Effects of macromolecular crowding and small ions on the folding, structure, and stability of Desulfovibrio desufuricans flavodoxin

The intracellular environment in which most proteins fold and function contains a range of biomolecules that results in significant volume exclusion, thus contrasting to the dilute buffer conditions common to most in vitro studies. In addition to intracellular macromolecular crowding, cells are ionic in nature, and although the Hofmeister series of ions has its origin in a work from 1888, much is still unclear concerning how small, charged ions affect protein properties. This thesis summarizes in vitro work assessing the effects of macromolecular crowding and small ions on the biophysical properties of a model protein -- Desulfovibrio desulfuricans flavodoxin. Flavodoxin is a small (15.7 kDa), single domain, cytoplasmic protein with alpha-helical and parallel beta-sheet secondary structural elements arranged in one of the five most common protein folds (the flavodoxin-like fold). Using a range of biophysical/spectroscopic methods (e.g., circular dichroism (CD), fluorescence, calorimetry, stopped-flow mixing) along with synthetic crowding agents (e.g., Ficoll and dextran), I have found that macromolecular crowding increases the secondary structural content of folded flavodoxin (toward that found in the crystal structure), increases flavodoxin thermal stability, and affects both the accumulation of a misfolded intermediate and the rate of proper protein folding. Collaborative in silico simulations employing Go-like modeling of apoflavodoxin in the presence of large, inert crowding agents agrees with my in vitro work and provides structural and mechanistic information with residue-specific resolution. We also found that small cations and anions in physiologically relevant concentrations (≤ 250 mM) increase flavodoxin thermal stability significantly. Both cations and anions in higher concentrations (300 mM-.75 M) affect oppositely charged proteins similarly suggesting that surface electrostatic charge plays only a minor role in mediating ionic effects on protein thermal stability. At all ion concentrations, ionic effects on protein stability are correlated to ion hydration (and thus the Hofmeister series). Our work suggests a dominant role for the peptide bond in coordinating ions at higher concentrations. This thesis work suggests that the crowded and ionic nature of the intracellular milieu can elicit changes to the structure, dynamics, stability, and folding mechanism of proteins which may not be captured in vitro using dilute buffer conditions

Bioinorganic Chemistry

This book covers material that could be included in a one-quarter or one-semester course in bioinorganic chemistry for graduate students and advanced undergraduate students in chemistry or biochemistry. We believe that such a course should provide students with the background required to follow the research literature in the field. The topics were chosen to represent those areas of bioinorganic chemistry that are mature enough for textbook presentation. Although each chapter presents material at a more advanced level than that of bioinorganic textbooks published previously, the chapters are not specialized review articles. What we have attempted to do in each chapter is to teach the underlying principles of bioinorganic chemistry as well as outlining the state of knowledge in selected areas. We have chosen not to include abbreviated summaries of the inorganic chemistry, biochemistry, and spectroscopy that students may need as background in order to master the material presented. We instead assume that the instructor using this book will assign reading from relevant sources that is appropriate to the background of the students taking the course. For the convenience of the instructors, students, and other readers of this book, we have included an appendix that lists references to reviews of the research literature that we have found to be particularly useful in our courses on bioinorganic chemistry

Spectroscopic and mechanistic investigation of two flavin-dependent enzymes: nitronate monooxygenase and choline oxidase

Propionate 3-nitronate (P3N) is a natural toxin that irreversibly inhibits mitochondrial succinate dehydrogenase. P3N poisoning leads to a variety of neurological disorders and even death. Nitronate monooxygenase (NMO) from Cyberlindnera saturnus (CsNMO) and Pseudomonas aeruginosa PAO1 (PaNMO) serve as paradigms for Class I NMO, which catalyze the oxidation of P3N involving single electron transfer. In this dissertation, the crystallographic structure of CsNMO was solved and demonstrated a highly conserved three-dimensional structure and active site with respect to NMO from PaNMO. The role of conserved residues in the active site of Class I NMO, e.g. Y109, Y254, Y299, Y303, and K307 in PaNMO in substrate binding and catalysis were investigated using site-directed mutagenesis, steady-state kinetics and pH effects on the UV-visible absorption spectrum. The study revealed that a protonated tyrosine is required for binding of the negatively charged P3N substrate. We also report that PaNMO can stabilize both the neutral and anionic semiquinones anaerobically for hours, providing a constant protein environment to study their photochemical and photophysical properties. Choline oxidase catalyzes two-step oxidation of choline to glycine betaine with betaine aldehyde as an intermediate. The FAD cofactor is covalently attached to the choline oxidase via H99 through an 8α-N3-histidyl linkage. In the active site of choline oxidase, S101 and H466 are located on two extent loops, ~ 4 Å from the flavin C4a atom. In this dissertation, a charge-induced, reversible C4a-S-cysteinyl-8α-N3-histidyl FAD was engineered by replacing S101 with a cysteine. The mechanistic rationale for the stabilization of de novo C4a-S-cysteinyl-flavins was illustrated with rapid kinetics, pH, kinetic isotope effects and proton inventory. A photoinduced transient C4a-N-histidyl-8α-N3-histidyl FAD in choline oxidase wild-type was also observed with the aid of fluorescence excitation spectroscopy. Site-directed mutagenesis, solvent equilibrium isotope effects and pH effects on the stoke shifts of flavin in choline oxidase wild-type demonstrated H466 as the adduct on the C4a atom of flavin upon excitation, and provided a mechanistic rationale involving photoinduced electron transfer (PET) for the formation of the novel photoinduced transient flavin C4a adduct

Metal ions and protein folding: conformational and functional interplay

Dissertation presented to obtain a PhD degree in Biochemistry at Instituto de Tecnologia Química e Biológica, Universidade Nova de LisboaMetal ions are cofactors in about 30% of all proteins, where they fulfill catalytical and structural roles. Due to their unique chemistry and coordination properties they effectively expand the intrinsic polypeptide properties (by participating in catalysis or electron transfer reactions), stabilize protein conformations (like in zinc fingers) and mediate signal transduction (by promoting functionally relevant protein conformational changes). However, metal ions can also exert have deleterious effects in living systems by incorporating in non-native binding sites, promoting aberrant protein aggregation or mediating redox cycling with generation of reactive oxygen and nitrogen species. For this reason, the characterization of the roles of metal ions as modulators of protein conformation and stability provides fundamental knowledge on protein folding properties and is instrumental in establishing the molecular basis of disease. In this thesis we have analyzed protein folding processes using model protein systems incorporating covalently bound metal cofactors – iron-sulfur (FeS) proteins – or where metal ion binding is reversible and associated conformational readjustments – the S100 proteins.(...