A convenient solid phase synthesis of S-palmitoyl transmembrane peptides

S-Palmitoylated peptides are important tools as models for integral membrane proteins to study peptide–lipid interactions. Herein, we report a convenient solid phase synthesis of S-palmitoyl transmembrane peptides. The highly acid labile S-(4-methoxytrityl) group is preferred over the S-(tert-butylsulfanyl) group for protection of the cysteine side chain since the latter gives rise to quantitative desulfurization during on-resin deprotection. The resulting free thiol function is modified with palmitic acid via a carbodiimide-mediated coupling and the title compounds are obtained in good yields and purity

Sensitivity of single membrane-spanning alpha-helical peptides to hydrophobic mismatch with a lipid bilayer: Effects on backbone structure, orientation, and extent of membrane incorporation

The extent of matching of membrane hydrophobic thickness with the hydrophobic length of transmembrane protein segments potentially constitutes a major director of membrane organization. Therefore, the extent of mismatch that can be compensated, and the types of membrane rearrangements that result, can provide valuable insight into membrane functionality. In the present study, a large family of synthetic peptides and lipids is used to investigate a range of mismatch situations. Peptide conformation, orientation, and extent of incorporation are assessed by infrared spectroscopy, tryptophan fluorescence, circular dichroism, and sucrose gradient centrifugation. It is shown that peptide backbone structure is not significantly affected by mismatch, even when the extent of mismatch is large. Instead, this study demonstrates that for tryptophan-flanked peptides the dominant response of a membrane to large mismatch is that the extent of incorporation is reduced, when the peptide is both too short and too long. With increasing mismatch, a smaller fraction of peptide is incorporated into the lipid bilayer, and a larger fraction is present in extramembranous aggregates. Relatively long peptides that remain incorporated in the bilayer have a small tilt angle with respect to the membrane normal. The observed effects depend on the nature of the flanking residues: long tryptophan-flanked peptides do not associate well with thin bilayers, while equisized lysine-flanked peptides associate completely, thus supporting the notion that tryptophan and lysine interact differently with membrane-water interfaces. The different properties that aromatic and charged flanking residues impart on transmembrane protein segments are discussed in relation to protein incorporation in biological systems

The role of the disulfide bond in the interaction of islet amyloid polypeptide with membranes

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus. It has been suggested that the N-terminal part, which contains a conserved intramolecular disulfide bond between residues 2 and 7, interacts with membranes, ultimately leading to membrane damage and b-cell death. Here, we used variants of the hIAPP1–19 fragment and model membranes of phosphatidylcholine and phosphatidylserine (7:3, molar ratio) to examine the role of this disulfide in membrane interactions. We found that the disulfide bond has a minor effect on membrane insertion properties and peptide conformational behavior, as studied by monolayer techniques, 2H NMR, ThT-fluorescence, membrane leakage, and CD spectroscopy. The results suggest that the disulfide bond does not play a significant role in hIAPP–membrane interactions. Hence, the fact that this bond is conserved is most likely related exclusively to the biological activity of IAPP as a hormon

The role of the disulfide bond in the interaction of islet amyloid polypeptide with membranes

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus. It has been suggested that the N-terminal part, which contains a conserved intramolecular disulfide bond between residues 2 and 7, interacts with membranes, ultimately leading to membrane damage and b-cell death. Here, we used variants of the hIAPP1–19 fragment and model membranes of phosphatidylcholine and phosphatidylserine (7:3, molar ratio) to examine the role of this disulfide in membrane interactions. We found that the disulfide bond has a minor effect on membrane insertion properties and peptide conformational behavior, as studied by monolayer techniques, 2H NMR, ThT-fluorescence, membrane leakage, and CD spectroscopy. The results suggest that the disulfide bond does not play a significant role in hIAPP–membrane interactions. Hence, the fact that this bond is conserved is most likely related exclusively to the biological activity of IAPP as a hormon

Effects of linker variation on the in vitro and in vivo characteristics of an 111In-labeled RGD peptide.

Contains fulltext : 52604.pdf (publisher's version ) (Closed access)INTRODUCTION: Due to the selective expression of the alpha(v)beta3 integrin in tumors, radiolabeled arginine-glycine-aspartic acid (RGD) peptides are attractive candidates for tumor targeting. Minor modifications of these peptides could have a major impact on in vivo characteristics. In this study, we systematically investigated the effects of linker modification between two cyclic RGD sequences and DOTA (1,4,7,10-tetraazadodecane-N,N',N",N'''-tetraacetic acid) on the in vitro and in vivo characteristics of the tracer. METHODS: A dimeric RGD peptide was synthesized and conjugated either directly with DOTA or via different linkers: PEG4 (polyethylene glycol), glutamic acid or lysine. The RGD peptides were radiolabeled with 111In, and their in vitro and in vivo alpha(v)beta3-binding characteristics were determined. RESULTS: LogP values varied between -2.82+/-0.06 and -3.95+/-0.33. The IC50 values for DOTA-E-[c(RGDfK)]2, DOTA-PEG4-E-[c(RGDfK)]2, DOTA-E-E-[c(RGDfK)]2 and DOTA-K-E-[c(RGDfK)]2 were comparable. Two hours after injection, the tumor uptakes of the 111In-labeled compounds were not significantly different. The kidney accumulation of [111In]-DOTA-K-E-[c(RGDfK)]2 [4.05+/-0.20% of the injected dose per gram (ID/g)] was significantly higher as compared with that of [111In]-DOTA-E-[c(RGDfK)]2 (2.63+/-0.19% ID/g; P<.05) as well as that of [111In]-DOTA-E-E-[c(RGDfK)]2 (2.16+/-0.21% ID/g; P<.01). The liver uptake of [111In]-DOTA-E-E-[c(RGDfK)]2 (2.12+/-0.09% ID/g) was significantly higher as compared with that of [111In]-DOTA-E-[c(RGDfK)]2 (1.64+/-0.1% ID/g; P<.05) as well as that of [111In]-DOTA-K-E-[c(RGDfK)]2 (1.52+/-0.04% ID/g; P<.01). CONCLUSIONS: Linker variation did not affect affinity for alpha(v)beta3 and tumor uptake. Insertion of lysine caused enhanced kidney retention; that of glutamic acid also resulted in enhanced retention in the kidneys. PEG4 appeared to be the most suitable linker as compared with glutamic acid and lysine because it has the highest tumor-to-blood ratio and the lowest uptake in the kidney and liver

Synthesis and biological evaluation of potent alphavbeta3-integrin receptor antagonists.

Item does not contain fulltextINTRODUCTION: alpha(v)beta(3) Integrin is expressed in sprouting endothelial cells in growing tumors, whereas it is absent in quiescent blood vessels. In addition, various tumor cell types express alpha(v)beta(3) integrin. alpha(v)beta(3) Integrin, a transmembrane heterodimeric protein, binds to the arginine-glycine-aspartic acid (RGD) amino acid sequence of extracellular matrix proteins such as vitronectin and plays a pivotal role in invasion, proliferation and metastasis. Due to the selective expression of alpha(v)beta(3) integrin in tumors, radiolabeled RGD peptides and peptidomimetics are attractive candidates for tumor targeting. METHODS: A cyclic RGD peptide, a peptoid-peptide hybrid, an all-peptoid and a peptidomimetic compound were synthesized, conjugated with 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA) and radiolabeled with (111)In. Their in vitro and in vivo alpha(v)beta(3)-binding characteristics were determined. RESULTS: IC(50) values were 236 nM for DOTA-E-c(RGDfK), 219 nM for DOTA-peptidomimetic, >10 mM for DOTA-all-peptoid and 9.25 mM for the peptoid-peptide hybrid DOTA-E-c(nRGDfK). (111)In-labeled compounds, except for [(111)In]DOTA-all-peptoid, showed specific uptake in human alpha(v)beta(3)-expressing tumors xenografted in athymic mice. Tumor uptake for [(111)In]DOTA-E-c(RGDfK) was 1.73+/-0.4% ID/g (2 h postinjection) and that of [(111)In]DOTA-peptidomimetic was 2.04+/-0.3% ID/g. Tumor uptake for the peptoid-peptide hybrid [(111)In]DOTA-E-c(nRGDfK) was markedly lower (0.45+/-0.07% ID/g). The all-peptoid [(111)In]DOTA-E-c(nRGnDnFnK) did not show specific uptake in tumors (0.11+/-0.04% ID/g). CONCLUSIONS: The peptidomimetic compound and the cyclic RGD peptide have a high affinity for alpha(v)beta(3) integrin, and these compounds have better tumor-targeting characteristics than the peptoid-peptide hybrid and the all-peptoid

Chemical-biological exploration of the limits of the ras de- and repalmitoylating machinery

A dynamic de-/repalmitoylation cycle determines localization and activity of H- and N-Ras. This combined cellular de- and repalmitoylation machinery has been shown to be substrate tolerant--it accepts variation of amino acid sequence, structure and configuration. Here, semisynthetic Ras-proteins in which the C-terminal amino acids are replaced by peptoid residues are used to reveal the first limitations of substrate recognition by the de- and repalmitoylating machinery