München, Techn. Univ., Diss., 2002. / Computerdatei im Fernzugriff.

München, Techn. Univ., Diss., 2002. / Computerdatei im Fernzugriff.

München, Techn. Universiẗat, Diss., 2002.

No description available.

Universiẗat, Diss., 2003--Marburg.

No description available.

Repair mechanisms for damaged DNA are essential for the proliferation of nearly all forms of life. Although DNA is quite robust, the vital information-storing molecule can often be damaged from environmental exposures such as ultra-violet (UV) light. Exposure to UV light can result in various types of mutagens creating structural damages. One specific type of UV-induced damage is the creation of a cyclobutylpyrimidine dimer (CPD). This specific type of lesion can be efficiently repaired by the flavoenzyme DNA photolyase (PL). DNA photolyase is an ancient protein found across kingdoms and plays a crucial role in preventing mutagenesis and cell death. DNA photolyase is a monomeric flavoprotein that utilizes blue light to repair UV-induced CPD lesions in DNA via an electron transfer mechanism. All photolyases contain at least one flavin adenine dinucleotide (FAD) molecule as the catalytic cofactor responsible for initiating the electron transfer induced repair process. Flavin cofactors are intriguing because of their unique ability to donate one or two electrons. The conservation of FAD and the unique U-shaped configuration of FAD in PL led researchers to question if the adenine moiety of the FAD molecule was essential in the DNA repair mechanism and generated a spectral signature indicative of a radical adenine species. The importance of the adenine moiety could be linked to structural changes associated with environmental temperature. The rate of electron transfer is exponentially dependent on temperature and DNA photolyase is found in organisms which thrive in harsh environments that vary in temperature, pH, ionic strength etc. Photolyase presents a unique opportunity to study the adaptations that are required for proteins to function in extreme environments where temperature dependent processes should show dramatic differences. We have used ultrafast transient absorption spectroscopy to compare the similarities and differences in excited state dynamics of the FAD cofactor. Photolyase isolated from the hyperthermophilic archaea Sulfolobus solfataricus (SsPL) is compared to PL isolated from the mesophilic E. coli (EcPL). These results indicate differences in the dynamics of fully reduced flavin between enzymes as a function of temperature. We present evidence for charge separation in the FAD cofactor in the thermophilic enzyme previously seen in computation studies of photolyase. To investigate the excited state charge redistribution of flavin which is critical to its role in nature, the charge redistribution of the precursors to flavin biosynthesis were examined. Lumazine is a precursor in the biosynthetic pathway of flavins. As such, lumazine could have served as an enzymatic cofactor prior to flavins. Lumazine has been identified in biological processes, however it is not as prevalent as flavins. We utilize Stark spectroscopy to examine the charge redistribution in excited state lumazine to understand / Chemistry

ABSTRACT Many enzymes require cofactors in order to carry out specific functions. Flavins, which are naturally fluorescent, compose a unique group of redox cofactors because they have the ability to transfer one or two electrons and are therefore found in three different oxidation states. A specific flavin, flavin adenine dinucleotide (FAD), is a crucial cofactor that facilitates electron transfer in many flavoproteins involved in DNA repair, photosynthesis, and regulatory pathways. One example of a FAD-containing DNA repair protein is DNA Photolyase (PL). E. coli PL is a monomeric flavoprotein that facilitates DNA repair via a photoinduced electron transfer reaction. The catalytic cofactor, FAD, transfers an electron to a thymidine dimer lesion, to cleave the cyclobutane ring and restore the DNA strand. Although the mechanism of repair has been partially elucidated by our group, it is still unclear whether or not the electron is transferred directly from the isoalloxazine moiety to the dimer or if the electron hops from the isoalloxazine moiety to the adenine moiety to the dimer. This sequential hopping mechanism should have excited state absorption features for the reduced flavin species, an adenine radical anion, and the semiquinone flavin species. To investigate the mechanistic role of adenine, E. coli PL has been reconstituted with -FAD, an FAD analogue in which the adenine was substituted via chemical means with 1,N6 – ethenoadenine dinucleotide. -FAD was selected due to its ease of synthesis and because its structure changes the thermodynamic driving force for the electron transfer reaction, by lowering the energetic gap (LUMO-LUMO) between the isoalloxazine ring and the modified adenine. In order to characterize the excited state dynamics of the mutant chromophore, the transient absorption measurements were made of each free flavin in solution. These measurements indicate the pathway of electron transfer must be mediated via superexchange rather than a hopping mechanism. This important result shows that the role of adenine in photolyase is to facilitate a superexchange electron transfer mechanism, and a modified flavin can act as a reporter under these experimental conditions. By exploiting Corynebacterium ammoniagenes FAD synthetase adenylation promiscuity, we have enzymatically-synthesized and purified a novel dually fluorescent flavin cofactor. This new flavin adenine dinucleotide (FAD) analogue, flavin 2-aminopurine (2Ap) dinucleotide (F2ApD), can be selectively excited through the 2Ap moiety at 310 nm, a wavelength at which flavins have intrinsically low extinction. The dinucleotide 2Ap emits at 370 nm with high efficiency. This emission has excellent overlap with the absorption spectra of both oxidized and reduced hydroquinone flavin (FlOX and FlHQ respectively), which emit at ~525 and ~505 nm respectively. We have characterized the optical properties of this dually fluorescent flavin, iFAD. Steady state fluorescence excitation and emission spectra were obtained and contrasted with the other flavins. Temperature- and solvent-dependent emission spectra suggest that F2ApD stacking interactions are significantly different compared to FAD and etheno-FAD (FAD). The optical absorption spectra of these dinucleotides were compared with FMN to explore electronic interactions between the flavin and nucleobase moieties. To probe the evolution of the different excited state populations, femtosecond transient absorption measurements were made on the iFADs, revealing that F2ApD exhibited unique transient spectra as compared to either FAD or FAD. The significance of these results to flavins, flavoprotein function, and bioimaging are discussed. The reconstituted -FAD in E.coli photolyase was catalytically active and actually repaired more efficiently than the FAD-reconstituted photolyase. To validate that an enzymatically synthesized iFAD could be reconstituted into a flavoprotein, this work shows a DNA repair assay using F2ApD that was reconstituted into E. coli photolyase, generating the reconstituted analogue, ApPL. Activity assays were compared between FAD-PL and ApPL. This comparison further elucidates the importance of the driving force on the electron transfer reaction in PL. A comparison of fluorescence spectroscopies between the reconstituted PLs highlights their applicability as biosensors and/or mechanistic reporters. / Chemistry

Ultraviolet light damages genomic material by inducing the formation of covalent bonds between adjacent pyrimidines. Cis-syn cyclobutane pyrimidine dimers (CPD)constitute the most abundant primary lesion in DNA. Photolyase, a light-activated enzyme, catalytically repairs these lesions. Although many steps in the photolyase-mediated repair process have been mapped, details of the mechanism remain cryptic. Difference FT-IR spectroscopy was employed to obtain new mechanistic information about photorepair. Purified oligonucleotides, containing a central diuracil, dithymidine, or cyclobutane thymidine dimer, were monitored using vibrational methods. Construction of difference infrared data between undamaged and damaged DNA permitted examination of nucleic acid changes upon formation of the CPD lesion; these experiments indicated that C=O and C-H frequencies can be used as markers for DNA damage. Furthermore, in purified photolyase containing isotopically-labeled aromatic amino acids, we observed that tryptophan residues in photolyase underwent structural changes during photorepair. These data indicate that electron transfer during DNA repair occurs through-bond, and that redox-active, aromatic residues form the pathway for electron transfer. / Master of Science

The short answer to the title question is: "a lot". It was transient absorption spectroscopy on geminate recombination in myoglobin that led Hans Frauenfelder to constructing his picture of protein's hierarchical energy landscape [1]. And even before that (in 1973), Joseph Lakowicz and Gregorio Weber at UIUC used quenching of tryptophan fluorescence by oxygen diffusing to solvent-inaccessible protein regions to conclude that "proteins, in general, undergo rapid structural fluctuations on the nanosecond time scale " [2]. The not-so-short answer is that the present text is written at a point where, after a decade of applying transient absorption spectroscopy to understand light induced electron transfer in a variety of enzymes, I am about to change the angle of attack and ask how these techniques and enzymes could be of help to solve some problems that are addressed in the IBS environment, namely protein dynamics, both structural and functional. It is for this reason that the answer will have to be delayed to the third and final part of this opus, "future", that deals with the perspectives. Meanwhile, the first part, "past", will be dedicated to showing on the example of the "paradigm" enzyme -DNA photolyase (the yellow egg hereunder)-, what transient absorption spectroscopy is capable of and the middle part, "present" dresses a short review into various experimental approaches currently used to obtain insight into protein dynamics. In the final section, I will delineate ways how optical spectroscopy could interact with projects existing or emerging in the protein dynamics community at IBS and thus contribute elements of an answer to the title question.