Gαs protein C‐terminal α‐helix at the interface: does the plasma membrane play a critical role in the Gαs protein functionality?

The heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins, Galphabetagamma) mediate the signalling process of a large number of receptors, known as G protein-coupled receptors. The C-terminal domain of the heterotrimeric G protein alpha-subunit plays a key role in the selective activation of G proteins by their cognate receptors. The interaction of this domain can take place at the end of a cascade including several successive conformational modifications. Galpha(s)(350-394) is the 45-mer peptide corresponding to the C-terminal region of the Galpha(s) subunit. In the crystal structure of the Galpha(s) subunit it encompasses the alpha4/beta6 loop, the beta6 beta-sheet segment and the alpha5 helix region. Following a previous study based on the synthesis, biological activity and conformational analysis of shorter peptides belonging to the same Galpha(s) region, Galpha(s)(350-394) was synthesized and investigated. The present study outlines the central role played by the residues involved in the alpha4/beta6 loop and beta6/alpha5 loops in the stabilization of the C-terminal Galpha(s)alpha-helix. H(2)O/(2)H(2)O exchange experiments, and NMR diffusion experiments show interesting evidence concerning the interaction between the SDS micelles and the polypeptide. These data prompt intriguing speculations on the role of the intracellular environment/cellular membrane interface in the stabilization and functionality of the C-terminal Galpha(s) region.     

A bioactive somatostatin analog without a type II' beta-turn: synthesis and conformational analysis in solution.

A cyclic somatostatin analog [structure: see text] (1) has been synthesized. Biological assays show that this compound has strong binding affinities to somatostatin hsst2 and hsst5 receptor subtypes (5.2 and 1.2 nM, respectively, and modest affinity to hsst4 (41.1 nM)). Our conformational analysis carried out in DMSO-d6 indicates that this compound exists as two structures arising from the trans and cis configurations of the peptide bond between Phe7 and N-alkylated Gly8. However, neither conformer exhibits a type II' beta-turn. This is the first report of a potent bioactive somatostatin analog that does not exhibit a type II' beta-turn in solution. Molecular dynamics simulations (500 ps) carried out at 300 K indicate that the backbone of compound 1 is more flexible than other cyclic somatostatin analogs formed by disulfide bonds.     

Synthesis and secondary structure of oligo(alpha-isobutyl beta,L-aspartate)s

The oligo(beta-peptide)s, hexa(alpha-isobutyl beta,L-aspartate) (Hex-AIBLA) and octa(alpha-isobutyl beta,L-aspartate) (Oct-AIBLA), were synthesized in solution by using standard coupling methods. Powder x-ray diffraction showed that the octamer crystallized in the two helical crystal forms known to exist in the homologous poly(beta-peptide), whereas the hexamer seemed to adopt an extended conformation. Both CD and 1H-NMR spectra of Oct-AIBLA in MeOH revealed the presence of a regularly folded conformation in this solvent, presumably the 3(14) helix. The helix-to-coil transition of Oct-AIBLA was observed to take place upon heating in both MeOH and CHCl3, in the second case associated with a not-well-defined aggregation-disaggregation process. The spectroscopic evidence obtained on the presence of folded structures in Hex-AIBLA were much weaker than for the octamer.     

Structure and hydration of the amylopectin trisaccharide building blocks--Synthesis, NMR, and molecular dynamics

To gain insight into the molecular details and hydration of amylopectin, the five constituting trisaccharides have been chemically synthesized as their methyl alpha-glycosides. All five trisaccharides were subjected to 950 MHz NMR spectroscopy for complete assignment and nanosecond molecular dynamics trajectories were calculated to study the structure and dynamics of the trisaccharides in aqueous solution. Systematic analysis of the simulation data revealed several examples of bridging water molecules playing an important role in the stabilization of specific amylopectin conformations, which was also supported by the experimental NMR data such as interresidue NOE's and heteronuclear scalar couplings between nuclei from neighboring residues. Although alpha-maltotriose, alpha-iso-maltotriose, alpha-panose and alpha-isopanose are relatively well characterized structures, the study also includes one less characterized trisaccharide with the structure alphaGlcp(1-->4)alphaGlcp(1-->6)alphaGlcp. This trisaccharide, tentatively labelled alpha-forkose, is located at the branch point of amylopectin, forking the amylopectin into two strands that align into double-helical segments. The results show that the conformation of alpha-forkose takes a natural bend form which fits well into the structure of the double-helical segment of amylopectin. As the only trisaccharide in this study the structure of alpha-forkose is not significantly influenced by the hydration. In contrast, alpha-isopanose takes a restricted, but rather extended form due to an exceptionally strong localized water density. The two homo-linkage oligomers, alpha-maltotriose and alpha-iso-maltotriose, showed to be the most extended and the most flexible trimers, respectively, providing regular structure for crystalline domains and maximum linker flexibility for amorphous domains.     

Molecular determinants of the aggregation behavior of alpha- and beta-synuclein

Alpha- and beta-synuclein are closely related proteins, the first of which is associated with deposits formed in neurodegenerative conditions such as Parkinson's disease while the second appears to have no relationship to any such disorders. The aggregation behavior of alpha- and beta-synuclein as well as a series of chimeric variants were compared by exploring the structural transitions that occur in the presence of a widely used lipid mimetic, sodium dodecyl sulfate (SDS). We found that the aggregation rates of all these protein variants are significantly enhanced by low concentrations of SDS. In particular, we inserted the 11-residue sequence of mainly hydrophobic residues from the non-amyloid-beta-component (NAC) region of alpha-synuclein into beta-synuclein and show that the fibril formation rate of this chimeric protein is only weakly altered from that of beta-synuclein. These intrinsic propensities to aggregate are rationalized to a very high degree of accuracy by analysis of the sequences in terms of their associated physicochemical properties. The results begin to reveal that the differences in behavior are primarily associated with a delicate balance between the positions of a range of charged and hydrophobic residues rather than the commonly assumed presence or absence of the highly aggregation-prone region of the NAC region of alpha-synuclein. This conclusion provides new insights into the role of alpha-synuclein in disease and into the factors that regulate the balance between solubility and aggregation of a natively unfolded protein.     

Assignment of absolute configuration of 3-hydroxy beta-lactams by the NMR "mix and shake" method

The absolute configurations of several 3-hydroxy beta-lactams were assigned by the NMR "mix and shake" methodology developed by Riguera and co-workers. The results from the NMR study correlated perfectly with the absolute configurations obtained from X-ray crystallographic structure analyses and chiral-phase HPLC data.     

Conformational flexibility and loss of structural rigidity for a model hexapeptide,GRGDTP: 1H‐NMR and molecular dynamics studies

The NMR and molecular dynamics methods are used to study the conformations of a hexapeptide, GRGDTP, which has been shown to be accessible to various types of cell‐adhesion based cellular behaviors such as cell‐to‐matrix interactions, cell differentiation, immunogenicity development, gene expression, angiogenesis, metastasis, sex determination and gamete fusion. ¹H‐NMR results indicate the existence of weak 5→2 hydrogen bonded β‐turn type‐III. Molecular simulation studies using a mixed protocol of distance geometry, constrained minimization, restrained molecular dynamics followed by energy minimization resulted additional conformations that include about 64% of population of inverse γ‐turn (HB, 3→1) and about 35% population of γ‐turn (HB, 4→2). The inter‐proton distances observed in γ‐and inverse γ‐turns are also consistent with the NMR constraints. The variable internal hydrogen bonding due to γ‐turns initiated at Gly 1 and Arg 2 , and its tendency to inter‐convert between γ‐and inverse γ‐turn conformations imply that the peptide is flexible in nature. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 460–471, 2013.     

NMR study and molecular dynamics simulations of optimized beta-hairpin fragments of protein G

The stability and structure of several beta-hairpin peptide variants derived from the C-terminus of the B1 domain of protein G were investigated by a number of experimental and computational techniques. Our analysis shows that the structure and stability of this hairpin can be greatly affected by one or a few simple mutations. For example, removing an unfavorable charge near the N-terminus of the peptide (Glu42 to Gln or Thr) or optimization of the N-terminal charge-charge interactions (Gly41 to Lys) both stabilize the peptide, even in water. Furthermore, a simple replacement of a charged residue in the turn (Asp47 to Ala) changes the beta-turn conformation. Finally, we show that the effects of combining these single mutations are additive, suggesting that independent stabilizing interactions can be isolated and evaluated in a simple model system. Our results indicate that the structure and stability of this beta-hairpin peptide can be modulated in numerous ways and thus contributes toward a more complete understanding of this important model beta-hairpin as well as to the folding and stability of larger peptides and proteins.     

Synthesis and immunosuppressive activity of new cyclolinopeptide A analogs modified with beta-prolines

Immune response suppressors are used in the medical praxis to prevent graft rejection after organ transplantation and in the therapy of some autoimmune diseases. As a continuation of our previous work searching for new, effective suppressors devoid of toxicity, we present the synthesis, conformational analysis, and biological activity of nonapeptides 1-6, analogs of naturally existing immunomodulatory peptide CLA. New CLA analogs were modified with (S)-beta(2)-iso-proline 7 or (S)-beta(3)-homo-proline 8, respectively. The conformational influence of the beta-iso-proline and beta-homo-proline building blocks was analyzed by NMR spectroscopy. Peptides 1-6 exist as a mixture of four isomers due to cis/trans isomerization of the Xxx-Pro peptide bond. The major isomers of peptides 1, 3, and 4 contain all peptide bonds of the trans geometry. The geometry of the proline-proline bond of the second populated isomer of peptides 3 and 4 is cis. The proline-proline peptide bond is cis for the major isomers of peptides 2, 5, and 6. The peptides were tested for their ability to suppress the proliferative response of mouse splenocytes to T- and B-cell mitogens and the secondary humoral immune response to sheep erythrocytes in vitro in parallel with a reference drug-cyclosporine A. The immunoregulatory actions of the peptides depended on the position and content of proline isomers and were, with some exceptions, strongly inhibitory at the highest dose tested (100 microg/ml). In addition, the peptides were practically devoid of toxicity at that dose. In conclusion, the replacement of Pro by beta-Pro may be useful for fine-tuning CLA immunosuppressive potency and undesirable toxicity.     

NMR secondary structure and interactions of recombinant human MOZART1 protein,a component of the gamma‐tubulin complex

Mitotic‐spindle organizing protein associated with a ring of γ‐tubulin 1 (MOZART1) is an 8.5 kDa protein linked to regulation of γ‐tubulin ring complexes (γTuRCs), which are involved in nucleation of microtubules. Despite its small size, MOZART1 represents a challenging target for detailed characterization in vitro. We described herein a protocol for efficient production of recombinant human MOZART1 in Escherichia coli and assessed the properties of the purified protein using a combination of size exclusion chromatography coupled with multiangle light scattering (SEC‐MALS), dynamic light scattering (DLS), and nuclear magnetic resonance (NMR) experiments. MOZART1 forms heterogeneous oligomers in solution. We identified optimal detergent and buffer conditions for recording well resolved NMR experiments allowing nearly full protein assignment and identification of three distinct alpha‐helical structured regions. Finally, using NMR, we showed that MOZART1 interacts with the N‐terminus (residues 1–250) of GCP3 (γ‐tubulin complex protein 3). Our data illustrate the capacity of MOZART1 to form oligomers, promoting multiple contacts with a subset of protein partners in the context of microtubule nucleation.     

NMR assignment of iso-alpha-acids from isomerised extracts of Humulus lupulus L. cones

Iso-alpha-acids are known to contribute to the characteristic bitter taste of beer. Six iso-alpha-acids were isolated from a carbon dioxide extract of the cones of Humulus lupulus L. by centrifugal partition chromatography followed by separation through beta-cyclodextrin binding. This method overcame the low yield issue of most isolation procedures that results from the low stability of these compounds to light and oxygen. Their full identification was performed using one- and two-dimensional NMR spectrometry, including (1)H- and (13)C-NMR, (1)H-(1)H COSY, HMQC and HMBC and electrospray ionisation mass spectrometry. The results confirmed the structures of the isolated compounds as trans-isocohumulone, cis-isocohumulone, trans-isohumulone, cis-isohumulone, trans-isoadhumulone and cis-isoadhumulone. Epimers can be easily distinguished by observing the chemical shift differences of the H-5, H-1', H-2' and C-5 signals and the different splitting pattern of H-5 and H-2'.     

Steroidal constituents of rice (Rryza sativa) hulls with algicidal and herbicidal activity against blue-green algae and duckweed

Two new compounds, 14-methyl stigmast-9(11)-en-3alpha-ol-3beta-D-glucopyranoside (1) and cholest-11-en-3beta, 6beta, 7alpha, 22beta-tetraol-24-one-3beta-palmitoleate (2), along with the known compound beta-sitosteryl-3beta-D-glucopyranosyl-6'-linoleiate (3), were isolated from the methanolic extract of rice (Oryza sativa) hulls. The structures of the two new compounds were elucidated using one- and two-dimensional NMR in combination with IR, EI/MS, FAB/MS, HR-EI/MS and HR-FAB/MS. In bioassays with blue-green algae, Microcystis aeruginosa UTEX 2388 and duckweed, Lemna paucicostata Hegelm 381, the efficacy of bioactivity of the two new compounds linearly increased as the concentration increased from 0.3 to 300 IgM. Compared with momilactone A, compounds 1 and 2 showed similar and higher inhibitory activities against the growth of M. aeruginosa at a concentration of 300 microM. However, compound 2 was similar to momilactone A in inhibiting L. paucicostata growth at a concentration of 300 microM. As a result, compound 2 appears to have a strong potential for the environmentally friendly control of weed and algae that are harmful to water-logged rice.     

Selection and structural analysis of de novo proteins from an α3β3 genetic library

The construction of novel functional proteins has been a key area of protein engineering. However, there are few reports of functional proteins constructed from artificial scaffolds. Here, we have constructed a genetic library encoding α3β3 de novo proteins to generate novel scaffolds in smaller size using a binary combination of simplified hydrophobic and hydrophilic amino acid sets. To screen for folded de novo proteins, we used a GFP‐based screening system and successfully obtained the proteins from the colonies emitting the very bright fluorescence as a similar intensity of GFP. Proteins isolated from the very bright colonies (vTAJ) and bright colonies (wTAJ) were analyzed by circular dichroism (CD), 8‐anilino‐1‐naphthalenesulfonate (ANS) binding assay, and analytical size‐exclusion chromatography (SEC). CD studies revealed that vTAJ and wTAJ proteins had both α‐helix and β‐sheet structures with thermal stabilities. Moreover, the selected proteins demonstrated a variety of association states existing as monomer, dimer, and oligomer formation. The SEC and ANS binding assays revealed that vTAJ proteins tend to be a characteristic of the folded protein, but not in a molten‐globule state. A vTAJ protein, vTAJ13, which has a packed globular structure and exists as a monomer, was further analyzed by nuclear magnetic resonance. NOE connectivities between backbone signals of vTAJ13 suggested that the protein contains three α‐helices and three β‐strands as intended by its design. Thus, it would appear that artificially generated α3β3 de novo proteins isolated from very bright colonies using the GFP fusion system exhibit excellent properties similar to folded proteins and would be available as artificial scaffolds to generate functional proteins with catalytic and ligand binding properties.     

Evaluation of chiral discrimination of highly negatively charged enantiomers by quaternary ammonium beta-cyclodextrin using 1H NMR

The positively charged quaternary ammonium cyclodextrin, QA-beta-CD, was previously used as a chiral selector to achieve baseline resolution of two of the dianionic enantiomers of disodium 3-(p-isothiocyanatophenoxy)-3-(p-isothiocyanatophenyl)propane-1,2-disulfate by capillary electrophoresis. The basis of the chiral discrimination between QA-beta-CD and the enantiomers was investigated by (1)H NMR spectroscopy. COSY and NOESY spectra were used to infer the role that molecular interactions and the stereocenters have upon association of QA-beta-CD with the enantiomers. A parallel two-step complexation model is used to rationalize the NMR and the chiral discrimination observed during separation of the enantiomers.     

19F NMR studies of ��-synuclein-membrane interactions

α‐Synuclein function is thought to be related to its membrane binding ability. Solution NMR studies have identified several α‐synuclein‐membrane interaction modes in small unilamellar vesicles (SUVs), but how membrane properties affect binding remains unclear. Here, we use ¹⁹F NMR to study α‐synuclein‐membrane interactions by using 3‐fluoro‐L‐tyrosine (3FY) and trifluoromethyl‐L‐phenylalanine (tfmF) labeled proteins. Our results indicate that the affinity is affected by both the head group and the acyl chain of the SUV. Negatively charged head groups have higher affinity, but different head groups with the same charge also affect binding. We show that the saturation of the acyl chain has a dramatic effect on the α‐synuclein‐membrane interactions by studying lipids with the same head group but different chains. Taken together, the data show that α‐synuclein's N‐terminal region is the most important determinate of SUV binding, but its C‐terminal region also modulates the interactions. Our data support the existence of multiple tight phospholipid‐binding modes, a result incompatible with the model that α‐synuclein lies solely on the membrane surface.     

Folding of peptides characterized by c3Val,a highly constrained analogue of valine

Using a combined chemical/chiral chromatographic approach we synthesized an N-protected derivative of (R)-c(3)Val, a severely conformationally restricted C(alpha)-tetrasubstituted alpha-amino acid characterized by a C(beta,beta)-dimethylated cyclopropane system. A set of terminally protected derivatives and model peptides (to the heptamer level), containing one or two (R)-c(3)Val residues in combination with either Aib or Gly residues, was prepared by solution methods. A detailed solution and crystal-state conformational investigation, based on Fourier transform infrared (FTIR) absorption, (1)H-NMR, and x-ray diffraction techniques, performed in comparison with a similar study on related derivatives and peptides rich in (alphaMe)Val, the prototype of C(alpha)-tetrasubstituted alpha-amino acids of this subfamily, allowed us to conclude the following: (a) c(3)Val is a good beta-bend and helix former, although less efficient than (alphaMe)Val. (b) The relationship between alpha-carbon chirality and screw sense of the folded structure formed is the same as that of (alphaMe)Val, i.e., the (R)-enantiomer has a strong left-handed bias. (c) c(3)Val seems more prone than (alphaMe)Val to fold into a gamma-bend conformation. The conformational propensities of C(beta,beta)-disubstituted Ac(3)c residues are also discussed in comparison with those of the parent cyclopropane residue.     

Role of tumor necrosis factor-alpha and interferon-gamma in Helicobacter pylori infection

Immune responses to Helicobacter pylori infection play important roles in gastroduodenal diseases. The contributions of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) to the induction of gastric inflammation and to the protection from H. pylori infection were investigated using TNF-alpha geneknockout (TNF-alpha(-/-)) mice and IFN-gamma gene-knockout (IFN-gamma(-/-)) mice. We first examined the colonizing ability of H. pylori strain CPY2052 in the stomach of C57BL/6 wild-type and knockout mice. The number of H. pylori colonized in the stomach of IFN-gamma(-/-) and TNF-alpha(-/-) mice was higher than that of wild-type mice. These findings suggest that TNF-alpha and IFN-gamma may play a protective role in H. pylori infection. Furthermore, we examined the contribution of TNF-alpha and IFN-gamma to gastric inflammation. The CPY2052-infected TNF-alpha(-/-) mice showed a moderate infiltration of mononuclear cells in the lamina propria and erosions in the gastric epithelium as did wild-type mice, whereas the CPY2052-infected IFN-gamma(-/-) mice showed no inflammatory findings even 6 months after infection. These results demonstrate that IFN-gamma may play an important role in gastric inflammation induced by H. pylori infection, whereas TNF-alpha may not participate in the development of inflammatory response.     

Chemical constituents of rice (Oryza sativa) hulls and their herbicidal activity against duckweed (Lemna paucicostata Hegelm 381)

Four new compounds, stigmastanol-3beta-p-glyceroxydihydrocoumaroate (1), stigmastanol-3beta-p-butanoxydihydrocoumaroate (2), lanast-7,9(11)-dien-3alpha,15alpha-diol-3alpha-D-glucofuranoside (3) and 1-phenyl-2-hydroxy-3,7-dimethyl-11-aldehydic-tetradecane-2-beta-D-glucopyranoside (4), along with several known compounds were isolated from the methanol extract of hulls of Oryza sativa. The new structures were established by one- and two-dimensional NMR and in combination with IR, EI/MS, FAB/MS and HR-FAB/ MS. Compound (3) strongly inhibited the growth of duckweed (Lemna paucicostata Hegelm 381), whilst compounds (2) and (4) exhibited weak inhibition.     

1H-NMR study of the inclusion processes for alpha- and gamma-cyclodextrin with fenbufen

1H-NMR spectra of aqueous solutions of fenbufen and two cyclodextrins (alpha- or gamma-cyclodextrin, respectively) mixtures confirm the formation of an inclusion complex if gamma-cyclodextrin is used, whereas in the case of alpha-cyclodextrin no inclusion complex was obtained. The stoichiometry of the fenbufen/gamma-cyclodextrin complex is of the [2:1] type. The geometry of this supramolecular architecture was established through MM+ molecular mechanics calculations.     

Utilization of (18‐Crown‐6)‐2,3,11,12‐tetracarboxylic Acid as a Chiral NMR Solvating Agent for Diamines and β‐Amino Acids

The compound (18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid was evaluated as a chiral nuclear magnetic resonance (NMR) solvating agent for a series of diamines and bicyclic β‐amino acids. The amine must be protonated for strong association with the crown ether. An advantage of (18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid over many other crown ethers is that it undergoes a neutralization reaction with neutral amines to form the protonated species needed for binding. Twelve primary diamines in neutral and protonated forms were evaluated. Diamines with aryl and aliphatic groups were examined. Some are atropisomers with equivalent amine groups. Others have two nonequivalent amine groups. Association equilibria for these systems are complex, given the potential formation of 2:1, 1:1, and 1:2 crown‐amine complexes and given the various charged species in solution for mixtures of the crown ether with the neutral amine. The crown ether produced enantiomeric differentiation in the ¹H NMR spectrum of one or more resonances for every diamine substrate. Also, a series of five bicyclic β‐amino acids were examined and (18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid caused enantiomeric differentiation in the ¹H NMR spectrum of three or more resonances of each compound. Chirality 27:708–715, 2015. © 2015 Wiley Periodicals, Inc.