Mimicry by asx- and ST-turns of the four main types of beta-turn in proteins

Hydrogen-bonded beta-turns in proteins occur in four categories: type I (the most common), type II, type II', and type I'. Asx-turns resemble beta-turns, in that both have an NH. . .OC hydrogen bond forming a ring of 10 atoms. Serine and threonine side chains also commonly form hydrogen-bonded turns, here called ST-turns. Asx-turns and ST-turns can be categorized into four classes, based on side chain rotamers and the conformation of the central turn residue, which are geometrically equivalent to the four types of beta-turns. We propose asx- and ST-turns be named using the type I, II, I', and II' beta-turn nomenclature. Using this, the frequency of occurrence of both asx- and ST-turns is: type II' > type I > type II > type I', whereas for beta-turns it is type I > type II > type I' > type II'. Almost all type II asx-turns occur as a recently described three residue feature named an asx-nest.     

Design,synthesis and biological evaluation of cyclic angiotensin II analogues with 3,5 side-chain bridges. Role of C-terminal aromatic residue and ring cluster for activity and implications in the drug design of AT1 non-peptide antagonists

The novel amide linked angiotensin II (ANG II) cyclic analogues: gamma, epsilon -cyclo(3, 5)-[Sar(1)-Glu(3)-Lys(5)-Ile(8)] ANG II (I) and gamma, epsilon -cyclo(3, 5)-[Sar(1)-Glu(3)-Lys(5)-Phe(8)] ANG II (II) have been designed, synthesized and bioassayed in anesthetized rabbits in order to unravel structural ring cluster characteristics important for receptor activation. Analogue I with Ile at position 8 was an inhibitor of Angiotensin II while analogue II with Phe at position 8 was found to be an agonist. Similar results were reported for cyclic compounds that have reversed the linking between positions 3 and 5. The overall results show that positions 3 and 5 do not govern the biological activity of the synthetic analogues. It also appears that the aromatic ring cluster (Tyr-His-Phe) in agonist peptides is an essential stereo-electronic feature for Angiotensin II to exert its biological activity. A non-peptide mimetic of ANG II, 1-[2'-[(N-benzyl)tetrazol-5-yl]biphenyl-4-yl]methyl]-2-hydroxymethylbenzimidazole (BZI8) has been designed and synthesized. This molecule is more rigid and much less active than AT(1) non-peptide mimetic losartan probably because it lacks to mimic the orientation of tetrazole and the pharmacophore segments of butyl chain and imidazole ring.     

Structure-function relationship studies of PTH(1-11) analogues containing sterically hindered dipeptide mimetics.

The N-terminal 1-34 fragment of parathyroid hormone (PTH) is fully active in vitro and in vivo and reproduces all biological responses characteristic of the native intact PTH. In order to develop safer and non-parenteral PTH-like bone anabolic agents, we have studied the effect of introducing conformationally constrained dipeptide mimetics into the N-terminal portion of PTH in an effort to generate miniaturized PTH-mimetics. To this end, we have synthesized and conformationally and biologically characterized PTH(1-11) analogues containing 3R-carboxy-6S-amino-7,5-bicyclic thiazolidinlactam (7,5-bTL), a rigidified dipeptide mimetic unit. The wild type sequence of PTH(1-11) is H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-NH(2). The following pseudo-undecapeptides were prepared: [Ala(1), 7,5-bTL(3, 4), Nle(8), Arg(11)]hPTH(1-11)NH(2) (I); [Ala(1), 7,5-bTL(6, 7), Nle(8), Arg(11)]hPTH(1-11)NH(2) (II); [Ala(1), Nle(8), 7,5-bTL(9, 10), Arg(11)]hPTH(1-11)NH(2) (III). In aqueous solution containing 20% TFE, only analogue I exhibited the typical CD pattern of the alpha-helical conformation. NMR experiments and molecular dynamics calculations located the alpha-helical stretch in the sequence Ile(5)-His(9). The dipeptide mimetic unit 7,5-bTL induces a type III beta-turn, occupying the positions i - 1 and i of the turn. Analogue II exhibited an equilibrium between a type I beta-turn and an alpha-helix, and analogue III did not show any ordered structure. Biological tests revealed poor activity for all analogues (EC(50) > 0.1 mM). Apparently, the relative side-chain orientation of Val(2), Ile(5) and Met(8) can be critical for effective analogue-receptor interaction. Considering helicity as an essential property to obtain active PTH agonists, one must decorate the correctly positioned dipeptide mimetic azabicycloalkane scaffold with substitutions corresponding to the displaced amino acids.     

Conformational constraint of mammalian,chicken, and salmon GnRHs,but not GnRH II,enhances binding at mammalian and nonmammalian receptors: evidence for preconfiguration of GnRH II

Mammalian GnRH (mGnRH) is believed to interact with mGnRH type I receptors in a beta-II' turn conformation involving residues 5-8. This conformation can be constrained by substitution of a D-amino acid at position 6 or by a lactam ring involving residues 6 and 7, thereby increasing receptor binding affinity. It has been proposed that this is not the case for non-mGnRH receptors. However, we show that this conformational constraint increases the binding affinity of mammalian, chicken, and salmon GnRH for the chicken and catfish receptors, as well as for the mouse receptor. Therefore, we conclude that the beta-II' turn conformation enhances ligand binding for non-mGnRH as well as mGnRH type I receptors. In contrast, most substitutions of a D-amino acid in position 6 have limited effect on binding affinity for GnRH II. We suggest that this ligand is preconfigured through intramolecular interactions, which accounts for its high binding affinity and total conservation of primary structure over 500 million years of evolution.     

Nanosecond time scale folding dynamics of a pentapeptide in water

Reverse turns, four-residue sections of polypeptides where the chain changes direction by about 180 degrees, are thought to be important protein folding initiation structures. However, the time scale and mechanism for their formation have yet to be determined experimentally. To develop a microscopic picture of the formation of protein folding initiation structures, we have carried out a pair of 2.2-ns molecular dynamics simulations of Tyr-Pro-Gly-Asp-Val, a peptide which is known to form a high population of reverse turns in water. In the first simulation, which was started with the peptide in an ideal type II reverse turn involving the first four residues, the turn unfolded after about 1.4 ns. After about 0.6 ns in the second simulation, which was started with the peptide in a fully extended conformation, the peptide folded into a type II turn which had a transient existence before unfolding. The peptide remained unfolded for another 0.9 ns before folding into a type I turn involving the last four residues. The type I turn lasted for about 0.2 ns before unfolding. Thus, these simulations showed that protein folding initiation structures can form and dissolve on the nanosecond time scale. Furthermore, the atomic-level detail of the simulations allowed us to identify some of the interactions which can stabilize the folded structures. The type II turns were stabilized by either a salt bridge between the terminal groups or a backbone-C-terminal group hydrogen bond, and the type I turns were stabilized by a hydrophobic interaction between the proline and valine-side chains.     

Activation of cyclic AMP-dependent protein kinases I and II by cyclic 3',5'-phosphates of 9-beta-d-ribofuranosylpurine and 2-beta-D-ribofuranosylbenzimidazole

Analogs of cyclic AMP lacking the 6-amino group—9-β-D-ribofuranosylpurine cyclic 3′,5′-phosphate (I)—or the 1- and 3-nitrogens as well as the 6-amino group—1-β-D-ribofuranosylbenzimidazole cyclic 3′,5′-phosphate (II)—were effective activators of both type I (cAKI) and type II (cAKII) isozymes of cAMP-dependent protein kinase. An analog with a pyrimidine ring fused to the benzimidazole ring system of II—3-β-D-ribofuranosyl-8-aminoimidazo[4,5-g]-quinazoline cyclic 3′,5′-phosphate (III)—was equipotent to I or II as an activator of cAKII but only $\text{1}\text{10}$ as potent as I or II as an activator of cAKII. The results show that neither cAKI nor cAKII requires the 6-amino group and that they may have different sensitivities to the effects of alterations in the electron distribution in the pyrimidine ring.     

Complexation of copper(II) by a peptide hybrid of bleomycin and amsacrine. Circular dichroism and electron spin resonance studies

A model incorporating the metal chelating moiety of bleomycin and an anilinoacridine ring able to intercalate in DNA has been synthesized. The copper(II) complex of that molecule has been studied using circular dichroism and electron spin resonance by comparison with bleomycin. The introduction of the anilinoacridine ring involves a modification in the geometry of the complex. A distortion of the square-pyramidal form (type II complex) gives rise to a type I complex in which the metallic atom is drawn out of the plane of the four square-planar ligands and displaced slightly towards the fifth ligand.     

Dynamic apical surface rings in superficial layer cells of koi Cyprinus carpio scale epidermis

This study examined the novel ring‐shaped structures found in the apical surface of individual cells of the scale epidermis of koi Cyprinus carpio. These apical rings are highly dynamic structures with lifetimes ranging from a few to several minutes. While several ring forms were observed, the predominant ring morphology is circular or oval. Two distinct ring forms were identified and designated type I and type II. Type I rings have a well‐defined outer border that encircles the surface microridges. Type II rings are smooth‐surfaced, dinner‐plate‐like structures with membranous folds or compressed microridges in the centre. Type II rings appear less frequently than type I rings. Type I rings form spontaneously, arising from swollen or physically interrupted microridges but without initially perturbing the encircled microridges. After persisting for up to several minutes the ring closes in a centripetal movement to form a circular or irregular‐shaped structure, the terminal disc. The terminal disc eventually disappears, leaving behind a submembranous vesicle‐like structure, the terminal body. Type I rings can undergo multiple cycles of formation and closing. Recycling epidermal apical rings form through centrifugal expansion from the terminal disc followed by apparent contraction back to the disc structure, whereupon the cycle may repeat or cease. The findings demonstrate a novel skin surface structure in fishes and are discussed with respect to communication with the external aqueous environment.     

Centromere Co-orientation at metaphase I in interchange heterozygotes of rye,Secale cereale L.

Three plants of cultivated rye heterozygous for reciprocal translocations have been used to analyze the centromere co-orientation in ring quadrivalent associations at metaphase I. New cytological evidence of the existence of the alternate-II configuration is presented. From the statistical analysis of the data one can deduce that: (i) the four configurations (adjacent-I, adjacent-II, alternate-I and alternate-II) appear with different frequencies, (ii) alternate co-orientations are much more frequent than adjacent ones, (iii) co-orientations of type I are more frequent than those of type II, (iv) among the alternate configurations those of type I are much more frequent than those of type II, (v) among the type I co-orientations the alternate configuration is much more frequent than the adjacent one, (vi) there is no correlation between the percentages of type I and type II classes within the adjacent and alternate co-orientations.     

Kinetic Analysis of Chemokinesis of Paramecium1

Paramecia detect and accumulate in or disperse from some chemicals. Cells do this by changing frequency of turning and speed of swimming. There are at least two mechanisms by which cells respond: one dependent on ability to turn, one dependent on speed modulation. There are also two classes of chemicals: those that require the cells' ability to turn in order to cause accumulation and dispersal (type I), and those that apparently require only speed modulation (type II). Attractants of type I cause qualitatively similar changes in behavior to repellents of type II and the converse; therefore, assays are needed to distinguish between these two classes of chemicals, despite qualitatively similar behavior of some attractants and repellents. We examined two assays of paramecium chemoresponse, T-maze assay and well test, to understand how the T-maze distinguishes between attractants of type I and repellents of type II and why the well test does not.     

Membrane changes under oxidative stress: the impact of oxidized lipids

Studying photosensitized oxidation of unsaturated phospholipids is of importance for understanding the basic processes underlying photodynamic therapy, photoaging and many other biological dysfunctions. In this review we show that the giant unilamellar vesicle, when used as a simplified model of biological membranes, is a powerful tool to investigate how in situ photogenerated oxidative species impact the phospholipid bilayer. The extent of membrane damage can be modulated by choosing a specific photosensitizer (PS) which is activated by light irradiation and can react by either type I and or type II mechanism. We will show that type II PS generates only singlet oxygen which reacts to the phospholipid acyl double bond. The byproduct thus formed is a lipid hydroperoxide which accumulates in the membrane as a function of singlet oxygen production and induces an increase in its area without significantly affecting membrane permeability. The presence of a lipid hydroperoxide can also play an important role in the formation of the lipid domain for mimetic plasma membranes. Lipid hydroperoxides can be also transformed in shortened chain compounds, such as aldehydes and carboxylic acids, in the presence of a PS that reacts via the type I mechanism. The presence of such byproducts may form hydrophilic pores in the membrane for moderate oxidative stress or promote membrane disruption for massive oxidation. Our results provide a new tool to explore membrane response to an oxidative stress and may have implications in biological signaling of redox misbalance.     

Identification ofClostridium histolyticum collagenase hyperreactive sites in type I,II, and III collagens: Lack of correlation with local triple helical stability

The class I and IIClostridium histolyticum collagenases (CHC) have been used to identify hyperreactive sites in rat type I, bovine type II, and human type III collagens. The class I CHC attack both collagens at loci concentrated in the N-terminal half of these collagens starting with the site closest to the N-terminus. The class II CHC initiate collagenolysis by attacking both collagens in the interior to produce a mixture of C-terminal 62,000 and a N-terminal 36,000 fragments. Both fragments are next shortened by removal of a 3000 fragment. These results are very similar to those reported earlier for the hydrolysis of rat type I collagen by these CHC, indicating that the three collagens share many hyperreactive sites. Similar reactions carried out with the respective gelatins show that they are cleaved at many sites at approximately the same rate. Thus, the hyperreactivity of the sites identified must be attributed to their environment in the native collagens. N-terminal sequencing of the fragments produced in these reactions has allowed the identification of 16 cleavage sites in the α1(I), α2(I), α1(II), and α1(III) collagen chains. An analysis of the triple helical stabilities of these cleavage site regions as reflected by their imino acid contents fails to yield a correlation between reactivity and triple helical stability. The existence of these hyperreactive CHC cleavage sites suggests that type I, II, and III collagens contain regions that have specific nontriple helical conformations. The sequence of these sites presented here now makes it possible to investigate these conformations by computational and peptide mimetic techniques.     

Probing the cyclic nucleotide binding sites of cAMP-dependent protein kinases I and II with analogs of adenosine 3',5'-cyclic phosphorothioates

A set of cAMP analogs were synthesized that combined exocyclic sulfur substitutions in the equatorial (Rp) or the axial (Sp) position of the cyclophosphate ring with modifications in the adenine base of cAMP. The potency of these compounds to inhibit the binding of [3H]cAMP to sites A and B from type I (rabbit skeletal muscle) and type II (bovine myocardium) cAMP-dependent protein kinase was determined quantitatively. On the average, the Sp isomers had a 5-fold lower affinity for site A and a 30-fold lower affinity for site B of isozyme I than their cyclophosphate homolog. The mean reduction in affinities for the equivalent sites of isozyme II were 20- and 4-fold, respectively. The Rp isomers showed a decrease in affinity of approximately 400-fold and 200-fold for site A and B, respectively, of isozyme I, against 200-fold and 45-fold for site A and B of isozyme II. The Sp substitutions therefore increased the relative preference for site A of isozyme I and site B of isozyme II. The Rp substitution, on the other hand, increased the relative preference for site B of both isozymes. These data show that the Rp and Sp substitutions are tolerated differently by the two intrachain sites of isozymes I and II. They also support the hypothesis that it is the axial, and not the previously proposed equatorial oxygen that contributes the negative charge for the ionic interaction with an invariant arginine in all four binding sites. In addition, they demonstrate that combined modifications in the adenine ring and the cyclic phosphate ring of cAMP can enhance the ability to discriminate between site A and B of one isozyme as well as to discriminate between isozyme I and II. Since Rp analogs of cAMP are known to inhibit activation of cAMP-dependent protein kinases, the findings of the present study have implications for the synthesis of analogs having a very high selectivity for isozyme I or II.     

Succinate Dehydrogenase and Other Respiratory Pathways in Thylakoid Membranes of Synechocystis sp. Strain PCC 6803: Capacity Comparisons and Physiological Function

Respiration in cyanobacterial thylakoid membranes is interwoven with photosynthetic processes. We have constructed a range of mutants that are impaired in several combinations of respiratory and photosynthetic electron transport complexes and have examined the relative effects on the redox state of the plastoquinone (PQ) pool by using a quinone electrode. Succinate dehydrogenase has a major effect on the PQ redox poise, as mutants lacking this enzyme showed a much more oxidized PQ pool. Mutants lacking type I and II NAD(P)H dehydrogenases also had more oxidized PQ pools. However, in the mutant lacking type I NADPH dehydrogenase, succinate was essentially absent and effective respiratory electron donation to the PQ pool could be established after addition of 1 mM succinate. Therefore, lack of the type I NADPH dehydrogenase had an indirect effect on the PQ pool redox state. The electron donation capacity of succinate dehydrogenase was found to be an order of magnitude larger than that of type I and II NAD(P)H dehydrogenases. The reason for the oxidized PQ pool upon inactivation of type II NADH dehydrogenase may be related to the facts that the NAD pool in the cell is much smaller than that of NADP and that the NAD pool is fully reduced in the mutant without type II NADH dehydrogenase, thus causing regulatory inhibition. The results indicate that succinate dehydrogenase is the main respiratory electron transfer pathway into the PQ pool and that type I and II NAD(P)H dehydrogenases regulate the reduction level of NADP and NAD, which, in turn, affects respiratory electron flow through succinate dehydrogenase.     

The design and synthesis of a potent Angiotensin II cyclic analogue confirms the ring cluster receptor conformation of the hormone Angiotensin II

The novel amide linked Angiotensin II potent cyclic analogue, c-[Sar1,Lys3,Glu5] ANG II 19 has been designed and synthesized in an attempt to test the aromatic ring clustering and the charge relay bioactive conformation we have recently suggested for ANG II. This constrained cyclic analogue was synthesized by connecting the Lys3 amino and Glu5 carboxyl side chain groups, and it was found to be potent in the rat uterus assay and in anesthetized rabbits. The central part of the molecule is fixed covalently in the conformation predicted according to the backbone bend conformational model proposed for Angiotensin II. The obtained results using a combination of 2D NMR, 1D NOE spectroscopy and molecular modeling revealed a similar Tyr4-Ile5-His6 bend, a His6-Pro7 trans configuration and a side chain aromatic ring cluster of the key aminoacids Tyr4, His6, Phe8 for c-[Sar1,Lys3,Glu5] ANG II as it has been found for ANG II (Matsoukas, J. H.; Hondrelis, J.; Keramida, M.; Mavromoustakos, T.; Markriyannis, A.; Yamdagni, R.; Wu, Q.; Moore, G. J. J. Biol. Chem. 1994, 269, 5303). Previous study of the conformational properties of the Angiotensin II type I antagonist [Hser(gamma-OMe)8] ANG II (Matsoukas, J. M.; Agelis, G.; Wahhab, A.; Hondrelis, J.; Panagiotopoulos. D.; Yamdagni, R.; Wu, Q.; Mavromoustakos, T.; Maia, H.; Ganter, R.; Moore, G. J. J. Med. Chem. 1995, 38, 4660) using 1-D NOE spectroscopy coupled with the present study of the same type of lead antagonist Sarilesin revealed that the Tyr4-Ile5-His6 bend, a conformational property found in Angiotensin II is not present in type I antagonists. The obtained results provide an important conformational difference between Angiotensin II agonists and type I antagonists. It appears that our synthetic attempt to further support our proposed model was successful and points out that the charge relay system and aromatic ring cluster are essential stereoelectronic features for Angiotensin II to exert its biological activity.     

Inhibition by enkephalin of medial vestibular nucleus neurons responding to horizontal pendular rotation

Electrophysiological studies were performed to determine whether or not enkephalin modulates the activities of medial vestibular nucleus (MVN) neurons responding to horizontal pendular rotation using alpha-chloralose anesthetized cats. The effects of microiontophoretically applied drugs were examined in type I and type II neurons identified according to responses to horizontal, sinusoidal rotation; type I and type II neurons showed an increase and decrease in firing with rotation ipsilateral to the recording site and vice versa with contralateral rotation, respectively. Iontophoretic application of enkephalin suppressed spike firing induced by rotation of the animals in type I neuron, but not in type II neuron. The spike firing induced by iontophoretically applied glutamate was also inhibited during the application of enkephalin. The inhibition by enkephalin of both rotation- and glutamate-induced firing was antagonized by naloxone which was given simultaneously. These results suggest that enkephalin acts on MVN type I neuron to inhibit transmission from the vestibule, thereby controlling vestibulo-ocular reflex.     

Electron-microscopic study of the development of an equine adenovirus in cultured fetal equine kidney cells

Sequential changes induced by an equine adenovirus in cultured fetal equine kidney cells were studied by electron microscopy. The first morphological change was the appearance of type I inclusions. These inclusions developed to type II inclusions which appeared as ring forms. Type III inclusions were formed within the central part of type II inclusions and finally filled up most of the nuclear space. As the infection proceeded, type IV inclusions which appeared as dense dark-staining spheres were formed at the center of the type III inclusions and also inside the cytoplasm. These dark-staining spheres developed and their center was filed with light-staining material and virus particles which eventually resulted in the formation of type V inclusions. Autoradiography study showed that types I, II, and III were composed of nucleoprotein and type IV was composed of protein.     

Discovery of a new type inhibitor of human glyoxalase I by myricetin-based 4-point pharmacophore

The human glyoxalase I (hGLO I), which is a rate-limiting enzyme in the pathway for detoxification of apoptosis-inducible methylglyoxal (MG), has been expected as an attractive target for the development of new anti-cancer drugs. We have previously identified a natural compound myricetin as a substrate transition-state (Zn²⁺-bound MG-glutathione (GSH) hemithioacetal) mimetic inhibitor of hGLO I. Here, we constructed a hGLO I/inhibitor 4-point pharmacophore based on the binding mode of myricetin to hGLO I. Using this pharmacophore, in silico screening of chemical library was performed by docking study. Consequently, a new type of compound, which has a unique benzothiazole ring with a carboxyl group, named TLSC702, was found to inhibit hGLO I more effectively than S-p-bromobenzylglutathione (BBG), a well-known GSH analog inhibitor. The computational simulation of the binding mode indicates the contribution of Zn²⁺-chelating carboxyl group of TLSC702 to the hGLO I inhibitory activity. This implies an important scaffold-hopping of myricetin to TLSC702. Thus, TLSC702 may be a valuable seed compound for the generation of a new lead of anti-cancer pharmaceuticals targeting hGLO I.     

Mapping adenosine cyclic 3',5'-phosphate binding sites on type I and type II adenosine cyclic 3',5'-phosphate dependent protein kinases using ribose ring and cyclic phosphate ring analogues of adenosine cyclic 3',5'-phosphate

A series of adenosine cyclic 3',5'-phosphate (cAMP) derivatives containing modifications or substitutions in either the 2',3',4', or 5' position or the phosphate were examined for their abilities to activate type I isozymes of cAMP-dependent protein kinase (PK I) from rabbit or porcine skeletal muscle and type II isozymes of cAMP-dependent protein kinase (PK II) from bovine brain and heart. The studies revealed that the activation of both PK I and PK II isozymes requires a 2'-hydroxyl group in the ribo configuration, a 3' oxygen in the ribo configuration, and a charged cyclic phosphate. The two isozymes appeared to differ in those portions of their respective cAMP-binding sites that are adjacent to the 4' position of the ribose ring and the 3' position, 5' position, and phosphate portion of the cyclic phosphate ring.     

Imidazoline pseudodipeptides as mimics of reverse turn structures.

The synthesis of an imidazoline dipeptide mimetic (a 4,5-dihydroimidazole-4-carboxylic acid) is reported that displays an intramolecular hydrogen-bond consistent with a turn conformation in solution.