To investigate the determinants of protein order and disorder, three primary and one derivative database of intrinsically disordered proteins were compiled. The segments in each primary database were characterized by one of the following: X-ray crystallography, nuclear magnetic resonance (NMR), or circular dichroism (CD). The derivative database was based on homology. The three primary disordered databases have a combined total of 157 proteins or segments of length à "à vuà ' à rvqrà uvyrà urà qrvhvrà qhhihrà phvà $&! proteins from 32 families with 52,688 putatively disordered residues. For the four disordered databases, the amino acid compositions were compared with those from a database of ordered structure. Relative to the ordered protein, the intrinsically disordered segments in all four databases were significantly depleted in W, C, F, I, Y, V, L and N, significantly enriched in A, R, G, Q, S, P, E and K, and inconsistently different in H, M, T, and D, suggesting that the first set be called order-promoting and the second set disorder-promoting. Also, 265 amino acid properties were ranked by their ability to discriminate order and disorder and then pruned to remove the most highly correlated pairs. The 10 highest-ranking properties after pruning consisted of 2 residue contact scales, 4 hydrophobicity scales, 3 scales associated vuà urrà hqà rà yhvà phyrà à Vvtà urrà à rvrà sà phvà sà urà " primary databases suggests that disorder in all 3 databases is very similar, but with those characterized by NMR and CD being the most similar, those by CD and X-ray being next, and those by NMR and X-ray being the least similar.

stimulates breathing via medullary and spinal pathways. J Appl Physiol 98: 1387-1395, 2005. First published November 19, 2004 doi:10.1152/japplphysiol.00914.2004.-A central neuronal network that regulates respiration may include hypothalamic neurons that produce orexin, a peptide that influences sleep and arousal. In these experiments, we investigated 1) projections of orexin-containing neurons to the pre-Bötzinger region of the rostral ventrolateral medulla that regulates rhythmic breathing and to phrenic motoneurons that innervate the diaphragm; 2) the presence of orexin A receptors in the pre-Bötzinger region and in phrenic motoneurons; and 3) physiological effects of orexin administered into the pre-Bötzinger region and phrenic nuclei at the C3-C4 levels. We found orexin-containing fibers within the pre-Bötzinger complex. However, only 0.5% of orexincontaining neurons projected to the pre-Bötzinger region, whereas 2.9% of orexin-containing neurons innervated the phrenic nucleus. Neurons of the pre-Bötzinger region and phrenic nucleus stained for orexin receptors, and activation of orexin receptors by microperfusion of orexin in either site produced a dose-dependent, significant (P Ͻ 0.05) increase in diaphragm electromyographic activity. These data indicate that orexin regulates respiratory activity and may have a role in the pathophysiology of sleep-related respiratory disorders.hypothalamus; pre-Bötzinger region; phrenic motor neurons; orexin-1 receptors; sleep apnea BREATHING IS AN ACTIVE NEURALLY controlled process that is regulated by neural mechanisms that adjust respiratory-related drive to the behavioral state and to the metabolic demands of an organism. Hypothalamic neurons are part of this controlling system and play an important role in the regulation of breathing rate and depth (14,26).Neurons in the lateral hypothalamus synthesize orexin A and orexin B, also called hypocretin-1 (hcrt-1) and hypocretin-2 (hcrt-2). These peptide neurotransmitters are processed from a common precursor, prepro-orexin, encoded by a gene localized to human chromosome 17q2. Orexin-containing neurons affect autonomic, neuroendocrine, and sleep-wakefulness neuroregulatory systems that in turn could potentially influence breathing (9,13,16,19).The orexins stimulate target cells via two orexin G proteincoupled receptors, orexin R1 and orexin R2 (40). It has been proposed that orexin-containing neurons promote wakefulness by excitation of cholinergic neurons in the basal forebrain, which release acetylcholine and thereby contribute to the cortical activation of wakefulness. However, the causality of these associations remains to be determined because wakefulness is often accompanied by behavioral activation. Suppression of rapid eye movement sleep occurs through an inhibition of the cholinergic neurons in the laterodorsal tegmental and pedunculopontine nuclei (49).In the central nervous system, orexin-containing neurons innervate multiple sites, including cell groups in the brain stem and spinal cord that are involved in t...

We present what we believe to be the first documented example of an inducement of distinctly different secondary structure types onto agonists and antagonists selective for the same G-coupled protein receptor using the same membrane-model matrix wherein the induced structures are consistent with those suggested to be biologically active by extensive analogue studies and conventional binding assays. 1H NMR chemical shift assignments for the mammalian NK1 receptor-selective agonists alpha-neurokinin (NKA) and beta-neurokinin (NKB) as well as the mammalian NK1 receptor-selective antagonists [d-Pro2,d-Phe7,d-Trp9]SP and [d-Arg1, d-Pro2,d-Phe7,d-His9]SP have been determined at 600 MHz in sodium dodecyl sulfate (SDS) micelles. The SDS micelle system simulates the membrane-interface environment the peptide experiences when in the proximity of the membrane-embedded receptor, allowing for conformational studies that are a rough approximation of in vivo conditions. Two-dimensional NMR techniques were used to assign proton resonances, and interproton distances were estimated from the observed nuclear Overhauser effects (NOEs). The experimental distances were used as constraints in a molecular dynamics and simulated annealing protocol using the modeling package DISCOVER to generate three-dimensional structures of the two agonists and two antagonists when present in a membrane-model environment to determine possible prebinding ligand conformations. It was determined that (1) NKA is helical from residues 6 to 9, with an extended N-terminus; (2) NKB is helical from residues 4 to 10, with an extended N-terminus; (3) [d-Pro2,d-Phe7,d-Trp9]SP has poorly defined helical properties in the midregion and a beta-turn structure in the C-terminus (residues 6-9); and (4) [d-Arg1,d-Pro2, d-Phe7,d-His9]SP has a helical structure in the midregion (residues 4-6) and a well-defined beta-turn structure in the C-terminus (residues 6-10). Attempts have been made to correlate the observed conformational differences between the agonists and antagonists to their binding potencies and biological activity.

To investigate the molecular mechanisms involved in paramyxovirus-induced cell fusion, the function and structure of a peptide with a 20-amino-acid sequence from the leucine zipper region (heptad repeat region 2) of the Newcastle disease virus fusion protein (F) were characterized. A peptide with the sequence ALDKLEESNSKLDKVNVKLT (amino acids 478-497 of the F protein) was found to inhibit syncytia formation after virus infection and after transfection of Cos cells with the HN (hemagglutinin-neuraminidase) and F protein cDNAs. Using an F protein gene that requires addition of exogenous trypsin for cleavage, it was shown that the peptide exerted its inhibitory effect prior to cleavage. The three-dimensional conformation of the peptide in aqueous solution was determined through the use of NMR and molecular modeling. Results showed that the peptide formed a helix with properties between an alpha-helix and a 3(10)-helix and that leucine residues aligned along one face of the helix. Side chain salt bridges and hydrogen bonds likely contributed to the stability of the peptide secondary structure. Analysis of the aqueous solution conformation of the peptide suggested mechanisms for specificity of interaction with the intact F protein.

The linear nonapeptide hormone bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) is involved, either directly or indirectly, in a wide variety of physiological processes, particularly pain and hyperanalgesia. Additional evidence suggests that bradykinin also plays a major role in inflammatory response, asthma, sepsis, and symptoms associated with the rhinoviral infection. It has long been speculated that a beta-turn at the C-terminus of bradykinin plays a major role in the biological activity of the neuropeptide. The beta-turn forming potential of bradykinin in three vastly different local chemical environments, DMSO, 9:1 dioxane/water, and in the presence of 7.4 mM lyso phosphatidylcholine micelles, was investigated using two-dimensional homonuclear nmr experiments coupled with simulated annealing calculations. The results of these investigations show that in all three systems residues 6-9 of the C-terminus adopt very similar beta-turn like structures. These results suggest that the beta-turn at the C-terminus of bradykinin is an important secondary structural feature for receptor recognition and binding.

The interactions between the positively charged neuropeptides substance P (SP), bradykinin (BK), and zwitterionic Met-enkephalin (ME) neuropeptides, and negatively charged SDS and zwitterionic lysophosphatidylcholine (LPC) membrane model systems, have been investigated using one- and two-dimensional nmr experiments. Proton longitudinal relaxation studies were used to characterize these interactions as intrinsic or extrinsic. An extrinsic interaction are similar to those observed for extrinsic membrane proteins. An intrinsic interaction are similar to those observed for intrinsic membrane proteins, and would require that the hydrophobic residues penetrate or insert into the hydrophobic core of the membrane. The interactions between both SP and BK and SDS, based on nmr results, may be characterized as intrinsic, and the interaction between ME and SDS may be characterized as extrinsic. Two-dimensional nuclear Overhauser enhancement spectroscopy experiments proved the insertion of the phenylalanine residues on both SP and BK into the hydrophobic core of SDS micelles. The interaction between SP and BK with LPC based on nmr results are characterized as extrinsic, with the interaction between ME and SDS characterized as weakly intrinsic.

The pursuit for more sensitive NMR probes culminated with development of the cryogenic cooled NMR probe. A key factor for the sensitivity is the overall resistance of RF circuitry and sample. Lowering the coil temperature to approximately 25 K and the use of superconducting coil material has greatly reduced the resistance contribution of the hardware. However, the resistance of a salty sample remains the same and evolves as the major factor determining the signal-to-noise ratio. Several approaches have been proposed to reduce the resistance contribution of the sample. These range from encapsulating proteins in a water cavity formed by reverse micelles in low viscosity fluids to the optimal selection of low mobility, low conductivity buffer ions. Here we demonstrate that changing the sample diameter has a pronounced effect on the sample resistance and this results in dramatic improvements of the signal-to-noise ratio and shorter pi/2 pulses. We determined these parameters for common 5 mm NMR tubes under different experimental conditions and compared them to the 2, 3 and 4 mm tubes, in addition, 5mm Shigemi tubes were included since these are widely used. We demonstrate benefits and applicability of studying NMR samples with up to 4M salt concentrations in cryogenic probes. Under high salt conditions, best results in terms of short pi/2 pulses and high signal-to-noise ratios are obtained using 2 or 3mm NMR tubes, especially when limited sample is available. The 4 mm tube is preferred when sample amounts are abundant at intermediate salt conditions.

The retinal pigment epithelium (RPE) is a highly specialized CNS tissue that plays crucial roles in retinal homeostasis. Age-related morphological changes in the RPE have been associated with retinal degenerative disorders; our understanding of the underlying molecular mechanisms, however, remains incomplete. Here we report on a key role of Klotho (Kl), an aging-suppressor gene, in retinal health and RPE physiology. Kl Ϫ / Ϫ mice show RPE and photoreceptor degeneration, reduced pigment synthesis in the RPE, and impaired phagocytosis of the outer segment of the photoreceptors. Klotho protein (KL) is expressed in primary cultured human RPE, and regulates pigment synthesis by increasing the expression of MITF (microphthalmia transcription factor) and TYR (tyrosinase), two pivotal genes in melanogenesis. Importantly, KL increases phagocytosis in cultured RPE by inducing gene expression of MERTK/AXL/TYRO3. These effects of KL are mediated through cAMP-PKA-dependent phosphorylation of transcription factor CREB. In cultured human RPE, KL increases the L-3,4-dihydroxyphenylalanine synthesis and inhibits vascular endothelial growth factor (VEGF) secretion from basal membrane by inhibiting IGF-1 signaling and VEGF receptor 2 phosphorylation. KL also regulates the expression of stress-related genes in RPE, lowers the production of reactive oxygen species, and thereby, protects RPE from oxidative stress. Together, our results demonstrate a critical function for KL in mouse retinal health in vivo, and a protective role toward human RPE cells in vitro. We conclude that KL is an important regulator of RPE homeostasis, and propose that an age-dependent decline of KL expression may contribute to RPE degeneration and retinal pathology.