Peptide design: influence of a guest Aib-Pro segment on the stereochemistry of an oligo-val sequence--solution conformations and crystal structure of Boc-(Val)2-Aib-Pro-(Val)3-OMe

The peptide Boc-Val-Val-Aib-Pro-Val-Val-Val-OMe has been synthesized to investigate the effect of introduction of a strong beta-turn promoting guest segment into an oligopeptide with a tendency to form extended structures. 1H-nmr studies in solution using analysis of NH group solvent accessibility and nuclear Overhauser effects suggest an appreciable solvent dependence of conformations. In chloroform a 3(10)-helical structure is favored, while in dimethylsulfoxide an Aib-Pro beta-turn with extended arms on either side is suggested. In the crystal, the backbone forms a somewhat distorted 3(10)-helix despite the presence of a Pro residue in the middle. Among the four possible intrahelical hydrogen bonds three are of the 4----1 type and one 5----1. Head-to-tail NH...O = C hydrogen bonds link the helical molecules into continuous columns. The space group is P2(1)2(1)2(1) a = 11.320(2), b = 19.889(3), and c = 21.247(3) A.     

Functional coupling of human beta 3-adrenoreceptors to the KvLQT1/MinK potassium channel

The slow component of the delayed rectifier potassium current (IKs) plays an important role during repolarization in the human heart. Life-threatening arrhythmias can be triggered by sympathetic stimulation, presumably acting on IKs. The ion channel responsible for the IKs current is made of two proteins, the KvLQT1 protein and the MinK protein. In this study, we investigated the effects of adrenergic stimulation on the KvLQT1/MinK channel by coexpressing KvLQT1/MinK channels with the human beta(3)-adrenoreceptor subunit heterologously in Xenopus oocytes. Western blot experiments revealed that beta(3)-adrenoreceptor proteins appear in the cell membrane of Xenopus oocytes, when the corresponding cRNA was injected. In electrophysiological measurements we found that stimulation with the beta-adrenergic agonist isoproterenol increased the current amplitude of the beta(3)/KvLQT1/MinK complex up to 237% with an ED(50) of 8 nm, a value similar to that found on IKs in guinea pig cardiomyocytes. When oocytes with beta(3)/KvLQT1/MinK were preincubated with cholera toxin (2 microg/ml), an activator of G(S) proteins, the basal current amplitude of the beta(3)/KvLQT1/MinK complex was increased 3.1-fold, and the current amplitude increase by isoproterenol was drastically reduced, indicating that the signal transduction cascade was mediated via G(s) proteins. The knowledge about functional coupling of the human beta(3)-adrenoreceptor to KvLQT1/MinK channels reveals interesting aspects about the genesis and therapy of arrhythmias.     

Design and synthesis of AB(3)-type (A = 1,3,5-benzenetricarbonyl unit; B = Glu diOME or Glu(7) octa OMe) peptide dendrimers: crystal structure of the first generation

The first generation molecule of glutamic acid-based dendrons on a 1, 3,5-benzenetricarbonyl core leads to a cylindrical assembly as demonstrated by single crystal x-ray diffraction. The benzene pi-pi stack (A) is stabilized by vertical NH...O===C hydrogen bonding with each subunit participating in three intermolecular hydrogen bonds related by three-fold rotation symmetry.     

Broadly distributed nucleophilic reactivity of proteins coordinated with specific ligand binding activity

Covalent nucleophile-electrophile interactions have been established to be important for recognition of substrates by several enzymes. Here, we employed an electrophilic amidino phosphonate ester (EP1) to study the nucleophilic reactivity of the following proteins: albumin, soluble epidermal growth factor receptor (sEGFR), soluble CD4 (sCD4), calmodulin, casein, alpha-lactalbumin, ovalbumin, soybean trypsin inhibitor and HIV-1 gp120. Except for soybean trypsin inhibitor and alpha-lactalbumin, these proteins formed adducts with EP1 that were not dissociated by denaturing treatments. Despite their negligible proteolytic activity, gp120, sEGFR and albumin reacted irreversibly with EP1 at rates comparable to the serine protease trypsin. The neutral counterpart of EP1 reacted marginally with the proteins, indicating the requirement for a positive charge close to the electrophilic group. Prior heating resulted in altered rates of formation of the EP1-protein adducts accompanied by discrete changes in the fluorescence emission spectra of the proteins, suggesting that the three-dimensional protein structure governs the nucleophilic reactivity. sCD4 and vasoactive intestinal peptide (VIP) containing phosphonate groups (EP3 and EP4, respectively) reacted with their cognate high-affinity binding proteins gp120 and calmodulin, respectively, at rates exceeding the corresponding reactions with EP1. Reduced formation of EP3-gp120 adducts and EP4-calmodulin adducts in the presence of sCD4 and VIP devoid of the phosphonate groups was evident, suggesting that the nucleophilic reactivity is expressed in coordination with non-covalent recognition of peptide determinants. These observations suggest the potential of EPs for specific and covalent targeting of proteins, and raise the possibility of nucleophile-electrophile pairing as a novel mechanism stabilizing protein-protein complexes.     

Effects of hydrogen-bond deletion on peptide helices: structural characterization of depsipeptides containing lactic acid

The insertion of alpha-hydroxy acids into peptide chains provides a convenient means for investigating the effects of hydrogen bond deletion on polypeptide secondary structures. The crystal structures of three oligopeptides containing L-lactic acid (Lac) residue have been determined. Peptide 1, Boc-Val-Ala-Leu-Aib-Val-Lac-Leu-Aib-Val-Ala-Leu-OMe (Boc: tert-butyloxycarbonyl; Aib: alpha- aminoisobutyric acid; OMe: methyl ester), and peptide 2, Boc-Val-Ala-Leu-Aib-Val-Lac-Leu-Aib-Val-Leu-OMe, adopt completely helical conformations in the crystalline state with the Lac(6) residue comfortably accommodated in the center of a helix. The distance between the O atoms of Leu(3) CO group and the Lac(6) O (ester) in both the structures is 3.1-3.3 A. The NMR and CD studies of peptide 1 and its all-amide analogue 4, Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe, provide firm evidence for a continuous helical conformation in solution in both the cases. In a 14-residue peptide 3, Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Val-Ala-Leu-Aib-Val-Lac-Leu-OMe, residues Val(1)-Leu(10) adopt a helical conformation. Aib(11) is the site of chiral reversal resulting in helix termination by formation of a Schellman motif. Residues 12-14 adopt nonhelical conformations. The loss of the hydrogen bond near the C-terminus appears to facilitate the chiral reversal at Aib(11). Published 2001 John Wiley & Sons, Inc. Biopolymers 59: 276-289, 2001     

Nitration of the pollen allergen bet v 1.0101 enhances the presentation of bet v 1-derived peptides by HLA-DR on human dendritic cells

Nitration of pollen derived allergens can occur by NO(2) and ozone in polluted air and it has already been shown that nitrated major birch (Betula verrucosa) pollen allergen Bet v 1.0101 (Bet v 1) exhibits an increased potency to trigger an immune response. However, the mechanisms by which nitration might contribute to the induction of allergy are still unknown. In this study, we assessed the effect of chemically induced nitration of Bet v 1 on the generation of HLA-DR associated peptides. Human dendritic cells were loaded with unmodified Bet v 1 or nitrated Bet v 1, and the naturally processed HLA-DR associated peptides were subsequently identified by liquid chromatography-mass spectrometry. Nitration of Bet v 1 resulted in enhanced presentation of allergen-derived HLA-DR-associated peptides. Both the copy number of Bet v 1 derived peptides as well as the number of nested clusters was increased. Our study shows that nitration of Bet v 1 alters antigen processing and presentation via HLA-DR, by enhancing both the quality and the quantity of the Bet v 1-specific peptide repertoire. These findings indicate that air pollution can contribute to allergic diseases and might also shed light on the analogous events concerning the nitration of self-proteins.     

An asymmetric conformation of 1,3,5-benzene tricarbonyl [Aib4OMe]3.

Molecules of 1,3,5-benzene tricarbonyl [Aib(4)OMe](3) do not possess any internal symmetry, neither exact nor approximate, in the crystalline state. The Aib(4)OMe moieties each form a 3(10)-helix with an appropriate pair of hydrogen bonds but the sense of rotation is right-handed for two of the helices and left-handed for the third one. The helices are not evenly positioned around the benzene ring, and their helix axes are inclined toward one side of the plane of the benzene ring, giving the molecule the shape of a shallow bowl with an irregular periphery. The molecules are largely surrounded by water and dimethyl sulfoxide (DMSO) solvent molecules that form hydrogen bonds with the CO and NH moieties that protrude from the surfaces of the peptide molecule. The space group is Cc with a = 23.618(4) A, b = 19.708(6) A, c = 17.939(7) A and beta = 100.09(3) degrees.     

Dual regulation of renal Kir7.1 potassium channels by protein Kinase A and protein Kinase C

The renal inward rectifier potassium channel Kir7.1 has been proposed to be functionally important for tubular K⁺ recycling and secretion. This study investigated the regulation of Kir7.1 by PKA and PKC.Cloned human Kir7.1 channels were expressed heterologously in Xenopus oocytes. After pharmacological PKC activation, Kir7.1 currents were strongly inhibited. Co-application of PKC inhibitors attenuated this effect. Inactivation of PKC consensus sites also strongly attenuated the effect with a single site (²⁰¹S) being essential for almost the total PKC sensitivity. In contrast, PKA activation induced an increase of Kir7.1 currents. This effect was absent in mutant Kir7.1 channels lacking PKA consensus site ²⁸⁷S.In summary, this study demonstrates the dual regulation of Kir7.1 channel function by PKA and PKC. Structurally, these regulations depend on two key residues in the C-terminal channel domain (^Ser201 for PKC and ^Ser287 for PKA).     

Deletion of protein kinase A phosphorylation sites in the HERG potassium channel inhibits activation shift by protein kinase A.

We investigated the role of protein kinase A (PKA) in regulation of the human ether-a-go-go-related gene (HERG) potassium channel activation. HERG clones with single mutations destroying one of four consensus PKA phosphorylation sites (S283A, S890A, T895A, S1137A), as well as one clone carrying all mutations with no PKA phosphorylation sites (HERG 4M) were constructed. These clones were expressed heterologously in Xenopus oocytes, and HERG potassium currents were measured with the two microelectrode voltage clamp technique. Application of the cAMP-specific phosphodiesterase (PDE IV) inhibitor Ro-20-1724 (100 microM), which results in an increased cAMP level and PKA stimulation, induced a reduction of HERG wild type outward currents by 19.1% due to a shift in the activation curve of 12.4 mV. When 100 microM Ro-20-1724 was applied to the HERG 4M channel, missing all PKA sites, there was no significant shift in the activation curve, and the current amplitude was not reduced. Furthermore, the adenylate cyclase activator forskolin that leads to PKA activation (400 microM, 60 min), shifted HERG wild type channel activation by 14.1 mV and reduced currents by 39.9%, whereas HERG 4M channels showed only a small shift of 4.3 mV and a weaker current reduction of 22.3%. We conclude that PKA regulates HERG channel activation, and direct phosphorylation of the HERG channel protein has a functional role that may be important in regulation of cardiac repolarization.     

Effects of End Group and Aggregation on Helix Conformation: Crystal Structure of Ac-(Aib-Val-Ala-Leu)2-Aib- OMe

The role of end groups in determining stereochemistry and packing in hydrophobic helical peptides has been investigated using an α-aminosobutyric acid (Aib) containing model nonapeptide sequence. In contrast to the Boc-analogue, Ac-(Aib-Val-Ala-Leu)₂-Aib-OMe crystallizes with two independent molecules in a triclinic cell. The cell parameters are: space group P1, a=10.100(2)Å, b=15.194(4) Å, c=19.948(5) Å, α=63.12(2)°, β=88.03(2)°, γ=88.61(2)°, Z=2, R=7.96% for 5140 data where |F_o|>3σ(F). The two independent molecules alternate in infinite columns formed by head-to-tail hydrogen bonding. The helices in the two independent molecules are quite similar to each other but one molecule is rotated ≈?123° about its helix axis with respect to the other. All the helical columns pack parallel to each other in the crystal. Replacement of the bulky Boc group does not lead to any major changes in conformation. Packing characteristics are also similar to those observed for similar helical peptides.