Peptide purification using the chemoselective reaction between N-(methoxy)glycine and isothiocyanato-functionalized resin.

An efficient peptide purification strategy is established, comprising the selective reaction of an N-terminal N-(methoxy)glycine residue of the peptide and isothiocyanato-functionalized resins, and subsequent Edman degradation. These reactions take place in acidic media; in particular, the Edman degradation proceeds smoothly in media containing more than 50% trifluoroacetic acid (v/v). These acidic conditions offer increased solubility, making them advantageous for the purification of hydrophobic and aggregation-prone peptides. The effectiveness of this method, together with scope and limitations, is demonstrated using model peptides and the practical purification of the loop region of the human dopamine D2 receptor long isoform (residues 240-272). Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Site-specific labeling of synthetic peptide using the chemoselective reaction between N-methoxyamino acid and isothiocyanate.

Site-specific labeling of synthetic peptides carrying N-methoxyglycine (MeOGly) by isothiocyanate is demonstrated. A nonapeptide having MeOGly at its N-terminus was synthesized by the solid-phase method and reacted with phenylisothiocyanate under various conditions. In acidic solution, the reaction specifically gave a peptide having phenylthiourea structure at its N-terminus, leaving side chain amino group intact. The synthetic human ß-defensin-2 carrying MeOGly at its N-terminus or the side chain amino group of Lys(10) reacted with phenylisothiocyanate or fluorescein isothiocyanate also at the N-methoxyamino group under the same conditions, demonstrating that this method is generally useful for the site-specific labeling of linear synthetic peptides as well as disulfide-containing peptides.

Coupling of transmembrane helix orientation to membrane release of the juxtamembrane region in FGFR3.

Activation of the protein tyrosine kinase receptors requires the coupling of ligand binding to a change in both the proximity and orientation of the single transmembrane (TM) helices of receptor monomers to allow transphosphorylation of the receptor kinase domain. We make use of peptides corresponding to the TM and juxtamembrane (JM) regions of the fibroblast growth factor receptor 3 to assess how mutations in the TM region (G380R and A391E), which lead to receptor activation, influence the orientation of the TM domain and interactions of the intracellular JM sequence with the membrane surface. On the basis of fluorescence and Fourier transform infrared spectroscopy, we find that both activating mutations change the TM helix tilt angle relative to the membrane normal and release the JM region from the membrane. These results suggest a general mechanism regarding how the TM-JM region functionally bridges the extracellular and intracellular regions for these receptors.

Differential effects of the peptides Stomagen, EPF1 and EPF2 on activation of MAP kinase MPK6 and the SPCH protein level.

The positioning and density of leaf stomata are regulated by three secretory peptides, EPIDERMAL PATTERNING FACTOR 1 (EPF1), EPF2 and stomagen. Several lines of published evidence have suggested a regulatory pathway as follows. EPF1 and EPF2 are perceived by receptor complexes consisting of a receptor-like protein, TOO MANY MOUTHS (TMM), and receptor kinases, ERECTA (ER), ERECTA-LIKE (ERL) 1 and ERL2. These receptors activate a mitogen-activated protein (MAP) kinase module. MAP kinases phosphorylate and destabilize the transcription factor SPEECHLESS (SPCH), resulting in a decrease in the number of stomatal lineage cells. Stomagen acts antagonistically to EPF1 and EPF2. However, there is no direct evidence that EPF1 and EPF2 activate or that stomagen inactivates the MAP kinase cascade, through which they might regulate the SPCH level. Experimental modulation of these peptides in Arabidopsis thaliana would change the number of stomatal lineage cells in developing leaves, which in turn would change the expression of SPCH, making the interpretation difficult. Here we reconstructed this signaling pathway in differentiated leaf cells of Nicotiana benthamiana to examine signaling without the confounding effect of cell type change. We show that EPF1 and EPF2 are able to activate the MAP kinase MPK6, and that both EPF1 and EPF2 are able to decrease the SPCH level, whereas stomagen is able to increase it. Our data also suggest that EPF1 can be recognized by TMM together with any ER family receptor kinase, whereas EPF2 can be recognized by TMM together with ERL1 or ERL2, but not by TMM together with ER.

Effect of amino acid distribution of amphipathic helical peptide derived from human apolipoprotein A-I on membrane curvature sensing.

Amphipathic helix, which senses membrane curvature, is of growing interest. Here we explore the effect of amino acid distribution of amphipathic helical peptide derived from the C-terminal region (residues 220-241) of human apolipoprotein (apo) A-I on membrane curvature sensing. This peptide preferred a curved membrane in a manner similar to full-length apoA-I, although its model peptide did not sense membrane curvature. Substitution of several residues both on the polar and non-polar faces of the amphipathic helix had no significant effect on sensing, suggestive of the elaborate molecular architecture in the C-terminal helical region of apoA-I to exert lipid efflux function.

Transmembrane helix orientation influences membrane binding of the intracellular juxtamembrane domain in Neu receptor peptides.

The transmembrane (TM) and juxtamembrane (JM) regions of the ErbB family receptor tyrosine kinases connect the extracellular ligand-binding domain to the intracellular kinase domain. Evidence for the role of these regions in the mechanism of receptor dimerization and activation is provided by TM-JM peptides corresponding to the Neu (or rat ErbB2) receptor. Solid-state NMR and fluorescence spectroscopy show that there are tight interactions of the JM sequence with negatively charged lipids, including phosphatidylinositol 4,5-bisphosphate, in TM-JM peptides corresponding to the wild-type receptor sequence. We observe a release of the JM sequence from the negatively charged membrane surface using peptides containing an activating V664E mutation within the TM domain or in peptides engineered to form TM helix dimers with Val664 in the interface. These results provide the basis of a mechanism for coupling ligand binding to kinase activation in the full-length receptor.

Hinge and chromoshadow of HP1α participate in recognition of K9 methylated histone H3 in nucleosomes.

The majority of the genome in eukaryotes is packaged into transcriptionally inactive chromatin. Heterochromatin protein 1 (HP1) is a major player in the establishment and maintenance of heterochromatin. HP1 specifically recognizes a methylated lysine residue at position 9 in histone H3 through its N-terminal chromo domain (CD). To elucidate the binding properties of HP1α to nucleosomes in vitro, we reconstituted nucleosomes containing histone H3 trimethylated at lysine 9. HP1α exhibited high-affinity binding to nucleosomes containing methylated histone H3 in a nucleosome core-number-dependent manner. The hinge region (HR) connecting the CD and C-terminal chromoshadow domain (CSD), and the CSD contributed to the selective binding of HP1α to histone H3 with trimethylated lysine 9 through weak DNA binding and by suppressing the DNA binding, respectively. We propose that not only the specific recognition of lysine 9 methylation of histone H3 by the CD but also the HR and the CSD cooperatively contribute to the selective binding of HP1α to histone H3 lysine 9 methylated nucleosomes.

Epimerization in peptide thioester condensation.

Peptide segment couplings are now widely utilized in protein chemical synthesis. One of the key structures for the strategy is the peptide thioester. Peptide thioester condensation, in which a C-terminal peptide thioester is selectively activated by silver ions then condensed with an amino component, is a powerful tool. But the amino acid adjacent to the thioester is at risk of epimerization. During the preparation of peptide thioesters by the Boc solid-phase method, no substantial epimerization of the C-terminal amino acid was detected. Epimerization was, however, observed during a thioester-thiol exchange reaction and segment condensation in DMSO in the presence of a base. In contrast, thioester-thiol exchange reactions in aqueous solutions gave no epimerization. The epimerization during segment condensation was significantly suppressed with a less polar solvent that is applicable to segments in thioester peptide condensation. These results were applied to a longer peptide thioester condensation. The epimer content of the coupling product of 89 residues was reduced from 27% to 6% in a condensation between segments of 45 and 44 residues for the thioester and the amino component, respectively.

Synthetic disulfide-bridged cyclic peptides mimic the anti-angiogenic actions of chondromodulin-I.

Chondromodulin-I (ChM-I) is a 25-kDa glycoprotein in cartilage matrix that inhibits angiogenesis. It contains two distinctive structural domains: the N-terminal third of the molecule is a hydrophilic domain that contains O-linked and N-linked oligosaccharide chains, and the C-terminal two-thirds is a hydrophobic domain that contains all of the cysteine residues. In the present study, we have attempted to further uncover the structural requirements for ChM-I to exert anti-angiogenic activity by monitoring its inhibition of the vascular endothelial growth factor (VEGF)-A-induced migration of HUVEC in vitro. Site-directed mutagenesis experiments revealed that the cyclic structure formed by the disulfide bridge between Cys(83) and Cys(99) in human ChM-I is indispensable for its anti-angiogenic function. Moreover, the C-terminal hydrophobic tail (from Trp(111) to Val(120) ) was found to play an important role in ensuring the effectiveness of ChM-I activity on HUVEC. A synthetic cyclic peptide corresponding to the ChM-I region between Ile(82) to Arg(100) also inhibited the migration of HUVEC, while replacing the Cys(83) and Cys(99) residues in this peptide with Ser completely negated this inhibitory activity. An additional synthetic cyclic peptide harboring the hydrophobic C-terminal tail of ChM-I clearly mimicked the inhibitory action of this protein on the migration of HUVEC and successfully inhibited tumor angiogenesis and growth in a xenograft mouse model of human chondrosarcoma.

Charge state-selective separation of peptides by reversible modification of amino groups and strong cation-exchange chromatography: evaluation in proteomic studies using peptide-centric database searches.

Here we describe an integrated approach for the selective separation of peptides from complex mixtures using strong cation-exchange chromatography. The procedure exploits the charge differences produced by reversible modification of primary amino groups in peptides, enabling their separation into three major fractions: 1) neutral peptides 2) peptides with one positive charge and 3) peptides with 2 or more positive charges. The procedure demonstrated an excellent selectivity which allowed restricted MS/MS ion searches with peptide-centric databases.

Physicochemical mechanism for the enhanced ability of lipid membrane penetration of polyarginine.

Arginine-rich, cell-penetrating peptides (e.g., Tat-peptide, penetratin, and polyarginine) are used to carry therapeutic molecules such as oligonucleotides, DNA, peptides, and proteins across cell membranes. Two types of processes are being considered to cross the cell membranes: one is an endocytic pathway, and another is an energy-independent, nonendocytic pathway. However, the latter is still not known in detail. Here, we studied the effects of the chain length of polyarginine on its interaction with an anionic phospholipid large unilamellar vesicle (LUV) or a giant vesicle using poly-l-arginine composed of 69 (PLA69), 293 (PLA293), or 554 (PLA554) arginine residues, together with octaarginine (R8). ζ-potential measurements confirmed that polyarginine binds to LUV via electrostatic interactions. Circular dichroism analysis demonstrated that the transition from the random coil to the α-helix structure upon binding to LUV occurred for PLA293 and PLA554, whereas no structural change was observed for PLA69 and R8. Fluorescence studies using membrane probes revealed that the binding of polyarginine to LUV affects the hydration and packing of the membrane interface region, in which the degree of membrane insertion is greater for the longer polyarginine. Isothermal titration calorimetry measurements demonstrated that although the binding affinity (i.e., the Gibbs free energy of binding) per arginine residue is similar among all polyarginines the contribution of enthalpy to the energetics of binding of polyarginine increases with increasing polymer chain length. In addition, confocal laser scanning microscopy showed that all polyarginines penetrate across giant vesicle membranes, and the order of the amount of membrane penetration is R8 ≈ PLA69 < PLA293 ≈ PLA554. These results suggest that the formation of α-helical structure upon lipid binding drives the insertion of polyarginine into the membrane interior, which appears to enhance the membrane penetration of polyarginine.

Lysine proximity significantly affects glycation of lysine-containing collagen model peptides.

Advanced glycation end products (AGE) are known to cause diabetes complications in hyperglycemia patients. In this study we prepared hetero-trimers of collagen model peptides comprising Ac-(Pro-Hyp-Gly)(5)-Pro-Lys-Gly-(Pro-Hyp-Gly)(5)-Ala-NH(2) (4) and Ac-(Pro-Hyp-Gly)(11)-Ala-NH(2) (5) to investigate the clustering effect of lysine on AGE formation. The formation rate of carboxymethyllysine over several months was determined for the mixtures of peptides 4 and 5 at (3:0), (2:1) and (1:2) in the presence of glucose. The contents of carboxymethyllysine were significantly enhanced for (3:0) and (2:1) as compared with (1:2), suggesting that the proximity of lysine residues in the trimers accelerated formation of the AGE. Furthermore, a lysine dimerization moiety (GOLD) was identified for the first time from AGEs of glucose origin, which implied the significance of GOLD in oligomerization of collagens and other long-life proteins.

Structure analysis of membrane-reconstituted subunit c-ring of E. coli H+-ATP synthase by solid-state NMR.

The subunit c-ring of H(+)-ATP synthase (F(o) c-ring) plays an essential role in the proton translocation across a membrane driven by the electrochemical potential. To understand its structure and function, we have carried out solid-state NMR analysis under magic-angle sample spinning. The uniformly [(13)C, (15)N]-labeled F(o) c from E. coli (EF(o) c) was reconstituted into lipid membranes as oligomers. Its high resolution two- and three-dimensional spectra were obtained, and the (13)C and (15)N signals were assigned. The obtained chemical shifts suggested that EF(o) c takes on a hairpin-type helix-loop-helix structure in membranes as in an organic solution. The results on the magnetization transfer between the EF(o) c and deuterated lipids indicated that Ile55, Ala62, Gly69 and F76 were lined up on the outer surface of the oligomer. This is in good agreement with the cross-linking results previously reported by Fillingame and his colleagues. This agreement reveals that the reconstituted EF(o) c oligomer takes on a ring structure similar to the intact one in vivo. On the other hand, analysis of the (13)C nuclei distance of [3-(13)C]Ala24 and [4-(13)C]Asp61 in the F(o) c-ring did not agree with the model structures proposed for the EF(o) c-decamer and dodecamer. Interestingly, the carboxyl group of the essential Asp61 in the membrane-embedded EF(o) c-ring turned out to be protonated as COOH even at neutral pH. The hydrophobic surface of the EF(o) c-ring carries relatively short side chains in its central region, which may allow soft and smooth interactions with the hydrocarbon chains of lipids in the liquid-crystalline state.

Structural conversion of neurotoxic amyloid-beta(1-42) oligomers to fibrils.

The amyloid-beta(1-42) (Abeta42) peptide rapidly aggregates to form oligomers, protofibils and fibrils en route to the deposition of amyloid plaques associated with Alzheimer's disease. We show that low-temperature and low-salt conditions can stabilize disc-shaped oligomers (pentamers) that are substantially more toxic to mouse cortical neurons than protofibrils and fibrils. We find that these neurotoxic oligomers do not have the beta-sheet structure characteristic of fibrils. Rather, the oligomers are composed of loosely aggregated strands whose C termini are protected from solvent exchange and which have a turn conformation, placing Phe19 in contact with Leu34. On the basis of NMR spectroscopy, we show that the structural conversion of Abeta42 oligomers to fibrils involves the association of these loosely aggregated strands into beta-sheets whose individual beta-strands polymerize in a parallel, in-register orientation and are staggered at an intermonomer contact between Gln15 and Gly37.

Evaluation of retro-inverso modifications of HTLV-1 protease inhibitors containing a hydroxyethylamine isoster.

Effects of retro-inverso (RI) modifications of HTLV-1 protease inhibitors containing a hydroxyethylamine isoster backbone were clarified. Construction of the isoster backbone was achieved by a stereoselective aldol reaction. Four diastereomers with different configurations at the isoster hydroxyl site and the scissile site substituent were synthesized. Inhibitory activities of the new inhibitors suggest that partially modified RI inhibitors would interact with HTLV-1 protease in the same manner as the parent hydroxyethylamine inhibitor.

Proline cis/trans-isomerase Pin1 regulates peroxisome proliferator-activated receptor gamma activity through the direct binding to the activation function-1 domain.

The important roles of a nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) are widely accepted in various biological processes as well as metabolic diseases. Despite the worldwide quest for pharmaceutical manipulation of PPARgamma activity through the ligand-binding domain, very little information about the activation mechanism of the N-terminal activation function-1 (AF-1) domain. Here, we demonstrate the molecular and structural basis of the phosphorylation-dependent regulation of PPARgamma activity by a peptidyl-prolyl isomerase, Pin1. Pin1 interacts with the phosphorylated AF-1 domain, thereby inhibiting the polyubiquitination of PPARgamma. The interaction and inhibition are dependent upon the WW domain of Pin1 but are independent of peptidyl-prolyl cis/trans-isomerase activity. Gene knockdown experiments revealed that Pin1 inhibits the PPARgamma-dependent gene expression in THP-1 macrophage-like cells. Thus, our results suggest that Pin1 regulates macrophage function through the direct binding to the phosphorylated AF-1 domain of PPARgamma.

Synthesis of peptide thioesters via an N-S acyl shift reaction under mild acidic conditions on an N-4,5-dimethoxy-2-mercaptobenzyl auxiliary group.

An efficient method of peptide thioester synthesis is described. The reaction is based on an N-4,5-dimethoxy-2-mercaptobenzyl (Dmmb) auxiliary-assisted N-S acyl shift reaction after assembling a peptide chain by Fmoc-solid phase peptide synthesis. The Dmmb-assisted N-S acyl shift reaction proceeded efficiently under mildly acidic conditions, and the peptide thioester was obtained by treating the resulting S-peptide with sodium 2-mercaptoethanesulfonate. No detectable epimerization of the amino acid residue adjacent to the thioester moiety in the case of Leu was found. The reactions were also amenable to the on-resin preparation of peptide thioesters. The utility was demonstrated by the synthesis of a 41-mer peptide thioester, a phosphorylated peptide thioester and a 33-mer peptide thioester containing a trimethylated lysine residue.

Defining lipid-binding regions of human serum amyloid A using its fragment peptides.

Human serum amyloid A (SAA) protein is an apolipoprotein predominantly present in the high-density lipoprotein fraction of plasma. Despite its critical roles in lipid metabolism, especially in acute phases, systematic understanding of the lipid interaction of this protein is limited. Lipid-binding properties of synthetic fragment peptides corresponding to the N-terminal (residues 1-27), central (residues 43-63), and C-terminal (residues 77-104) parts of SAA molecule were examined. SAA (1-27) peptide binds to lipid forming an alpha-helical structure, whereas SAA (43-63) and (77-104) peptides do not display binding to lipid with any conformational changes. These results indicate that the N-terminal region of SAA is important for lipid interaction. In addition, the finding that deletion of or proline substitution in the most N-terminal region (residues 1-11) markedly decreased the binding to lipid further suggests that the alpha-helical structure in residues 1-11 is essential for lipid binding of SAA.

Synthesis of [19, 35, 36-(13)C(3)]-labeled TAK779 as a molecular probe.

N,N-Dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbonyl]amino]benzyl]tetrahydro-2H-pyran-4-aminium chloride (TAK779) is a potent and selective non-peptide CCR5 antagonist. To use a site-specifically labeled form as a molecular probe, TAK779 containing (13)C at positions C19, 35, and 36 was produced. A commercially available [(13)C]-methyl iodide was employed for the labeling. Starting from a known carboxylic acid segment containing no labeled carbon, the labeled TAK779 was constructed by the successive coupling of [(13)C]-labeled tolyl boronic ester by the Suzuki-Miyaura reaction and a [(13)C]-labeled aniline segment by amide bond formation.