A synchrotron-based hydroxyl radical footprinting analysis of amyloid fibrils and prefibrillar intermediates with residue-specific resolution.

Structural models of the fibrils formed by the 40-residue amyloid-ß (Aß40) peptide in Alzheimer's disease typically consist of linear polypeptide segments, oriented approximately perpendicular to the long axis of the fibril, and joined together as parallel in-register ß-sheets to form filaments. However, various models differ in the number of filaments that run the length of a fibril, and in the topological arrangement of these filaments. In addition to questions about the structure of Aß40 monomers in fibrils, there are important unanswered questions about their structure in prefibrillar intermediates, which are of interest because they may represent the most neurotoxic form of Aß40. To assess different models of fibril structure and to gain insight into the structure of prefibrillar intermediates, the relative solvent accessibility of amino acid residue side chains in fibrillar and prefibrillar Aß40 preparations was characterized in solution by hydroxyl radical footprinting and structural mass spectrometry. A key to the application of this technology was the development of hydroxyl radical reactivity measures for individual side chains of Aß40. Combined with mass-per-length measurements performed by dark-field electron microscopy, the results of this study are consistent with the core filament structure represented by two- and three-filament solid state nuclear magnetic resonance-based models of the Aß40 fibril (such as 2LMN , 2LMO , 2LMP , and 2LMQ ), with minor refinements, but they are inconsistent with the more recently proposed 2M4J model. The results also demonstrate that individual Aß40 fibrils exhibit structural heterogeneity or polymorphism, where regions of two-filament structure alternate with regions of three-filament structure. The footprinting approach utilized in this study will be valuable for characterizing various fibrillar and nonfibrillar forms of the Aß peptide.

Pefluorocarbon inhibition of bubble induced Ca2+ transients in an in vitro model of vascular gas embolism.

Endothelial injury resulting from deleterious interaction of gas microbubbles occurs in many surgical procedures and other medical interventions. The symptoms of vascular air embolism (VAE), while serious, are often difficult to detect, and there are essentially no pharmaceutical preventative or post-event treatments currently available. Perfluorocarbons (PFCs), however, have shown particular promise as a therapeutic option in reducing endothelial injury both in- and ex-vivo. Recently, we demonstrated the effectiveness of Oxycyte, a third-generation PFC formulated in a phosphotidylcholine emulsion, using an in vitro model of VAE developed in our laboratory. This apparatus allows live cell imaging concurrent with precise manipulation of physiologically sized microbubbles so that they may be brought into individual contact with human umbilical vein endothelial cells dye-loaded with the Ca(2+) sensitive Fluo-4. Herein, we expand use of this fluorescence microscopy-based cell culture model. Specifically, we examined the concentration dependence of Oxycyte in reducing both the amplitude and frequency of large intracellular Ca(2+) currents that are both a hallmark of bubble contact and a quantifiable indication that abnormal intracellular signaling has been triggered. We measured dose dependence curves and fit the resultant data using a modified Black and Leff operational model of agonism. The half maximal inhibitory concentrations of Oxycyte for (i) inhibition of occurrence and (ii) amplitude reduction were 229 ± 49 µM and 226 ± 167 µM, respectively. This investigation shows the preferential gas/liquid interface occupancy of the PFC component of Oxycyte over that of mechanosensing glycocalyx components and validates Oxycyte's specific surfactant mechanism of action. Further, no lethality was observed for any concentration of this bioinert PFC, as it acts as a competitive allosteric inhibitor of syndecan activation to ameliorate cell response to bubble contact.

Mechanotransductional basis of endothelial cell response to intravascular bubbles.

Vascular air embolism resulting from too rapid decompression is a well-known risk in deep-sea diving, aviation and space travel. It is also a common complication during surgery or other medical procedures when air or other endogenously administered gas is entrained in the circulation. Preventive and post-event treatment options are extremely limited for this dangerous condition, and none of them address the poorly understood pathophysiology of endothelial response to intravascular bubble presence. Using a novel apparatus allowing precise manipulation of microbubbles in real time fluorescence microscopy studies, we directly measure human umbilical vein endothelial cell responses to bubble contact. Strong intracellular calcium transients requiring extracellular calcium are observed upon cell-bubble interaction. The transient is eliminated both by the presence of the stretch activated channel inhibitor, gadolinium, and the transient receptor potential vanilliod family inhibitor, ruthenium red. No bubble induced calcium upsurge occurs if the cells are pretreated with an inhibitor of actin polymerization, cytochalasin-D. This study explores the biomechanical mechanisms at play in bubble interfacial interactions with endothelial surface layer (ESL) macromolecules, reassessing cell response after selective digestion of glycocalyx glycosoaminoglycans, hyaluran (HA) and heparin sulfate (HS). HA digestion causes reduction of cell-bubble adherence and a more rapid induction of calcium influx after contact. HS depletion significantly decreases calcium transient amplitudes, as does pharmacologically induced sydencan ectodomain shedding. The surfactant perfluorocarbon Oxycyte abolishes any bubble induced calcium transient, presumably through direct competition with ESL macromolecules for interfacial occupancy, thus attenuating the interactions that trigger potentially deleterious biochemical pathways.

Air bubble contact with endothelial cells in vitro induces calcium influx and IP3-dependent release of calcium stores.

Gas embolism is a serious complication of decompression events and clinical procedures, but the mechanism of resulting injury remains unclear. Previous work has demonstrated that contact between air microbubbles and endothelial cells causes a rapid intracellular calcium transient and can lead to cell death. Here we examined the mechanism responsible for the calcium rise. Single air microbubbles (50-150 µm), trapped at the tip of a micropipette, were micromanipulated into contact with individual human umbilical vein endothelial cells (HUVECs) loaded with Fluo-4 (a fluorescent calcium indicator). Changes in intracellular calcium were then recorded via epifluorescence microscopy. First, we confirmed that HUVECs rapidly respond to air bubble contact with a calcium transient. Next, we examined the involvement of extracellular calcium influx by conducting experiments in low calcium buffer, which markedly attenuated the response, or by pretreating cells with stretch-activated channel blockers (gadolinium chloride or ruthenium red), which abolished the response. Finally, we tested the role of intracellular calcium release by pretreating cells with an inositol 1,4,5-trisphosphate (IP3) receptor blocker (xestospongin C) or phospholipase C inhibitor (neomycin sulfate), which eliminated the response in 64% and 67% of cases, respectively. Collectively, our results lead us to conclude that air bubble contact with endothelial cells causes an influx of calcium through a stretch-activated channel, such as a transient receptor potential vanilloid family member, triggering the release of calcium from intracellular stores via the IP3 pathway.

Structural determination of estrogen-related receptor gamma in the presence of phenol derivative compounds.

We screened the ligand-binding domain of estrogen-related receptor (ERR) gamma in ThermoFluor, in an effort to develop chemical tools and decipher the biology of this orphan nuclear receptor. Several ligands were found to stabilize thermodynamically the protein. Amongst the ligands were bisphenol A (BPA) and 4-chloro-3-methyl phenol (ClCH3Ph). These ligands were further characterized and found to be competitive for 4-hydroxytamoxifen (4OHT) binding, a known reported antagonist ligand for ERRgamma, but functionally they did not enhance or disrupt affinity of the receptor for co-activator peptides. The preservation of the constitutive active conformation of the receptor in the presence of these two ligands was confirmed upon the determination of the co-crystal structures. The structures of BPA and ClCH3Ph were determined to a resolution of 2.1 and 2.3A, respectively, and the antagonist 4OHT was refined to 2.5A resolution. In the presence of BPA and ClCH3Ph the receptor maintained the transcriptional active conformation as reported previously for the apo-protein in the presence of a co-activator peptide fragment. In addition the ERRgamma-BPA structure identifies an interaction between the phenolic-OH and the side chain of N346. The preservation of the constitutive active conformation of the receptor in the presence of the small phenol compounds suggest that the biological activity of the receptor might be regulated by a natural occurring ligand.

Thermal stability landscape for Klenow DNA polymerase as a function of pH and salt concentration.

The thermal denaturation of Klenow DNA polymerase has been characterized over a wide variety of solution conditions to obtain a relative stability landscape for the protein. Measurements were conducted utilizing a miniaturized fluorescence assay that measures Tm based on the increase in the fluorescence of 1,8-anilinonaphthalene sulfonate (ANS) when the protein denatures. The melting temperature (Tm) for Klenow increases as the salt concentration is increased and as the pH is decreased. Klenow's Tm spans a range of over 20 degrees C, from 40 to 62 degrees C, depending upon the solution conditions. The landscape reconciles and extends previously measured Tm values for Klenow. Salt effects on the stability of Klenow show strong cation dependence overlaid onto a more typical Hofmeister anion type dependence. Cationic stabilization of proteins has been far less frequently documented than anionic stabilization. The monovalent cations tested stabilize Klenow with the following hierarchy: NH4+>Na+>Li+>K+. Of the divalent cations tested: Mg+2 and Mn+2 significantly stabilize the protein, while Ni+2 dramatically destabilizes the protein. Stability measurements performed in combined Mg+2 plus Na+ salts suggest that the stabilizing effects of these monovalent and divalent cations are synergistic. The cationic stabilization of Klenow can be well explained by a model postulating dampening of repulsion within surface anionic patches on the protein.

Inhibition of carbonic anhydrase-II by sulfamate and sulfamide groups: an investigation involving direct thermodynamic binding measurements.

This paper examines the relative effectiveness of bioisosteric sulfamate and sulfamide derivatives for inhibition of human carbonic anhydrase-II (CA-II) by using a direct binding assay based on the ThermoFluor method (Matulis et al. Biochemistry 2005, 44, 5258). Compounds 1-10, which represent five cognate sulfamate/sulfamide pairs, were studied by ThermoFluor to obtain binding affinities (K(a) values). The corresponding dissociation constants, K(d), provide an independent measure of CA-II activity relative to commonly used K(i) values from enzyme kinetics studies. There was a sizable difference in potency between the sulfamates and sulfamides, with the sulfamides being much less potent, by factors ranging from 25 (7/8) to 1,200 (3/4). These results are consistent with our recent report that sulfamides tend to be much weaker inhibitors of CA-II than their corresponding sulfamates (Maryanoff et al. J. Med. Chem. 2005, 48, 1941). Additionally, for arylsulfamides 10-12 the K(d) values determined by ThermoFluor and the K(i) values determined from enzyme kinetics are consistent. It appears that the sulfamide group is less suitable than the sulfamate group for obtaining potent inhibition of CA-II.

Asymmetric distribution of cooperativity in the binding cascade of normal human hemoglobin. 1. Cooperative and noncooperative oxygen binding in Zn-substituted hemoglobin.

The complete binding cascade of human hemoglobin consists of eight partially ligated intermediates and 16 binding constants. Each intermediate binding constant can be evaluated via dimer-tetramer assembly when ligand configurations within the tetramer are fixed through the use of hemesite analogs. The Zn/Fe analog, in which the nonbinding Zn2+ heme substitutes for deoxy Fe2+ heme, also permits direct measurement of O2 binding to the remaining Fe2+ hemesites within the symmetrically ligated Hb tetramers. Measurement of O2 binding over a range of Zn/Fe Hb concentrations to both alpha-subunits (species 23) or to both beta-subunits (species 24) shows noncooperative binding and incomplete saturation of the available Fe2+ hemesites. In contrast, the asymmetrically ligated Zn/FeO2 species 21, in which both oxygens are bound to one of the dimers within the tetramer, exhibits positive cooperativity and >90% ligation under atmospheric conditions. These properties are confirmed in the present study by measurement of the rate constant for tetramer dissociation to free dimer. The binding constants thus derived for these partially ligated intermediates are consistent with the stoichiometric constants measured for native hemoglobin by standard O2 binding techniques, providing additional evidence that Zn2+-heme substitution provides an excellent deoxy hemoglobin analog. There is no evidence that Zn-substitution stabilizes a low-affinity form of the tetramer, as previously suggested. These characterizations demonstrate distinct, nonadditive physical properties of the doubly ligated tetrameric species, yielding an asymmetric distribution of cooperativity within the cascade of O2 binding by human hemoglobin.