Structure of the HERG K+ channel S5P extracellular linker - Role of an amphipathic alpha-helix in C-type inactivation

The HERG K+ channel has very unusual kinetic behavior that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarization as well as in preventing lethal ventricular arrhythmias. Mutagenesis studies have shown that the extracellular peptide linker joining the fifth transmembrane domain to the pore helix is critical for rapid inactivation of the HERG K+ channel. This peptide linker is also considerably longer in HERG K+ channels, 40 amino acids, than in most other voltage-gated K+ channels. In this study we show that a synthetic 42-residue peptide corresponding to this linker region of the HERG K+ channel does not have defined structural elements in aqueous solution; however, it displays two well defined helical regions when in the presence of SDS micelles. The helices correspond to Trp(585)-Ile(593) and Gly(604)-Tyr(611) of the channel. The Trp(585)-Ile(593) helix has distinct hydrophilic and hydrophobic surfaces. The Gly(604)-Tyr(611) helix corresponds to an N-terminal extension of the pore helix. Electrophysiological studies of HERG currents following application of exogenous S5P peptides show that the amphipathic helix in the S5P linker interacts with the pore region of the channel in a voltage-dependent manner.

Microscopic structure of opalescent and nonopalescent pecans

The ultrastructure of pecans was investigated using light microscopy, environmental scanning electron microscopy, scanning electron microscopy, and transmission electron microscopy. Specific methodology for the sample preparation of pecans for electron microscopy investigations was developed. Electron microscopy of the ultrastructure of opalescent (discoloration of the interior) and nonopalescent kernels revealed that cellular damage was occurring in opalescent kernels. The damage was due to cell wall and membrane rupture, which accounted for the release of oil throughout the kernel. This rupture is due to the lower level of calcium in the cell membranes of opalescent pecans, as shown by energy dispersive X-ray spectrometry, making them more susceptible to damage.

Cortex, cognition and the cell: New insights into the pyramidal neuron and prefrontal function

Arguably the most complex conical functions are seated in human cognition, the how and why of which have been debated for centuries by theologians, philosophers and scientists alike. In his best-selling book, An Astonishing Hypothesis: A Scientific Search for the Soul, Francis Crick refined the view that these qualities are determined solely by cortical cells and circuitry. Put simply, cognition is nothing more, or less, than a biological function. Accepting this to be the case, it should be possible to identify the mechanisms that subserve cognitive processing. Since the pioneering studies of Lorent de No and Hebb, and the more recent studies of Fuster, Miller and Goldman-Rakic, to mention but a few, much attention has been focused on the role of persistent neural activity in cognitive processes. Application of modern technologies and modelling techniques has led to new hypotheses about the mechanisms of persistent activity. Here I focus on how regional variations in the pyramidal cell phenotype may determine the complexity of cortical circuitry and, in turn, influence neural activity. Data obtained from thousands of individually injected pyramidal cells in sensory, motor, association and executive cortex reveal marked differences in the numbers of putative excitatory inputs received by these cells. Pyramidal cells in prefrontal cortex have, on average, up to 23 times more dendritic spines than those in the primary visual area. I propose that without these specializations in the structure of pyramidal cells, and the circuits they form, human cognitive processing would not have evolved to its present state. I also present data from both New World and Old World monkeys that show varying degrees of complexity in the pyramidal cell phenotype in their prefrontal cortices, suggesting that cortical circuitry and, thus, cognitive styles are evolving independently in different species.

The mouse sulfate anion transporter gene Sat1 (Slc26a1): Cloning, tissue distribution, gene structure, functional characterization, and transcriptional regulation by thyroid hormone

Sulfate (SO42-) is required for bone/cartilage formation and cellular metabolism. sat-1 is a SO42- anion transporter expressed on basolateral membranes of renal proximal tubules, and is suggested to play an important role in maintaining SO42- homeostasis. As a first step towards studying its tissue-specific expression, hormonal regulation, and in preparation for the generation of knockout mice, we have cloned and characterized the mouse sat-1 cDNA (msat-1), gene (sat1; Slc26a1) and promoter region. msat-1 encodes a 704 amino acid protein (75.4 kDa) with 12 putative transmembrane domains that induce SO42- (also oxalate and chloride) transport in Xenopus oocytes. msat-1 mRNA was expressed in kidney, liver, cecum, calvaria, brain, heart, and skeletal muscle. Two distinct transcripts were expressed in kidney and liver due to alternative utilization of the first intron, corresponding to an internal portion of the 5'-untranslated region. The Sa1 gene (similar to6 kb) consists of 4 exons. Its promoter is similar to52% G+C rich and contains a number of well-characterized cis-acting elements, including sequences resembling hormone responsive elements T3REs and VDREs. We demonstrate that Sat1 promoter driven basal transcription in OK cells was stimulated by tri-iodothyronine. Site-directed mutagenesis identified an imperfect T3RE at -454-bp in the Sat1 promoter to be responsible for this activity. This study represents the first characterization of the structure and regulation of the Sat1 gene encoding a SO42-/chloride/oxalate anion transporter.

The 2.2 A crystal structure of a pocilloporin pigment reveals a nonplanar chromophore conformation

Reef-building corals contain host pigments, termed pocilloporins, that function to regulate the light environment of their resident microalgae by acting as a photoprotectant in excessive sunlight. We have determined the crystal structure of an intensely blue, non-fluorescent pocilloporin to 2.2 Angstrom resolution and a genetically engineered fluorescent variant to 2.4 Angstrom resolution. The pocilloporin chromophore structure adopts a markedly different conformation in comparison with the DsRed chromophore, despite the chromophore sequences (Gin-Tyr-Gly) being identical; the tyrosine ring of the pocilloporin chromophore is noncoplanar and in the trans configuration. Furthermore, the fluorescent variant adopted a noncoplanar chromophore conformation. The data presented here demonstrates that the conformation of the chromophore is highly dependent on its immediate environment.

Structure and metal binding studies of the second copper binding domain of the Menkes ATPase

Biological utilisation of copper requires that the metal, in its ionic forms, be meticulously transported, inserted into enzymes and regulatory proteins, and excess be excreted. To understand the trafficking process, it is crucial that the structures of the proteins involved in the varied processes be resolved. To investigate copper binding to a family of structurally related copper-binding proteins, we have characterised the second Menkes N-terminal domain (MNKr2). The structure, determined using H-1 and N-15 heteronuclear NMR, of the reduced form of MNKr2 has revealed two alpha-helices lying over a single beta-sheet and shows that the binding site, a Cys(X)(2)Cys pair, is located on an exposed loop. H-1-N-15 HSQC experiments demonstrate that binding of Cu(I) causes changes that are localised to conserved residues adjacent to the metal binding site. Residues in this area are important to the delivery of copper by the structurally related Cu(I) chaperones. Complementary site-directed mutagenesis of the adjacent residues has been used to probe the structural roles of conserved residues. (C) 2003 Published by Elsevier Inc.

West nile virus core protein: Tetramer structure and ribbon formation

We have determined the crystal structure of the core (C) protein from the Kunjin subtype of West Nile virus (WNV), closely related to the NY99 strain of WNV, currently a major health threat in the U.S. WNV is a member of the Flaviviridae family of enveloped RNA viruses that contains many important human pathogens. The C protein is associated with the RNA genome and forms the internal core which is surrounded by the envelope in the virion. The C protein structure contains four a. helices and forms dimers that are organized into tetramers. The tetramers form extended filamentous ribbons resembling the stacked alpha helices seen in HEAT protein structures.

Changes to peptide structure, not concentration, contribute to expansion of the lowest avidity cytotoxic T lymphocytes

The efficient in vitro expansion of antigen-specific CD8(+) cytotoxic T lymphocytes (CTL) for use in adoptive immunotherapy represents an important clinical goal. Furthermore, the avidity of expanded CTL populations often correlates closely with clinical outcome. In our study, high-avidity CTL lines could be expanded ex vivo from an antigen-primed animal using low peptide concentration, and intermediate peptide concentrations favored the generation of lower avidity CTL. Further increases in peptide concentration during culture inhibited the expansion of all peptide-specific CD8(+) cells. In contrast, a single amino acid variant peptide efficiently generated functional CTL populations at high or low peptide concentration, which responded to wild-type epitope with the lowest average avidity seen in this study. We propose that for some peptides, the efficient generation of low-avidity CTL responses will be favored by stimulation with altered peptide rather than high concentrations of wild-type epitope. In addition, some variant peptides designed to have improved binding to major histocompatibility complex class I may reduce rather than enhance the functional avidity for the wild-type peptide of ex vivo-expanded CTL. These observations are relevant to in vitro expansion of CTL for immunotherapy and strategies to elicit regulatory or therapeutic immunity to neo-self-antigen when central tolerance has eliminated high-avidity, cognate T cells.

Solution structure of the cyclotide palicourein: Implications for the development of a pharmaceutical framework

The cyclotides are a family of disulfide-rich proteins from plants. They have the characteristic structural features of a circular protein backbone and a knotted arrangement of disulfide bonds. Structural and biochemical studies of the cyclotides suggest that their unique physiological stability can be loaned to bioactive peptide fragments for pharmaceutical and agricultural development. In particular, the cyclotides incorporate a number of solvent-exposed loops that are potentially suitable for epitope grafting applications. Here, we determine the structure of the largest known cyclotide, palicourein, which has an atypical size and composition within one of the surface-exposed loops. The structural data show that an increase in size of a palicourein loop does not perturb the core fold, to which the thermodynamic and chemical stability has been attributed. The cyclotide core fold, thus, can in principle be used as a framework for the development of useful pharmaceutical and agricultural bioactivities.

Structure of the N-terminal domain of Escherichia coli glutamine synthetase adenylyltransferase

We report the crystal structure of the N-terminal domain of Escherichia coli adenylyltransferase that catalyzes the reversible nucleotidylation of glutamine synthetase (GS), a key enzyme in nitrogen assimilation. This domain (AT-N440) catalyzes the deadenylylation and subsequent activation of GS. The structure has been divided into three subdomains, two of which bear some similarity to kanamycin nucleotidyltransferase (KNT). However, the orientation of the two domains in AT-N440 differs from that in KNT. The active site of AT-N440 has been identified on the basis of structural comparisons with KNT, DNA polymerase beta, and polyadenylate polymerase. AT-N440 has a cluster of metal binding residues that are conserved in polbeta-like nucleotidyl transferases. The location of residues conserved in all ATase sequences was found to cluster around the active site. Many of these residues are very likely to play a role in catalysis, substrate binding, or effector binding.

Predicting function from structure: 3D structure studies of the mammalian golgi complex

3D electron tomography studies of the structure of the mammalian Golgi complex have led to four functional predictions (1). The sorting and exit site from the Golgi comprises two or three distinct trans-cisternae (2). The docking of vesicular-tubular clusters at the cis-face and the fragmentation of trans-cisternae are coordinated (3). The mechanisms of transport through, and exit from, the Golgi vary with physiological state, and in different cells and tissues (4). Specialized trans-ER functions in the delivery of ceramide to sphingomyelin synthase in the trans-Golgi membrane, for the regulated sorting via sphingolipid-cholesterol-rich domains. These structure-based predictions can now be tested using a variety of powerful cell and molecular tools.

Hypothalamic paraventricular nucleus neurons regulate medullary catecholamine cell responses to restraint stress

Both physical and psychological stressors recruit catecholamine cells (CA) located in the ventrolateral medulla (VLM) and the nucleus of the solitary tract (NTS). In the case of physical stressors, this effect is initiated by signals that first access the central nervous system at or below the level of the medulla. For psychological stressors, however, CA cell recruitment depends on higher structures within the neuraxis. Indeed, we have recently provided evidence of a pivotal role for the medial amygdala (MeA) in this regard, although such a role must involve a relay, as MeA neurons do not project directly to the medulla. However, some of the MeA neurons that respond to psychological stress have been found to project to the hypothalamic paraventricular nucleus (PVN), a structure that provides significant input to the medulla. To determine whether the PVN might regulate medullary CA cell responses to psychological stress, animals were prepared with unilateral injections of the neurotoxin ibotenic acid into the PVN (Experiment 1), or with unilateral injections of the retrograde tracer wheat germ agglutinin-gold (WGA-Au) into the CA cell columns of the VLM or NTS (Experiment 2). Seven days later, animals were subjected to a psychological stressor (restraint; 15 minutes), and their brains were subsequently processed for Fos plus appropriate cytoplasmic markers (Experiment 1), or Fos plus WGA-Au (Experiment 2). PVN lesions significantly suppressed the stress-related induction of Fos in both VLM and NTS CA cells, whereas tracer deposits in the VLM or NTS retrogradely labeled substantial numbers of PVN cells that were also Fos-positive after stress. Considered in concert with previous results, these data suggest that the activation of medullary CA cells in response to psychological stress may involve a critical input from the PVN. (C) 2004 Wiley-Liss, Inc.

Removal of the N-linked glycan structure from the peanut peroxidase prxPNC2: Influence on protein stability and activity

Lines of transgenic tobacco have been generated that are transformed with either the wild-type peanut peroxidase prxPNC2 cDNA, driven by the CaMV3 5S promoter (designated 35S::prxPNC2-WT) or a mutated PNC2 cDNA in which the asparagine residue (Asn(189)) associated with the point of glycan attachment (Asn(189)) has been replaced with alanine (designated 35S::prxPNC2-M). PCR, using genomic DNA as template, has confirmed the integration of the 35S::prxPNC2-WT and 35::prxPNC2-M constructs into the tobacco genome, and western analysis using anti-PNC2 antibodies has revealed that the prxPNC2-WT protein product (PNC2-WT) accumulates with a molecular mass of 34,670 Da, while the prxPNC2-M protein product (PNC2-M) accumulates with a molecular mass of 32,600 Da. Activity assays have shown that both PNC2-WT and PNC2-M proteins accumulate preferentially in the ionically-bound cell wall fraction, with a significantly higher relative accumulation of the PNC2-WT isoenzyme in the ionically-bound fraction when compared with the PNC2-M isoform. Kinetic analysis of the partially purified PNC2-WT isozyme revealed an affinity constant (apparent K-m) of 11.2 mM for the reductor substrate guaiacol and 1.29 mM for H2O2, while values of 11.9 mM and 1.12 mM were determined for the PNC2-M isozyme. A higher Arrenhius activation energy (E,,) was determined for the PNC2-M isozyme (22.9 kJ mol(-1)), when compared with the PNC2-WT isozyme (17.6 kJ mol(-1)), and enzyme assays have determined that the absence of the glycan influences the thermostability of the PNC2-M isozyme. These results are discussed with respect to the proposed roles of N-linked glycans attached to plant peroxidases. (c) 2005 Elsevier Ltd. All rights reserved.

The role of disulfide bonds in the structure and function of murine epidermal growth factor (mEGF)

A systematic study using solid phase peptide synthesis has been undertaken to examine the role of the disulfide bonds in the structure and function of mEGF. A combination of one, two and three native disulfide pair analogues of an active truncated (4-48) form of mEGF have been synthesised by replacing specific cysteine residues with isosteric alpha-amino-n-butyric acid (Abu). Oxidation of the peptides was performed using either conventional aerobic oxidation at basic pH, in DMSO under acidic conditions or via selective disulfide formation using orthogonal protection of the cysteine pairs. The contribution of individual, or pairs of, disulfide bonds to EGF structure was evaluated by CD and H-1-NMR spectroscopy. The mitogenic activity of each analogue was determined using Balb/c 3T3 mouse fibroblasts. As we have reported previously (Barnham et al. 1998), the disulfide bond between residues 6 and 20 can be removed with significant retention of biological activity (EC50 20-50 nM). The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. We now show that removal of any other disulfide bond, either singly or in pairs, results in a major disruption of the tertiary structure, and a large loss of activity (EC50>900 nM). Remarkably, the linear analogue appears to have greater activity (EC50 580 nM) than most one and two disulfide bond analogues although it does not have a definable tertiary structure.