The impact of small anions on the physical properties of gel-forming mucin has been almost overlooked relative to that of cations. Recently, based on the coincident abnormalities in HCO 3 Ϫ secretion and abnormal mucus formed in the hereditary disease cystic fibrosis (CF), HCO 3 Ϫ was hypothesized to be critical in the formation of normal mucus by virtue of its ability to sequester Ca 2ϩ from condensed mucins being discharged from cells.

pleckstrin homology (PH) domains are an important class of membrane targeting domains that specifically bind target phosphoinositides present at the surface of inner cell membranes. Aside from target lipid headgrouprecognition, the other protein-lipid interactions that occurduring membrane docking are not well defined. Currently, high-resolution structural characterization of protein-membrane interfaces is difficult to achieve while this information is crucial to a physical chemical understanding of reversible protein-membrane binding. In this study, site-directed spin-labeling and electron paramagnetic resonance (EPR) power saturation measurements were employed to determine membrane depth parameters for the PI(3,4,5)P3-specific GRP1-PH domain docked to synthetic bilayer membranes. A library of nitroxide spin-labeled PH domain mutants was generated using site-directed cysteine mutagenesis and disulfide coupling to a methanethiosulfonate spin label (MTSSL). Subsequently, membrane depth parameters were determined for each spin-labeled position in the membrane-docked state. The depth parameters were then used as constraints to model the angular orientation and depth of penetration that describes the membrane docking geometry. Our preliminary structural model identifies the membrane binding surface of GRP1-PH and characterizes its partitioning into the membrane bilayer. Ultimately, the results of this study will aid in understanding the molecular determinants of the electrostatic search mechanism this PH domain uses to rapidly find its rare target lipid on the plasma membrane surface. Supported by NIH GM063235 (J.J.F.). In site-directed spin labeling, structural and dynamic information on a parent macromolecule is obtained by monitoring a covalently linked nitroxide radical using electron paramagnetic resonance (EPR) spectroscopy. Our group have developed a method of attaching nitroxide species, such as 1-oxyl-4-bromo-2,2,5,5-tetramethylpyrroline (R5a), to a specific nucleotide position within a target DNA or RNA sequence. The method relies on site-specific introduction of a phosphorothiate during the solid phase chemical synthesis of nucleic acids, and at each given labeling site the nitroxide is attached to one of two phosphorothioate diastereomers (Rp or Sp) in an approximately 50/50 ratio. We have recently reported that variations in DNA structural and dynamic features at the level of an individual nucleotide can be detected using R5a attached to mixed phosphorothioate diastereomers, in which an observed EPR spectrum is presumably a sum of those obtained from either diastereomer (Popova et al., Biochemistry, 2009, 48, 8540-50). In this work, we report X-band EPR spectra of R5a attached to purified Rp and Sp diastereomers at different sites within a B-form DNA duplex. Results are compared to those obtained with mixed nitroxide diastereomers, and advantages and limitations are discussed regarding the necessity of diastereomer separation when probing DNA local environment. Our work is a further step forward in deve...