reaction mechanisms are able to explain side products, solvent effects, and the stereochemistry of a product. They are also the basis for potentially optimizing reactions with respect to yield, enhancing the stereoselectivity, or for modifying reactions in order to obtain other related products.
A key step to investigate reaction mechanisms is the identification and characterization of intermediates, which may be reactive, short-lived, and therefore only weakly populated. Nowadays, the structures of those can in most cases only be hypothesized based on products, side products, and isolable intermediates, because intermediates with a life time of less than a few microseconds are not accessible with the commonly used techniques for structure determination such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy.
In this thesis, two-dimensional infrared (2D-IR) spectroscopy is shown to be a powerful complement to the existing techniques for structure determination in solution. 2D-IR spectroscopy uses a femtosecond laser setup to investigate interactions between vibrations - analogous to 2D-NMR, which investigates the interactions between spins. Its ultrafast time resolution makes 2D-IR spectroscopy particularly well suited for the two topics investigated in this thesis: Structure Determination of Reactive Intermediates and Conformational Dynamics of Proteins.
Structure Determination of Reactive Intermediates: The focus of this thesis is using polarization-dependent 2D-IR (P2D-IR) spectroscopy for structure determination of N-crotonyloxazolidinone (referred to as 1), a small organic compound with a chiral oxazolidinone, known as Evans auxiliary, and its reactive complexes with the Lewis acids SnCl4 and Mg(ClO4)2. Chiral oxazolidinones in combination with Lewis acids have frequently been used in stereoselective synthesis for over 30 years. Nevertheless, the detailed mechanisms are in many cases xvi ABSTRACT still mere hypotheses and have not yet been experimentally proven. By accurately measuring the angles between the transition dipole moments in the molecules using an optimized P2D-IR setup and comparing the results to DFT calculations, the conformation of 1 and the conformation and coordination of the main complexes with SnCl4 and Mg(ClO4)2 are unequivocally identified and analyzed in depth. Structural details, such as a slight twist in the solution structure of 1, are detected using P2D-IR spectroscopy; these cannot be inferred from NMR spectroscopy or DFT calculations. In addition to the main Lewis acid complexes, complexes in low concentration are detected and tentatively assigned to different conformations and complexation geometries. The knowledge of those structures is essential for rationalizing the observed stereoselectivities. Additionally, a method is introduced that enables structure determination of molecules in complex mixtures and even in the presence of molecules with similar spectral properties and in high concentration. This work sets the stage for future studies of other substrate-catalyst complexes and reaction intermediates for which the structure determination has not been possible to date.
Conformational Dynamics of Proteins: Exchange 2D-IR spectroscopy allows the investigation of fast dynamics without disturbing the equilibrium of the exchanging species. It is therefore well suited to investigate fast dynamics of proteins and to reveal the speed limit of those. The temperature dependence of the conformational dynamics between the myoglobin substates A1 and A3 in equilibrium is analyzed. The various substates of myoglobin can be detected with FTIR spectroscopy, if carbon monoxide is bound to the heme. From previous studies it is known that the exchange rates at room temperature are in the picosecond time range, well suited to be investigated by 2D-IR spectroscopy. In the temperature range between 0 °C and 40 °C only a weak temperature dependence of the exchange rate in the myoglobin mutant L29I is observed in the present study. The exchange rate approximately doubles from 15 ns-1 at 0 °C to 31 ns-1 at 40 °C. It turned out that the conformational dynamics correlates linearly with the solvent viscosity, which itself is temperature dependent. Comparing our results to measurements at cryogenic temperatures, the linear relation between exchange time constant for this process and the viscosity is shown for the temperature range between -100 °C and 40 °C (corresponding to a viscosity change of 14 orders of magnitude). Thus, it is proven that the dynamics of the conformational switching are mainly determined by solvent dynamics, i.e., the protein dynamics are slaved to the solvent dynamics. This is the first time slaving is observed for such fast processes (in the picosecond time range). The observation implies a long-range structural rearrangement between the myoglobin substates A1 and A3. In addition, the exchange for other mutants and wild type myoglobin is analyzed qualitatively and found to agree with the conclusions drawn from L29I myoglobin.
Molecular dynamics simulations and docking of non-nucleoside reverse transcriptase inhibitors (NNRTIs): a possible approach to personalized HIV treatment
Florian D. Roessler
Peter J. Bond
- The human immunodeficiency virus (HIV) is currently ranked sixth in the worldwide causes of death . One treatment approach is to inhibit reverse transcriptase (RT), an enzyme essential for reverse transcription of viral RNA into DNA before integration into the host genome . By using non-nucleoside RT inhibitors (NNRTIs) , which target an allosteric binding site, major side effects can be evaded. Unfortunately, high genetic variability of HIV in combination with selection pressure introduced by drug treatment enables the virus to develop resistance against this drug class by developing point mutations. This situation necessitates treatment with alternative NNRTIs that target the particular RT mutants encountered in a patient.
Previously, proteochemometric approaches have demonstrated some success in predicting binding of particular NNRTIs to individual mutants; however a structurebased approach may help to further improve the predictive success of such models. Hence, our aim is to rationalize the experimental activity of known NNRTIs against a variety of RT mutants by combining molecular modeling, long-timescale atomistic molecular dynamics (MD) simulation sampling and ensemble docking. Initial control experiments on known inhibitor-RT mutant complexes using this protocol were successful, and the predictivity for further complexes is currently being evaluated. In addition to predictive power, MD simulations of multiple RT mutants are providing fundamental insight into the dynamics of the allosteric NNRTI binding site which is useful for the design of future inhibitors. Overall, work of this type is hoped to contribute to the development of predictive efficacy models for individual patients, and hence towards personalized HIV treatment options.
Entwicklung einer reagenzienfreien Methode zur quantitativen Bestimmung der Inhaltsstoffe von Körperflüssigkeiten mit ATR-FTIR-Spektroskopie
- Teile dieser Arbeit sind Thema folgender Publikationen:
Gamze Hosafci, Oliver Klein, Gerhard Oremek, Werner Mäntele: Clinical chemistry without reagents? An infrared spectroscopic technique for determination of clinically relevant constituents of body fluids Anal Bioanal Chem, DOI 10.1007/s00216-006-0841-3
Gamze Hosafci, Oliver Klein, Gerhard Oremek, Werner Mäntele: Ein Tropfen Blut genügt: Reagenzienfreie Labordiagnostik in der Medizin mittels Infrarotspektroskopie GIT Labor-Fachzeitschrift, Bd 50 (2006), H. 4, S. 322-325, GIT Darmstadt
Gamze Hosafci, Oliver Klein, Gerhard Oremek, Werner Mäntele: It Will Just Need a Drop of Blood: Reagent-free Laboratory Diagnostics in Medicine Based on Infrared Spectroscopy GIT Laboratory Journal, Bd 50 (2006), H. 2, S. 34-37, GIT Darmstadt
Patent: W. Mäntele, O. Klein, G. Hosafci, G. Oremek: Vorrichtung für die qualitative und/oder quantitative Bestimmung von IR-aktiven Inhaltsstoffen in Flüssigkeiten sowie ein Verfahren zur qualitativen und/oder quantitativen Bestimmung von IR-aktiven Inhaltsstoffen in Flüssigkeiten Schutzrecht: DE102005048807 16.11.2006
Stimulation of TRPV1 by Green Laser Light
- Low-level laser irradiation of visible light had been introduced as a medical treatment already more than 40 years ago, but its medical application still remains controversial. Laser stimulation of acupuncture points has also been introduced, and mast-cells degranulation has been suggested. Activation of TRPV ion channels may be involved in the degranulation. Here, we investigated whether TRPV1 could serve as candidate for laser-induced mast cell activation. Activation of TRPV1 by capsaicin resulted in degranulation. To investigate the effect of laser irradiation on TRPV1, we used the Xenopus oocyte as expression and model system. We show that TRPV1 can functionally be expressed in the oocyte by (a) activation by capsaicin (K 1/2 = 1.1 μM), (b) activation by temperatures exceeding 42°C, (c) activation by reduced pH (from 7.4 to 6.2), and (d) inhibition by ruthenium red. Red (637 nm) as well as blue (406 nm) light neither affected membrane currents in oocytes nor did it modulate capsaicin-induced current. In contrast, green laser light (532 nm) produced power-dependent activation of TRPV1. In conclusion, we could show that green light is effective at the cellular level to activate TRPV1. To which extend green light is of medical relevance needs further investigation.
Inhibition of Activity of GABA Transporter GAT1 by δ-Opioid Receptor
- Analgesia is a well-documented effect of acupuncture. A critical role in pain sensation plays the nervous system, including the GABAergic system and opioid receptor (OR) activation. Here we investigated regulation of GABA transporter GAT1 by δOR in rats and in Xenopus oocytes. Synaptosomes of brain from rats chronically exposed to opiates exhibited reduced GABA uptake, indicating that GABA transport might be regulated by opioid receptors. For further investigation we have expressed GAT1 of mouse brain together with mouse δOR and μOR in Xenopus oocytes. The function of GAT1 was analyzed in terms of Na(+)-dependent [(3)H]GABA uptake as well as GAT1-mediated currents. Coexpression of δOR led to reduced number of fully functional GAT1 transporters, reduced substrate translocation, and GAT1-mediated current. Activation of δOR further reduced the rate of GABA uptake as well as GAT1-mediated current. Coexpression of μOR, as well as μOR activation, affected neither the number of transporters, nor rate of GABA uptake, nor GAT1-mediated current. Inhibition of GAT1-mediated current by activation of δOR was confirmed in whole-cell patch-clamp experiments on rat brain slices of periaqueductal gray. We conclude that inhibition of GAT1 function will strengthen the inhibitory action of the GABAergic system and hence may contribute to acupuncture-induced analgesia.
Ion stopping in dense plasma target for high energy density physics
Dieter H. H. Hoffmann
Olga N. Rosmej
Naeem Ahmad Tahir
Boris Y. Sharkov
Alexey A. Golubev
Vladimir E. Fortov
- The basic physics of nonrelativistic and electromagnetic ion stopping in hot and ionized plasma targets is thoroughly updated. Corresponding projectile-target interactions involve enhanced projectile ionization and coupling with target free electrons leading to significantly larger energy losses in hot targets when contrasted to their cold homologues. Standard stoppping formalism is framed around the most economical extrapolation of high velocity stopping in cold matter. Further elaborations pay attention to target electron coupling and nonlinearities due to enhanced projectile charge state, as well. Scaling rules are then used to optimize the enhanced stopping of MeV/amu ions in plasmas with electron linear densities nel ~ 10 18 -10 20 cm -2 . The synchronous firing of dense and strongly ionized plasmas with the time structure of bunched and energetic multicharged ion beam then allow to probe, for the first time, the long searched enhanced plasma stopping and projectile charge at target exit. Laser ablated plasmas (SPQR1) and dense linear plasma columns (SPQR2) show up as targets of choice in providing accurate and on line measurements of plasma parameters. Corresponding stopping results are of a central significance in asserting the validity of intense ion beam scenarios for driving thermonuclear pellets. Other applications of note feature thorium induced fission, novel ion sources and specific material processing through low energy ion beams. Last but not least, the given ion beam-plasma target interaction physics is likely to pave a way to the production and diagnostics of warm dense matter (WDM).
Fachspezifischer Anhang zur SPoL (Teil III): Studienfach Physik im Studiengang L3
Fachspezifischer Anhang zur SPoL (Teil III): Studienfach Physik in den Studiengängen L2 und L5
Prüfungsordnung für die Bachelor- und Master-Studiengänge Physik der Informationstechnologie der Johann Wolfgang Goethe-Universität Frankfurt am Main mit dem Abschluss Bachelor of Science (B.Sc.) bzw. Master of Science (M.Sc.) vom 14.01.2004 in der Fassung vom 16. Juli 2007 : genehmigt mit Erlass vom 28.September 2007, Az.: III 1.3 - 422/13/10.010 – (0002)
Studienordnung für den Bachelor- und den Master-Studiengang Physik der Informationstechnologie an der Johann Wolfgang Goethe-Universität Frankfurt am Main mit dem Abschluss Bachelor of Science (B.Sc.) bzw. Master of Science (M.Sc.) vom 14. Januar 2004 in der Fassung vom 16. Juli 2007 : genehmigt mit Erlass vom 28. September 2007, Az.: III 1.3 – 422/13/10.010 – (0002)