The aspartimide problem in Fmoc-based SPPS. Part II.

The sequence dependence of base-catalysed aspartmide formation during Fmoc-based SPPS was systematically studied employing the peptide models H-Val-Lys-Asp-Xaa-Tyr-Ile-OH. The extent of formation of aspartimide and related by-products was determined by RP-HPLC. Considerable amounts of by-products were formed in the case of Xaa = Asp(OtBu), Arg(Pbf), Asn(Mtt), Cys(Acm) and unprotected Thr. Aspartimide formation could be diminished by incorporation of Asp(OMpe) or by employing milder methods for Fmoc cleavage, e.g. hexamethyleneimine/N-methylpyrrolidine/HOBt/NMP/DMSO 4:50:4:71:71 (v/v/w/v/v).

The aspartimide problem in Fmoc-based SPPS. Part I.

A variety of Asp beta-carboxy protecting groups, Hmb backbone protection and a range of Fmoc cleavage protocols have been employed in syntheses of the model hexapeptide H-VKDGYI-OH to investigate the aspartimide problem in more detail. The extent of formation of aspartimide and aspartimide-related by-products was determined by RP-HPLC. This study included three new Fmoc-Asp-OH derivatives: the beta-(4-pyridyl-diphenylmethyl) and beta-(9-phenyl-fluoren-9-yl) esters and also the orthoester Fmoc-beta-(4-methyl-2,6,7-trioxabicyclo[2.2.2]-oct-1-yl)-alanine. 3-Methylpent-3-yl protection of the Asp side chain resulted in significant improvements with respect to aspartimide formation. Complete suppression was achieved using the combination OtBu side chain protection and Hmb backbone protection for the preceding Gly residue.

Synthesis and application of Fmoc-His(3-Bum)-OH.

This paper presents a reevaluation of the synthesis and properties of Fmoc-His(3-Bum)-OH regarding its application in SPPS with minimal racemization of histidine residues during coupling and esterification reactions. By-product formation during the deprotection of the test peptides could be significantly reduced by scavenging the concomitantly formed HCHO, e.g. with methoxyamine.

NMR solution structure of a complex of calmodulin with a binding peptide of the Ca2+ pump.

The three-dimensional structure of the complex between calmodulin (CaM) and a peptide corresponding to the N-terminal portion of the CaM-binding domain of the plasma membrane calcium pump, the peptide C20W, has been solved by heteronuclear three-dimensional nuclear magnetic resonance (NMR) spectroscopy. The structure calculation is based on a total of 1808 intramolecular NOEs and 49 intermolecular NOEs between the peptide C20W and calmodulin from heteronuclear-filtered NOESY spectra and a half-filtered experiment, respectively. Chemical shift differences between free Ca(2+)-saturated CaM and its complex with C20W as well as the structure calculation reveal that C20W binds solely to the C-terminal half of CaM. In addition, comparison of the methyl resonances of the nine assigned methionine residues of free Ca(2+)-saturated CaM with those of the CaM/C20W complex revealed a significant difference between the N-terminal and the C-terminal domain; i.e., resonances in the N-terminal domain of the complex were much more similar to those reported for free CaM in contrast to those in the C-terminal half which were significantly different not only from the resonances of free CaM but also from those reported for the CaM/M13 complex. As a consequence, the global structure of the CaM/C20W complex is unusual, i.e., different from other peptide calmodulin complexes, since we find no indication for a collapsed structure. The fine modulation in the peptide protein interface shows a number of differences to the CaM/M13 complex studied by Ikura et al. [Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B., and Bax, A. (1992) Science 256, 632-638]. The unusual binding mode to only the C-terminal half of CaM is in agreement with the biochemical observation that the calcium pump can be activated by the C-terminal half of CaM alone [Guerini, D., Krebs, J., and Carafoli, E. (1984) J. Biol. Chem. 259, 15172-15177].

Application of the allyloxycarbonyl protecting group for the indole of Trp in solid-phase peptide synthesis.

The synthesis and stability of allyloxycarbonyl (Aloc) indole-protected Trp derivatives and their application in solid-phase peptide synthesis are reported. The study shows that the Aloc protection on the indole moiety is suitable for orthogonal protection in the Fmoc/tBu strategy if the Fmoc group is cleaved with DBU. Several tryptophan-containing peptides have been synthesized including dynorphin A-(1-13), which has been intensively studied with respect to side reactions during the final TFA cleavage procedure. The results demonstrate the protective function of the Aloc group on the Trp during final deprotection. Furthermore, it could be demonstrated that Trp(Aloc)-containing peptides can be isolated and that the Aloc group can then be removed in a second step. The synthesis of phosphorylated delta sleep inducing peptide (P-DSIP) using the global phosphorylation approach provides another example in which Trp indole protection by Aloc prevents the formation of oxidative side products.

Differential and multiple binding of signal transducing molecules to the ITAMs of the TCR-zeta chain.

A biotin-streptavidin-based technique was developed for high affinity, unidirectional, and specific immobilization of synthetic peptides to a solid phase. Biotinylated 23-mer carboxamide peptides corresponding to the three immunoreceptor tyrosine-based activation motifs (ITAMs) of the T cell antigen receptor associated zeta-chain (TCR-zeta) in their bis-, mono-, or unphosphorylated forms were used to study the binding of cellular proteins from human Jurkat T cells to these signal transduction motifs. The protein tyrosine kinase ZAP-70 bound specifically to all bisphosphorylated peptides but not to the mono- or unphosphorylated peptides. In contrast, Shc, phosphatidylinositol 3-kinase (Pl3K), Grb2, and Ras-GTPase activating protein (GAP) bound with different affinities to the bis- or monophosphorylated peptides, while the Src family protein tyrosine kinase (PTK) Fyn did not bind specifically to any of the tested peptides. The different preferences of the studied signaling molecules for distinct ITAMs, and in particular the binding of some of them preferentially to monophosphorylated peptides, suggests that the TCR-zeta may bind multiple signaling molecules with each ITAM binding a unique set of such molecules. In addition, partial phosphorylation of the ITAMs may result in recruitment of different proteins compared to double phosphorylation. This may be crucial for coupling of the TCR to various effector functions under different conditions of receptor triggering.

Identification of two distinctly localized mitochondrial creatine kinase isoenzymes in spermatozoa.

The creatine kinase (CK) isoenzyme system is essential for motility in rooster and sea urchin sperm. In the present study, biochemical characterization as well as immunofluorescence and confocal laser microscopy with highly specific antibodies against various chicken CK isoenzymes revealed that cytosolic brain-type CK isoenzyme (B-CK) is the only CK isoenzyme in rooster seminal plasma, while three isoenzymes, cytosolic B-CK, sarcomeric mitochondrial CK (Mib-CK), and a variant of ubiquitous Mi-CK ('Mia-CK variant'), are found in rooster spermatozoa. These three isoenzymes are localized in different regions of the sperm cell. B-CK and Mib-CK were localized along the entire sperm tail and in the mitochondria-rich midpiece, respectively. The 'Mia-CK variant', on the other hand, was found predominantly at the head-midpiece boundary, in a non-uniform manner in the midpiece itself and, surprisingly, at the distal end of the sperm tail as well as at the acrosome. Several lines of evidence show that the 'Mia-CK variant' shares some characteristics with purified Mia-CK from chicken brain, but also displays distinctive features. This is the first evidence for two different Mi-CK isoenzymes occurring in one cell and, additionally, for the co-expression of Mib-CK and cytosolic brain-type B-CK in the same cell. The relevance of these findings for sperm physiology and energetics is discussed.

H+-induced membrane insertion of influenza virus hemagglutinin involves the HA2 amino-terminal fusion peptide but not the coiled coil region.

Fusion of influenza virus with target membranes is induced by acid and involves complex changes in the viral envelope protein hemagglutinin (HA). In a first, kinetically distinct step, the HA polypeptide chain 2 (HA2) is inserted into the target membrane bilayer. Using hydrophobic photolabeling with the phospholipid analogue 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4(trifluoromethyl-3H-diazirin -3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, we identified the segment within HA2 that interacts with the membrane. The sole part of the HA2 ectodomain that was labeled with the membrane-restricted reagent is the NH2-terminal fusion peptide (residues 1-22). No labeling occurred within the long coiled coil region generated during the acid-induced conformational transition (Bullough, P. A., Hughson, F. M., Skehel, J. J., and Wiley, D. C. (1994) Nature 371, 37-43). These data strongly suggest that the coiled coil region of HA2 does not insert into the lipid bilayer. This conclusion is at variance with the recent suggestion (Yu, Y. G., King, D. S., and Shin, Y.-K.(1994) Science 266, 274-276) that the coiled coil of HA may splay apart and insert into the target membrane, providing a mechanism by which the viral and the target membrane may come in close apposition.

The secondary structure of phospholamban: a two-dimensional NMR study.

Phospholamban (PLN) is an intrinsic membrane protein of 52 amino acids which regulates the Ca2+ pump of the sarcoplasmic reticulum of heart, slow-twitch and smooth muscle (SR): it is normally assumed to exist in the membrane as a homopentamer. A monomeric analogue of phospholamban PLN(C41F), in which Cys41 was replaced by a Phe, was synthesized and its conformation studied by 1H NMR spectroscopy in a 1:1 mixture of chloroform/methanol. Most of the resonances in the 1H NMR spectra were assigned. The work has shown that the C-terminal hydrophobic portion forms a very stable alpha-helix. The hydrophilic N-terminal part adopts an alpha-helix configuration which is much less stable except for the stretch containing the phosphorylation sites.

The calmodulin-binding domain of the inducible (macrophage) nitric oxide synthase.

A domain in the inducible, macrophage nitric oxide (NO) synthase has been selected as the putative calmodulin-binding site. The domain was synthesized as a peptide of 29 residues [P29, NO synthase-(504-532)-peptide], having the accepted hydrophobic/basic composition of calmodulin-binding domains and containing, like most of them, an aromatic amino acid at its N-terminus and a long chain aliphatic residue 12 amino acids downstream of it. A 34-residue peptide from the synthase sequence [P34, NO synthase-(499-532)-peptide], consisting of peptide P29 and of the five extra N-terminal amino acids, three of them basic, was also synthesized. Both peptides bound calmodulin in the presence as well as in the absence of Ca2+ (i.e. in the presence of excess EGTA). The KD of the binding in the presence of Ca2+ was < or = 1 nM. The binding affinity was lower, but still remarkably high in the presence of EGTA. The peptides counteracted the stimulation by calmodulin of a classical calmodulin-target enzyme, the Ca2+ pump of the plasma membrane.

Characterization of a 21 amino acid peptide sequence of the laminin G2 domain that is involved in HNK-1 carbohydrate binding and cell adhesion.

The N-linked HNK-1 carbohydrate expressed by several recognition molecules mediates the adhesion of early post-natal cerebellar neurons to the G2 domain of the terminal globular domain of the laminin alpha 1 chain (H. Hall et al., submitted). To define this binding site more precisely, G2-derived synthetic peptides were used for binding and competition studies. Peptide 5-G2, comprising the amino acid residues 3431-3451 of G2, inhibited the interaction between the HNK-1-carrying glycolipid and laminin in a concentration-dependent and saturable manner. Peptides which overlap only partially with this sequence interfered less. Peptides comprising other amino acid sequences from G2, and peptides derived from G1 and G3 or a scrambled version of peptide 5-G2, did not show significant effects. Direct binding of peptide 5-G2 to the HNK-1 glycolipid was also demonstrated. Furthermore, peptide 5-G2 interfered in a concentration-dependent and saturable manner with the adhesion of early postnatal cerebellar neurons to laminin. These observations indicate that amino acid residues 3431-3451 of the laminin G2 domain are involved in HNK-1 carbohydrate-mediated cell adhesion.

Calmodulin-binding domains: just two faced or multi-faceted?

The Ca(2+)-binding protein calmodulin binds to and activates several cellular enzymes in response to a rise in Ca2+ concentration. It binds certain basic amphiphilic helices within these enzymes, which also act as autoinhibitory domains. The modulation of the binding equilibrium of these helices between intramolecular (inhibition) and intermolecular (activation) sites forms a focal point for crosstalk between various signalling pathways.

Phosphorylation of the calmodulin binding domain of the plasma membrane Ca2+ pump by protein kinase C reduces its interaction with calmodulin and with its pump receptor site.

Two versions of the calmodulin binding domain of the plasma membrane Ca2+ ATPase, a 24-amino acid peptide, C24W (Q-I-L-W-F-R-G-L-N-R-I-Q-T-Q-I-R-V-V-N-A-F-R-S-S-NH2), and the corresponding phosphothreonine containing peptide, C24W-P (Q-I-L-W-F-R-G-L-N-R-I-Q-T(phospho)-Q-I-R-V-V-N-A-F-R-S-S-NH2), were synthesized. They were used to investigate the effect of threonine phosphorylation by protein kinase C on the binding of calmodulin by the calmodulin binding domain and on the inhibitory role of the domain on the activity of the Ca2+ pump. The phosphopeptide C24W-P was obtained after global phosphorylation of the free Thr side chain on the protected resin bound peptide. The phosphorylated calmodulin binding domain failed to bind calmodulin; this was shown by gel shift experiments, by fluorescence energy transfer studies and by competition experiments against calmodulin stimulation of the pump. The inhibition of the Ca2+ pump activity by the calmodulin binding domain in the absence of calmodulin was also affected by the phosphorylation of the threonine; the inhibition of the fully active calpain-truncated pump by the phosphothreonine containing peptide was lower than that by the unphosphorylated synthetic domain.

Anti-CD3 and phorbol ester induce distinct phosphorylated sites in the SH2 domain of p56lck.

P56lck is a protein tyrosine kinase of the Src family specifically expressed in T lymphocytes. Triggering of T cells with anti-CD3 or with phorbol 12-myristate 13-acetate (PMA) results in the appearance of slower migrating forms (shift) of p56lck. To investigate the phosphorylation sites on the shifted forms of p56lck and to assess the role of protein kinase C in this phosphorylation, Jurkat cells were treated with a selective inhibitor of this kinase (GF 109203X). This inhibitor completely reversed the shift induced by PMA but only partially reversed the one induced after triggering with anti-CD3. To analyze the shift further, p56lck was immunoprecipitated from in vivo labeled cells treated either with anti-CD3 or with PMA. Tryptic phosphopeptides were generated and analyzed by using a combination of thin layer chromatography, high reticulation polyacrylamide gel electrophoresis, reverse phase chromatography, and phosphopeptide sequencing. We identified serine 158 as a newly phosphorylated site after PMA treatment and tyrosine 192 and serine 194 in the major tryptic phosphopeptide obtained after anti-CD3 triggering. The three sites identified are located in the SH2 domain of p56lck; this suggests that their phosphorylation may regulate the interaction with other proteins or with other internal domains in p56lck.

Synthetic peptides corresponding to the calmodulin-binding domains of skeletal muscle myosin light chain kinase and human erythrocyte Ca2+ pump interact with and permeabilize liposomes and cell membranes.

Synthetic calmodulin-binding (CaM-binding) peptides (CBPs) representing CaM-binding domains of Ca2+/CaM-dependent enzymes have been reported to interfere with the activity of the melanocyte-stimulating hormone (MSH) receptor function in melanoma cells [Gerst, J. E., & Salomon, Y. (1988) J. Biol. Chem. 263, 7073-7078]. We postulated that membrane lipids may play an important role in the mode of action of CBPs on cells. We therefore tested the ability of CBPs to interact with membrane bilayers. Using artificial phospholipid vesicles, or M2R melanoma cells and cell membranes derived therefrom, as models, we report here that synthetic peptides representing the CaM-binding domains of skeletal muscle myosin light chain kinase (M5) and the human erythrocyte calcium pump (C28W), as well as other CBPs, interact with lipid bilayers and cell membranes. Significant interactions of CBPs with the lipid bilayer were detected in both model systems. M5 and C28W were found to partition into the lipid bilayer of melanoma cell membranes and soybean lecithin vesicles, and surface partition constants obtained (for the liposome model) were in the range 10(3)-10(4) M-1. In addition, C28W and its N-modified NBD derivative were found to inhibit [125I]iodo-[Nle4,D-Phe7]alpha MSH binding to cultured M2R melanoma cells. These and other CBPs were also found to induce the release of cations and calcein from liposomes, suggesting that the interaction of CBPs with the lipid bilayer increases membrane permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

The calmodulin binding domain of nitric oxide synthase and adenylyl cyclase.

Peptides corresponding to regions of the calmodulin-activated NO-synthase and of the calmodulin dependent adenylyl cyclase, which could represent the calmodulin binding domains of the two proteins, have been synthesized and tested for calmodulin binding. The chosen peptides were those in the sequence of the two proteins which most closely corresponded to the accepted general properties of the calmodulin binding domains, i.e., a hydrophobic sequence containing basic amino acids. In the case of the NO-synthase, the putative high-affinity calmodulin binding domain was identified by urea gel electrophoresis and fluorescence spectroscopy with dansylcalmodulin as peptide NO-30 (amino acids 725-754). The highest affinity calmodulin binding site of the calmodulin-dependent adenylyl cyclase was assigned to peptide AC-28 (amino acids 495-522) by titration with dansylcalmodulin and by the ability to inhibit the calmodulin-stimulated activity of purified calmodulin-stimulated adenylyl cyclase. The sequence 495-522 is located in the unit protruding into the cytosol from the sixth putative transmembrane domain of the molecule. It has the typical hydrophobic/basic composition of canonical calmodulin binding domains, and also contains, like most calmodulin binding domains, an aromatic amino acid in its N-terminal portion. It also contains two Cys residues in the central portion, which is an unusual feature of the calmodulin binding domain of this enzyme.

The fourth immunoglobulin-like domain of NCAM contains a carbohydrate recognition domain for oligomannosidic glycans implicated in association with L1 and neurite outgrowth.

We have previously shown that the neural adhesion molecules L1 and NCAM interact with each other to form a complex which binds more avidly to L1 than L1 to L1 alone (Kadmon, G., A. Kowitz, P. Altevogt, and M. Schachner. 1990a. J. Cell Biol. 110:193-208). This cis-association between L1 and NCAM is carbohydrate-dependent (Kadmon, G., A. Kowitz, P. Altevogt, and M. Schachner. 1990b. J. Cell Biol. 110:209-218). In the present study, we report that L1 and NCAM bind to each other via oligomannosidic carbohydrates expressed by L1, but not by NCAM, as shown in several experiments: (a) complex formation between L1 and NCAM is inhibited by a mAb to oligomannosidic carbohydrates and by the oligosaccharides themselves; (b) NCAM binds to oligomannosidic carbohydrates; (c) within the L1/NCAM complex, the oligomannosidic carbohydrates are hidden from accessibility to a mAb against oligomannosidic carbohydrates; (d) the recombinant protein fragment of NCAM containing the immunoglobulin-like domains and not the fragment containing the fibronectin type III homologous repeats binds to oligomannosidic glycans. Furthermore, the fourth immunoglobulin-like domain of NCAM shows sequence homology with carbohydrate recognition domains of animal C-type lectins and, surprisingly, also with plant lectins. A peptide comprising part of the C-type lectin consensus sequence in the fourth immunoglobulin-like domain of NCAM interferes with the association between L1 and NCAM. The functional importance of oligomannosidic glycans at the cell surface was shown for neurite outgrowth in vitro. When neurons from early postnatal mouse cerebellum were maintained on laminin or poly-L-lysine, neurite outgrowth was inhibited by oligomannosidic glycans, by glycopeptides, glycoproteins, or neoglycolipids containing oligomannosidic glycans, but not by nonrelated oligosaccharides or oligosaccharide derivates. Neurite outgrowth was also inhibited by the peptide comprising part of the C-type lectin consensus sequence in the fourth immunoglobulin-like domain of NCAM. The combined results suggest that carbohydrate-mediated cis-associations between adhesion molecules at the cell surface modulate their functional properties.

The C-terminal domain of the plasma membrane Ca2+ pump contains three high affinity Ca2+ binding sites.

The C-terminal portion of the plasma membrane Ca(2+)-ATPase contains different regulatory domains. A recombinant C-terminal fragment of the human plasma membrane Ca(2+)-ATPase 1b isoform (E1079-P1180) was used to study the role of two acidic amino acid stretches located on either side of the calmodulin binding domain, corresponding to synthetic peptides A18 (Vorherr, T., James, P., Krebs, J., Enyedi, A., McCormick, D. J., Penniston, J. T., and Carafoli, E. (1990) Biochemistry 29, 355-365) and B28 (James, P., Pruschy, M., Vorherr, T., Penniston, J. T., and Carafoli, E. (1989) Biochemistry 28, 4253-4258), respectively. The molecular mass of the recombinant C-terminal fragment, as determined by electrospray ionization mass spectrometry, was higher by 39 mass units than the calculated value (12,055 Da). This difference was the result of an EGTA-insensitive Ca2+ ion, which was located by chymotryptic proteolysis in a fragment corresponding to the last 37 amino acids of the expressed protein. Fluorescence experiments on the dansylated recombinant C-terminal fragment titrated with increasing amounts of free Ca2+ revealed two additional Ca2+ binding sites with affinities corresponding to KD values of about 30 and 300 nM, respectively. Stains All spectra of different synthetic peptides, corresponding to subdomains of the expressed protein, indicated that the site with the KD of 30 nM was probably located in the acidic sequence on the N-terminal side of the calmodulin binding domain (peptide A18); the site with the 300 nM KD was apparently located on the C-terminal side of the calmodulin binding domain (peptide B28) or, alternatively, formed by the cooperation of distant residues of the domain.

Total synthesis and functional properties of the membrane-intrinsic protein phospholamban.

The membrane-intrinsic protein phospholamban (PLN), the regulatory protein of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, was chemically synthesized. The synthesis was accomplished by double couplings and efficient capping procedures, thus eliminating hydrophobic failure sequences. The crude peptide was purified by high-performance liquid chromatographic ion exchange and gel permeation chromatography in chloroform-methanol mixtures. Ion spray mass spectroscopy showed that the product had the correct molecular mass. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis runs produced the typical monomer-pentamer structural pattern. A predominantly helical CD spectrum was obtained in 0.075% C12E8 (67.9% helix, 1.8% beta, 12.2% turn, 18.1% random coil). Synthetic PLN was phosphorylated in detergent solutions by protein kinase A with a stoichiometry close to 1:1 (Pi to PLN monomer). Reconstitution of the isolated skeletal muscle SR Ca2+ ATPase in phosphatidylcholine membranes in the presence of PLN using the freezing and thawing technique yielded a preparation with lower Ca(2+)-dependent ATPase activity. The inhibition was mainly due to a decrease in the affinity (Km(Ca)) of the ATPase for Ca2+ and was partially reversed by PLN phosphorylation with protein kinase A. By contrast, addition of PLN to diluted intact SR vesicles uncoupled the Ca(2+)-transport reaction, suggesting an ionophoric effect of PLN. Because this effect was observed at very high PLN-to-SR vesicle ratios and was not influenced by PLN phosphorylation, its biological function is doubtful.

Study of calmodulin binding to the alternatively spliced C-terminal domain of the plasma membrane Ca2+ pump.

The C-terminal regions of the four human plasma membrane Ca2+ pump isoforms 1a-d generated from alternatively spliced RNA have been expressed in Escherichia coli, and the recombinant proteins have been purified to a very high degree. The C-termini of isoforms 1a, 1c, and 1d contain an insert encoded by an alternatively spliced exon which is homologous to the calmodulin binding domain of isoform 1b. In isoforms 1c and 1d (29 and 38 amino acid insertions, respectively), subdomain A of the original calmodulin binding site of isoform 1b is followed by the spliced-in domain, which is then followed by subdomain B of the original calmodulin binding site. The positive charges of histidine residues at positions 27, 28, and 38 of the alternatively spliced sequence are likely to be responsible for the observed pH-dependent calmodulin binding to the novel "duplicated" binding site. The affinity of calmodulin for the C-terminal domains of isoforms 1a, 1c, and 1d, which contain the histidine-rich inserts, is much higher at pH 5.9 than at pH 7.2. A synthetic peptide (I31) containing 31 amino acids of the alternatively spliced sequence (from residue 9 to 40) also binds calmodulin with strong pH dependency. Alternative splicing in the C-terminal domain is proposed to confer pH dependence to the regulation of the activity of Ca2+ pump isoforms.