Design of hydropathically complementary peptides for Big Endothelin affinity purification

Molecular recognition between Big Endothelin (Big ET) and a computer generated peptide hydropathically complementary to Big ET[16-29] sequence has been studied by analytical high performance liquid affinity chromatography (HPLAC), circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments. Specific binding was observed between solid support immobilized complementary peptide and Big ET[1-38], [1-32], and [16-32], but not with Big ET fragments [1-21], [16-21], [22-32], and [22-38], obtained by chymotrypsin proteolytic degradation. Selectivity in the recognition process was clearly demonstrated by the ability of complementary peptide affinity column to purify the Big ET molecule from complex peptide mixtures, even when present in very low concentrations. Similar selectivity was evidenced with the Big ET fragment [16-32], [NH₂-HLDIIWVNTPEHIVPYG-COOH] containing the entire hydropathically complementary sequence. Binding was followed by marked spectroscopic changes, as monitored by circular dichroism and one- and two-dimensional nuclear magnetic resonance experiments. The NMR spectra of the complementary peptides 1:1 mixture showed variations in the chemical shifts of proton resonances in several residues, both in the main chain (amide protons) and in the side chains (aliphatic and aromatic protons). These data support the hypothesis of a multilocalized type of interaction between complementary peptides, where many residues along the peptide chains participate in co-operative stabilizing contacts in the forming complex.     

Molecular basis of branched peptides resistance to enzyme proteolysis

We found that synthetic peptides in the form of dendrimers become resistant to proteolysis. To determine the molecular basis of this resistance, different bioactive peptides were synthesized in monomeric, two-branched and tetra-branched form and incubated with human plasma and serum. Proteolytic resistance of branched multimeric sequences was compared to that of the same peptides synthesized as multimeric linear molecules. Unmodified peptides and cleaved sequences were detected by high pressure liquid chromatography and mass spectrometry. An increase in peptide copies did not increase peptide resistance in linear multimeric sequences, whereas multimericity progressively enhanced proteolytic stability of branched multimeric peptides. A structure-based hypothesis of branched peptide resistance to proteolysis by metallopeptidases is presented.     

Kinetic analyses of Keap1-Nrf2 interaction and determination of the minimal Nrf2 peptide sequence required for Keap1 binding using surface plasmon resonance

The Keap1-Nrf2 interaction plays important roles in regulation of Nrf2 activity and induction of chemopreventive enzymes. To better understand the interaction and to determine the minimal Nrf2 sequence required for Keap1 binding, we synthesized a series of Nrf2 peptides containing ETGE motif and determined their binding affinities to the Kelch domain of Keap1 in solution using a surface plasmon resonance-based competition assay. The equilibrium dissociation constant for the interaction between 16mer Nrf2 peptide and Keap1 Kelch domain in solution (K(solution)(D)) was found to be 23.9 nM, which is 10× lower than the surface binding constant (K(surface)(D)) of 252 nM obtained for the direct binding of Keap1 Kelch domain to the immobilized 16mer Nrf2 peptide on a surface plasmon resonance sensor chip surface. The binding affinity of Nrf2 peptides to Keap1 Kelch domain was not lost until after deletion of eight residues from the N-terminus of the 16mer Nrf2 peptide. The 9mer Nrf2 peptide has a moderate binding affinity with a (K(solution)(D)) of 352 nM and the affinity was increased 15× upon removal of the positive charge at the peptide N-terminus by acetylation. These results suggest that the minimal Nrf2 peptide sequence required for Keap1 binding is the 9mer sequence of LDEETGEFL.     

Synthesis and evaluation of N,N-dialkyl enkephalin-based affinity labels for delta opioid receptors

To develop affinity labels for delta opioid receptors based on peptide antagonists, the Phe(4) residues of N,N-dibenzylleucine enkephalin and N,N-diallyl[Aib(2),Aib(3)]leucine enkephalin (ICI-174, 864) were substituted with either Phe(p-NCS) or Phe(p-NHCOCH(2)Br). A general synthetic method was developed for the conversion of small peptide substrates into potential affinity labels. The target peptides were synthesized using Phe(p-NH(2)) and a Boc/Fmoc orthogonal protection strategy which allowed for late functional group conversion of a p-amine group in the peptides to the desired affinity labeling moieties. A key step in the synthesis was the selective deprotection of a Boc group in the presence of a tert-butyl ester using trimethylsilyl trifluoromethanesulfonate (TMS-OTf). The target peptides were evaluated in radioligand binding experiments in Chinese hamster ovary (CHO) cells expressing delta or mu opioid receptors. The delta receptor affinities of the N, N-dibenzylleucine enkephalin analogues were 2.5-10-fold higher than those for the corresponding ICI-174,864 analogues. In general, substitution at the para position of Phe(4) decreased binding affinity at both delta and mu receptors in standard radioligand binding assays; the one exception was N, N-dibenzyl[Phe(p-NCS)(4)]leucine enkephalin (2) which exhibited a 2-fold increase in affinity for delta receptors (IC(50) = 34.9 nM) compared to N,N-dibenzylleucine enkephalin (IC(50) = 78.2 nM). The decreases in mu receptor affinities were greater than in delta receptor affinities so that all of the analogues tested exhibited significantly greater delta receptor selectivity than the unsubstituted parent peptides. Of the target peptides tested, only N, N-dibenzyl[Phe(p-NCS)(4)]leucine enkephalin (2) exhibited wash-resistant inhibition of radioligand binding to delta receptors. To our knowledge, 2 represents the first peptide-based affinity label to utilize an isothiocyanate group as the electrophilic affinity labeling moiety. As a result of this study, enkephalin analogue 2 emerges as a potential affinity label useful for the further study of delta opioid receptors.     

Synthesis, opiate receptor affinity, and conformational parameters of [4-tryptophan]enkephalin analogues.

A series of analogues of the opioid peptide enkephalin with tryptophan substituted for phenylalanine in position 4 was synthesized by the solid-phase method. The [Trp4]enkephalin analogues and the corresponding [Phe4]enkephalin analogues displayed nearly parallel affinities in the opiate receptor binding assay throughout the series. In a conformational study fluorescence parameters were measured and intramolecular Tyr-Trp distances were estimated on the basis of resonance energy transfer experiments. No gross conformational differences were observed between analogues with widely differing opiate receptor affinity; however, small but significant changes in the intramolecular distance between the phenol ring and the indole moiety and/or in their relative orientation became apparent in some compounds. Identical intramolecular distances of 9.3 +/- 0.2 angstrom between the two aromatic rings were obtained with [Trp4,Met5]enkephalin, [Trp4,Leu5]enkephalin, and the N-terminal tetrapeptide comprised in the latter two analogues, indicating the existence of folded conformationas in 2 X 10(-5) M aqueous solution and demonstrating conformational analogy between these three peptides. The conformational parameters are discussed in relation to the observed affinities and the putative opiate receptor topography.     

Novel,potent ORL-1 receptor agonist peptides containing alpha-Helix-promoting conformational constraints

The ORL-1 receptor has recently been cloned and is implicated in a wide variety of physiological and pathophysiological processes. Toward the goal of elucidating important features of the receptor-bound conformation of the endogenous ligand, nociceptin (NC), several conformationally constrained analogues were prepared. Either alpha-aminoisobutyric acid (Aib) or N-methylalanine (MeAla) were inserted as replacement(s) for Ala7, Ala11, or Ala15 in the native NC sequence (FGGFTGARKSARKLANQ). In vitro assays measuring human ORL-1 receptor affinity (competition binding against [3H] NC), functional potency ([35S]GTP gamma S), and efficacy (as compared to NC) were performed for each new peptide. The receptor affinities of the Aib-containing peptides generally matched NC, showing K(i)'s in the range of 0.1-0.5 nM. By comparison, the receptor affinities of the MeAla-containing peptides were significantly diminished. Peptide 14 (FGGFTG[Aib]RKS[Aib]RKLANQ-NH2), which contains two constrained alanine residues (positions 7 and 11) and a C-terminal amide modification, was found to be a very potent agonist with K(i) = 0.05 nM and EC50 = 0.08 nM in the human ORL-1 assays. The data support a hypothesis that the receptor-bound form of NC might adopt an amphipathic helix in the "address" segment of the sequence.     

Synthesis of peptidocalix[4]arene libraries and their application to the development of chemical sensors for oligopeptides

Calix[4]arene libraries consisting of ca. 50000 members substituted with peptides at the lower rim were synthesized using encoded spilt synthesis with 15 amino acids. Screening of the library for binding with a dye-labeled oligopeptide indicated that some peptidocalix[4]arenes selectively bind the oligopeptide. The binding constant was estimated to be approximately 2 x 10(4) mol dm(-3). In an attempt to develop chemical sensors for peptides, fluorescence-labeled peptidocalix[4]arene libraries consisting of ca. 3500 members were synthesized. The fluorescence spectrum of peptidocalix[4]arene, which was found in the screening of libraries against the target peptide, was dependent on the concentration of the peptide. The libraries substituted at the upper rim were also synthesized with the aim of developing more selective and sensitive chemical sensors. The binding selectivity of the library members for the target peptides was higher and the behavior of the sensing was markedly different from the lower rim-modified peptidocalixarene sensor.     

Templated assembly of the pH‐sensitive membrane‐lytic peptide GALA

Abstract: Delivery of protein or nucleic acid therapeutics into intracellular compartments may require facilitation to allow these macromolecules to cross otherwise impermeant cellular membranes. Peptides capable of forming membrane‐spanning channels hold promise as just such facilitators, although the requirement for peptide oligomerization to form these channels may limit their effectiveness. Synthetic molecules containing multiple copies of membrane‐active peptides attached to a template molecule in a pre‐oligomerized form have attracted interest for drug‐delivery applications. Using three template designs, we synthesized multimeric versions of the pH‐sensitive lytic peptide GALA and compared their performance to monomeric GALA. Template assembly stabilized helix formation: templated GALA retained α‐helical structure even at neutral pH, unlike monomeric GALA. In membrane leakage assays, templated GALA retained the pH sensitivity of the monomer, with improved leakage for dimeric GALA. Surprisingly, trimeric GALA was less effective, particularly when synthesized with a larger and more flexible spacer. Surface plasmon resonance analysis indicated that reversible binding of templated GALA to lipid surfaces at acidic conditions was greatly reduced compared with monomeric GALA, but that the amount of irreversibly bound material was similar. We interpreted these results to indicate that templated peptides may cyclize into ‘self‐satisfied’ oligomeric structures, incapable of further aggregation and subsequent pore formation. Future design of templated peptides must be carefully performed to avoid this unwanted consequence.     

Peptides as receptor selectivity modulators of opiate pharmacophores

In an effort to investigate whether "address" segments of endogenous opioid peptides, which are responsible for modulating receptor selectivity, also could modulate the selectivity of opioid alkaloid pharmacophores, we have synthesized analogues of leucine-enkephalin and dynorphin in which the N-terminal dipeptide "message" sequence has been replaced by oxymorphone or naltrexone. A hydrazone group was employed as a linkage between the alkaloids and peptides. The binding data for mu, kappa, and delta receptors indicate that peptide portions of the analogues can modulate the receptor selectivity of the attached alkaloid pharmacophores. The selectivity for different opioid receptor types depends on a balance between the affinities of the message and address components. In cases where these components have comparable receptor affinities, the address can significantly shift selectivity by increasing affinity to one receptor type while reducing affinity to other types. When the message component has high affinity for a particular receptor type, the modulatory role of the address is expressed mainly by reducing the affinity of the ligand for other opioid receptor types.     

Identification of structural requirements for analogues of atrial natriuretic peptide (ANP) to discriminate between ANP receptor subtypes

The structure-activity relationships for affinity and selective binding of atrial natriuretic peptide (ANP) and analogues to guanylate cyclase coupled (CC) and non-cyclase coupled (NC) receptors in rabbit lung membranes are described. We have designed a series of peptides to try to identify the minimal sequence involved in specific recognition of each receptor subtype. The affinity of the peptides was determined from competitive binding experiments. Several peptides derived from the rat ANP sequence, e.g., des-[Phe106, Gly107, Ala115, Gln116]ANP-(103-125)NH2 (4), des-[Cys105,121]ANP-(104-126) (5), and [Acm-Cys105]ANP-(105-114)NH2 (9) have high affinity and selectivity for the noncoupled site. Peptide 4 was the most selective ligand with an affinity superior to that of ANP-(103-126). This compound does not displace the radiolabeled ligand from the guanylate cyclase coupled receptor at the highest concentration tested (100 nM). The structure-activity relationship for affinity and selectivity is discussed. Comparison of the peptide sequences suggests that the structural feature responsible for recognition of the NC site resides in a single sequence of seven contiguous amino acids from the cyclic core of the hormone. The corresponding heptapeptide retains affinity to the guanylate cyclase uncoupled binding site and is proposed to encompass the minimal sequence for specific recognition of the non-guanylate cyclase coupled ANP receptor.     

Neuropeptide Y (NPY) receptors in HEL cells: comparison of binding and functional parameters for full and partial agonists and a non-peptide antagonist.

1. We have compared the binding and Ca2+ mobilizing properties of various full agonists, partial agonists and a non-peptide antagonist at the neuropeptide Y (NPY) receptor of human erythroleukemia (HEL) cells. 2. [125I]-NPY binding to intact HEL cells was rapid, saturable, of high affinity and with a specificity typical for the Y1-like subtype: NPY, peptide YY (PYY) and [Pro34]-NPY competed for [125I]-NPY binding with high affinity whereas NPY13-36 and NPY18-36 had only low affinity. 3. NPY, PYY and [Pro34]-NPY potently increased intracellular Ca2+ in HEL cells and had equal efficacy. NPY13-36, vasoactive intestinal peptide (VIP) and pancreatic polypeptide (PP) increased intracellular Ca2+ only poorly. 4. Whereas VIP and PP did not significantly affect NPY-stimulated Ca2+ mobilization, NPY13-36 inhibited NPY-stimulated Ca2+ increases and shifted the NPY concentration-response curve to the right without altering its maximal effect. 5. The agonist (pEC50) potencies of the various peptides corresponded well with the affinities of these compounds in the binding assay (pKi), whereas the antagonist potencies (pKb) of the peptide partial agonists and the pA2 value of the non-peptide NPY antagonist (He 90481), calculated from functional data, were lower than the respective affinities determined in the binding studies. 6. A plot of the fractional Ca2+ response vs the fractional receptor occupancy did not reveal any non-linear receptor-effector coupling for NPY or [Pro34]-NPY; a small receptor reserve might exist for PYY. 7. We conclude that the binding and functional properties of HEL cell NPY receptors are very similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine binding sites at S-adenosylhomocysteine hydrolase are controlled by the NAD+/NADH ratio of the enzyme

S-Adenosylhomocysteine hydrolase (AdoHcy hydrolase) catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) to adenosine (Ado) and homocysteine. On the basis of the kinetics of Ado binding to AdoHcy hydrolase we have shown that AdoHcy hydrolase binds Ado with different affinities [Kidney Blood Press. Res. 19 (1996) 100]. Since AdoHcy hydrolase in its totally reduced form binds Ado with high affinity we determined in the present study the Ado binding characteristics of purified AdoHcy hydrolase from bovine kidney (native form) and of reconstituted forms with defined NAD(+)/NADH ratios. AdoHcy hydrolase in its native form and at a ratio of 50% NAD(+) and 50% NADH exhibits two binding sites for Ado with a K(D1) of 9.2+/-0.6 nmol/L and a K(D2) of 1.4+/-0.1 micromol/L, respectively. Binding of Ado to AdoHcy hydrolase in its NADH form and in its NAD(+) form exhibits only one binding site with high affinity 48.3+/-2.7 nmol/L for the NADH form and with a low affinity of 4.9+/-0.3 micromol/L for the NAD(+) form. To identify these two Ado binding sites, AdoHcy hydrolase was covalently modified with [2-3H]-8-azido-Ado. After irradiation of the native AdoHcy hydrolase two different photolabeled peptides were isolated and identified as Asp(307)-Val(325) and Tyr(379)-Thr(410). When the reconstituted AdoHcy hydrolase in its NADH and in its NAD(+) form was irradiated with [2-3H]-8-azido-Ado only one peptide was identified as Asn(312)-Lys(318) from the NADH form and as Asp(391)-Ala(396) from the NAD(+) form. Based on the crystallographic data, the labeled peptide Asp(391)-Ala(396) (low affinity binding site), appears to belong to the catalytic domain of AdoHcy hydrolase, whereas the labeled peptide, identified as Asn(312)-Lys(318) (high affinity binding site), is located in the NAD domain. In conclusion, our data show that AdoHcy hydrolase has two different Ado binding sites which are dependent upon the enzyme-bound NAD(+)/NADH ratios.     

The binding of [125I]-angiotensin to rat renal epithelial cell membranes.

1 Specific high affinity binding sites for [125I]-angiotensin II have been identified in crude basolateral and brush border membranes from rat cortex. 2 A central high affinity site, KD 0.62 nM; Bmax 299 fmol/mg was identified as part of a complex multicomponent binding system. 3 This high affinity site was saturable and exhibited specificity for angiotensin II analogues and closely related peptides but not for bradykinin, substance P or peptide fragments of angiotensin II. 4 Specific [125I]-angiotensin II binding was partially dependent on NaCl. Absence of NaCl resulted in a decrease in Bmax, had no effect on the rate of association but increased the rate of dissociation of [125I]-angiotensin from its binding site.     

Development and evaluation of endothelin-A receptor (radio)ligands for positron emission tomography

The expression and function of endothelin (ET) receptors are abnormal in cardiovascular diseases, tumor progression, and tumor metastasis. A previously reported promising radioligand for positron emission tomography (PET) based on the non-peptide ET(A) receptor antagonist PD 156707 showed specific binding to target receptors in the myocardium but high accumulation in bile and intestine, probably because of its high lipophilicity. In this study we describe the synthesis of a series of fluorinated derivatives with hydrophilic building blocks. All compounds were evaluated as high affinity ET(A) receptor ligands (16, 17, 23-26, K(i) = 1.4-7.9 nM) with high subtype selectivity over the ET(B) receptor. [(18)F]3-Benzo[1,3]dioxol-5-yl-4-{3-[1-(2-{2-[2-(2-fluoroethoxy)ethoxy]ethoxy}ethyl)-1H-[1,2,3]triazol-4-ylmethoxy]-4,5-dimethoxybenzyl}-5-hydroxy-5-(4-methoxyphenyl)-5H-furan-2-one ([(18)F]17) was synthesized as one of the radioligands of this series that possesses a higher hydrophilicity and an excellent stability in human serum. Improved clearance properties and specific uptake in target organs have been confirmed by biodistribution studies and small animal PET imaging.     

Function of negative charge in the "address domain" of deltorphins.

Deltorphins A, B, and C exhibit high delta-affinities (Ki = 0.12-0.31 nM) and very high delta-receptor binding selectivities (Ki mu/Ki delta = 1800-4100). A study of the delta-receptor binding properties of 15 deltorphin analogues focused primarily on the influence of the anionic group in the C-terminal tetrapeptide. Amidation of the beta-carboxyl groups of [Asp7], [Glu4], or [Asp4] in deltorphins A, B, and C, respectively, yielded peptides with enhanced mu-receptor affinities and minor changes in delta-affinities (Ki delta = 0.20-0.65 nM), but 5-8-fold diminished delta-selectivities. A free C-terminal carboxyl group markedly reduced delta-affinities and decreased delta-selectivities 6-11-fold; thus, the C-terminal amide group critically facilitates delta-affinity. Modifications in the anionic charged group or hydrophobic residues in the C-terminal tetrapeptide address domain of deltorphin A altered spatial distributions critical for delta-affinity and selectivity; e.g., [Nle6]deltorphin A enhanced mu-affinity and lowered delta-selectivity by two-thirds; the progressive, step-wise repositioning of Asp in deltorphin C (from position 4 to 7) was accompanied by linear decreases in delta-affinities and -selectivities, and increased mu-affinities of these peptides; enhancement of the charge density to -2, in [Asp6, Asp-OH7]deltorphin A, decreased delta-affinity and -selectivity, while [Asp4,5,His7]deltorphin A bound to neither mu- nor delta-sites. These results demonstrate that while an anionic group may occasionally facilitate high delta-receptor affinity, it represents an absolute requirement for the high delta-binding selectivity of these peptides. The locations of the charged groups relative to hydrophobic residues in the address domain of the peptide are also critical determinants for both delta-affinity and -selectivity.     

Benzo[a]carbazole derivatives. Synthesis, estrogen receptor binding affinities, and mammary tumor inhibiting activity

A number of 11-alkylbenzo[a]carbazoles and their 5,6-dihydro derivatives with one or two hydroxy groups in the aromatic rings were synthesized and studied for their binding affinities for the estrogen receptor. Best conditions for the receptor binding are provided by one hydroxy group at C-3 and a second one at position 8 or 9. The binding affinities of the benzo[a]carbazoles are somewhat lower than those of the dihydro derivatives but still high regarding the planar structure of these molecules. The highest relative binding affinity (RBA) values (e.g., 30 for 13b, 13 for 16b, 20 for 25a; estradiol = 100) are close to those of the corresponding 2-phenylindole derivatives. Depending on the positions of the oxygen functions, the benzo[a]carbazoles behaved as strong estrogens (13c, 25a) or impeded estrogens (16c, 28a) in the immature mouse. Derivative 16c inhibited the growth of dimethylbenzanthracene-induced hormone-dependent mammary tumors of the rat at a dose of 6 X 1 mg/kg per week. In vitro, 16b and 28b showed inhibitory activity on estrogen receptor positive MCF-7 breast cancer cells. A mode of action involving the estrogen receptor system is assumed.     

Design and biological evaluation of linear and cyclic phosphopeptide ligands of the N-terminal SH2 domain of protein tyrosine phosphatase SHP-1

In an effort to gain further insight into the conformational and topographical requirements for recognition by the N-terminal SH2 domain of protein tyrosine phosphatase SHP-1, we synthesized a series of linear and cyclic peptides derived from the sequence surrounding phosphotyrosine 2267 in the receptor tyrosine kinase Ros (EGLNpYMVL). A molecular modeling approach was used to suggest peptide modifications sterically compatible with the N-SH2-peptide binding groove and possibly enhanced binding affinities compared to the parent peptide. The potencies of the synthesized compounds were evaluated by assaying their ability to stimulate phosphatase activity as well as by their binding affinities to the GST-fused N-SH2 domain of SHP-1. In the series of linear peptides, structural modifications of Ros pY2267 in positions pY + 1 to pY + 3 by amino acid residues structurally related to Phe, for example l-erythro/threo-Abu(betaPh) (5a, 5b), yielded ligands with increased binding affinity. The incorporation of d-amino acid residues at pY + 1 and pY + 3 led to inactive peptides. The replacement of Phe in both pY + 1 and pY + 3 by Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) was also not tolerated due to steric hindrance. Cyclic peptides (13, 14) that were linked via residues in positions pY - 1 (Lys) and pY + 2 (Asp/Glu) and contained a Gly residue in the bridging unit displayed much lower potencies for the stimulation of SHP-1 activity but increased binding affinities compared to Ros pY2267. They partially competed with Ros pY2267 in the activation assay. Such cyclic structures may serve as scaffolds for competitive SHP-1 inhibitor design targeting N-SH2 domain-protein interactions that block SHP-1 activation.     

Novel (E)-alpha-[(1H-indol-3-yl)methylene]benzeneacetic acids as endothelin receptor ligands

Endothelin-1 (ET-1), a peptide of 21 amino acid residues, is the most potent vasoconstrictor substance known and now it is understood to be one of a family of three mammalian vasoactive peptides that also includes ET-2 and ET-3. The endothelins (ETs) affect multiple organ systems and seem to be involved in the pathogenesis of many diseases such as hypertension, pulmonary hypertension, atherosclerosis, apoptosis inhibition and angiogenesis. The ETs exert their effects via activation of two distinct G-protein coupled receptor subtypes termed ET(A) and ET(B). To date a number of ET receptor ligands with good affinity and selectivity is known, nevertheless these compounds belong only to few chemical classes. The aim of this work was the identification of a "hit compound" with novel chemical structure, endowed with reasonable ET affinity and selectivity. Accordingly, a new class of (E)-alpha-[(1H-indol-3-yl)methylene]benzeneacetic acid derivatives (1-23) was synthesized for evaluation of their binding profiles.     

Design and Synthesis of Potent Bivalent Peptide Agonists Targeting the EphA2 Receptor

相似文献   

The role of the C-terminus of the insulin B-chain in modulating structural and functional properties of the hormone

In memoriam Howard S. Tager Within the scope of structure-function studies on the proteohormone insulin, the role of the C-terminal segment B26-B30 for self-association and receptor interaction was analyzed. Insulin derivatives with modifications in the region B26-B30 were synthesized by trypsin-catalyzed coupling reactions of des-(B23-B30)-insulin with synthetic peptides. The peptides were obtained by Fmoc solid-phase peptide synthesis. Insulins with multiple amino acid → glycine substitutions were examined to distinguish between the influence of the side chains and the influence of the main chain in positions B27-B30 on the self-association of the hormone. The analogues [Gly^{B27,B28,B29,B30}]insulin and [Gly^B27,B28,B30]insulin exhibit relative receptor affinities of 80% and self-associate. The successive extension of [Ala^B26]des-(B27-B30)-insulin-B26-amide (relative receptor binding 273%) with amino xids corresponding to the native sequence B27-B30 showed the influence of the length of the B-chain on receptor affinity: the extension by B27-threonine amide reduces receptor binding to 71%, all further prolongations have only small effects on the binding. The effect of the B28-side chain on main-chain conformation, self-association and receptor binding was examined with [X^B28]des-(B29-B30)-insulin-B28-amides (X = Phe, Gly, D-Pro). While the glycine and D-proline analogues (relative binding 104 and 143%, respectively) retain the self-association properties typical of insulin, [Phe^B28]des-(B29-B30)-insulin-B28-amide (relative binding 50%) shows diminished self-association. The backbone-modified insulin derivative [Sar^B26]des-(B27-B30)-insulin-B26-amide (sarcosine =N-methylglycine) exhibits an unexpectedly high receptor affinity of 1100% which demonstrates that the B26-amide hydrogen of the native hormone is not important for receptor binding. © Munksgaard 1995.