Bioisosterism: a hydrogen bonding study of methanesulfonanilides

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Towards understanding flavin reactivity : a structural study of cholesterol oxidase

Flavoenzymes catalyze a wide variety of biochemical reactions and are commonly observed as electron transport proteins. The redox reactive portion of the enzymes is the isoalloxazine ring system of the flavin cofactor. It is known that the protein environment modulates the redox potential of the flavin, for example, "tuning" its redox potential to favor either a one-electron transfer (electron transfer proteins) or a two-electron transfer (oxidation reactions). This thesis presents an in depth structural study of the flavoenzyme, cholesterol oxidase (EC 1.1.3.6) from Streptomyces sp. SA-COO (SCOA) a multifunctional enzyme that oxidizes and isomerizes 3-beta-hydroxysteroids. This work was pursued in order to further our understanding of the mechanisms through which the protein interacts with the isoalloxazine system and modulates reactivity. Previous kinetic experiments have identified an active site asparagine (N485) and a histidine residue (H447) both of which are critical to the oxidative activity of the enzyme. On an atomic scale the role of the asparagine residue was unknown. Using mutagensis and crystallographic techniques we have characterized this novel N-H ··· pi protein-flavin interaction. SCOA crystals diffract to sub-atomic resolution providing us with a unique view of the protein bound isoalloxazine system. These atomic resolution maps have revealed unexpected structural features that were not previously apparent in the 1.5 A resolution of SCOA. For example, a second narrow pathway leading directly to the isoalloxazine system was discovered, which has provided a more complete mechanistic understanding of the reactions catalyzed by SCOA. Five atomic resolution structures of SCOA at varying pH values are reported. Differences among these structures provide insight into the affect of pH on protein structure and have revealed structural differences resulting from an inadvertent reduction of the cofactor. For example, these str

Molecular features of the copper binding sites in the octarepeat domain of the prion protein.

Recent evidence suggests that the prion protein (PrP) is a copper binding protein. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds Cu2+ in vivo. In a previous study using peptide design, EPR, and CD spectroscopy, we showed that the HGGGW segment within each octarepeat comprises the fundamental Cu2+ binding unit [Aronoff-Spencer et al. (2000) Biochemistry 40, 13760-13771]. Here we present the first atomic resolution view of the copper binding site within an octarepeat. The crystal structure of HGGGW in a complex with Cu2+ reveals equatorial coordination by the histidine imidazole, two deprotonated glycine amides, and a glycine carbonyl, along with an axial water bridging to the Trp indole. Companion S-band EPR, X-band ESEEM, and HYSCORE experiments performed on a library of 15N-labeled peptides indicate that the structure of the copper binding site in HGGGW and PHGGGWGQ in solution is consistent with that of the crystal structure. Moreover, EPR performed on PrP(23-28, 57-91) and an 15N-labeled analogue demonstrates that the identified structure is maintained in the full PrP octarepeat domain. It has been shown that copper stimulates PrP endocytosis. The identified Gly-Cu linkage is unstable below pH approximately 6.5 and thus suggests a pH-dependent molecular mechanism by which PrP detects Cu2+ in the extracellular matrix or releases PrP-bound Cu2+ within the endosome. The structure also reveals an unusual complementary interaction between copper-structured HGGGW units that may facilitate molecular recognition between prion proteins, thereby suggesting a mechanism for transmembrane signaling and perhaps conversion to the pathogenic form

Improving biophysical properties of a bispecific antibody scaffold to aid developability: quality by molecular design

While the concept of Quality-by-Design is addressed at the upstream and downstream process development stages, we questioned whether there are advantages to addressing the issues of biologics quality early in the design of the molecule based on fundamental biophysical characterization, and thereby reduce complexities in the product development stages. Although limited number of bispecific therapeutics are in clinic, these developments have been plagued with difficulty in producing materials of sufficient quality and quantity for both preclinical and clinical studies. The engineered heterodimeric Fc is an industry-wide favorite scaffold for the design of bispecific protein therapeutics because of its structural, and potentially pharmacokinetic, similarity to the natural antibody. Development of molecules based on this concept, however, is challenged by the presence of potential homodimer contamination and stability loss relative to the natural Fc. We engineered a heterodimeric Fc with high heterodimeric specificity that also retains natural Fclike biophysical properties, and demonstrate here that use of engineered Fc domains that mirror the natural system translates into an efficient and robust upstream stable cell line selection process as a first step toward a more developable therapeutic. \ua9 2013 Landes Bioscience.Peer reviewed: YesNRC publication: Ye