Because of the serious neurologic consequences of iron deficiency and iron excess in the brain, interest in the iron status of the central nervous system has increased significantly in the past decade. While iron plays an important role in many physiological processes, its accumulation may lead to diseases such as Huntington's, Parkinson's, and Alzheimer's. Therefore, it is important to develop methodologies that can monitor the presence of iron in a selective and sensitive manner. In this paper, we first showed the synthesis and characterization of the iron-binding protein (FBP) from Haemophilus inf luenzae, specific for ferrous ions. Subsequently, we employed this protein in our nanopipette platform and utilized it in functionalized nanoprobes to monitor the presence of ferrous ions. A suite of characterization techniques: absorbance spectroscopy, dynamic light scattering, and small-angle X-ray scattering were used for FBP. The functionalized Fe-nanoprobe calibrated in ferrous chloride enabled detection from 0.05 to 10 μM, and the specificity of the modified iron probe was evaluated by using various metal ion solutions.

This study combines molecular dynamics(MD) simulations with small angle X-ray scattering(SAXS) measurements to investigate range of conformations that can be adopted by a pH/ionic strength(IS) sensitive protein and to quantify its distinct populations in solution.To explore how conformational distribution of proteins may be modified in the environmental niches of biological media,we focus on the periplasmic ferric binding protein A(FbpA) from H.influenzae involved in the mechanism by which bacteria capture iron from higher organisms.We examine iron-binding/release mechanisms of FbpA in varying conditions simulating its biological environment.While we show that these changes fall within the detectable range for SAXS as evidenced by differences observed in the theoretical scattering patterns calculated from the crystal structure models of apo and holo forms,detection of conformational changes due to point mutation D52A and changes in ionic strength(IS) from SAXS scattering profiles have been challenging.Here, statistical analyses with SAXS profiles and results from different techniques were combined. SAXS data complemented by size exclusion chromatography(SEC) and iron-binding assays point to multiple and/or alternative conformations at physiological IS whereas they are well-explained by single crystallographic structures in low IS buffers.By fitting the SAXS data with unique conformations sampled by a series of MD simulations under conditions mimicking the buffers, we quantify the populations of the occupied substates.We also find that the D52A mutant that we predicted by coarse-grained computational modeling to allosterically control the iron binding site in FbpA, responds to the environmental changes in our experiments with conformational selection scenarios that differ from those of the wild type.

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The binding of the Marburg virus protein VP24 to the human Keap1 protein allows the nuclear accumulation of Nrf2, activating cytoprotective antioxidant response pathways during the viral life cycle. We investigate the molecular level details of VP24-Keap1 interactions for both Marburg and Ebola VP24. Our results show that the presence of the cysteine residues in the K-loop region of mVP24 provides strong interfacial interactions with Keap1, including hydrogen bonding and S-HÁÁÁp interactions, facilitating the formation of a stable complex with Marburg VP24. These cysteine residues are not present in eVP24, which does form a stable complex with Keap1. These results provide insights into how the Marburg virus, but not Ebola, is able to activate the antioxidant response pathways through direct interactions with Keap1. 900-PosSolution NMR Structure of the Gtpase Activating (GAP) Domain of Vope, A Vibrio Cholerae T3Ss Effector Protein