Enhanced activity and stability of papain immobilized on CNBr-activated sepharose.

Immobilization of papain was carried out by covalent attachment on Sepharose 6B activated by using cyanogen bromide. Immobilization process brought about significant enhancement of storage and thermal stability, stability at extreme pHs, and resistance against the inhibitory effects of various bivalent metal ions with respect to papain. The optimum temperature of papain increased by 20 °C (from 60 to 80 °C) and its optimum pH was shifted from 6.5 to 8.0 upon immobilization. The activation energy of the enzymatic reaction for immobilized papain showed a significant increase as compared with its free form (1.87 kcal mol(-1) K(-1) for free and 4.69 kcal mol(-1) K(-1) for immobilized enzyme). The kinetic parameters, Km and kcat, were estimated to be 0.62 µM and 162 × 10(-4) s(-1) for free and 0.79 µM and 102 × 10(-4) s(-1) for immobilized papain, respectively.

[Efficient fusion expression of G13 domain derived from granulysin in Escherichia coli].

The G13 domain derived from granulysin shows high antimicrobial activities against Gram-positive and Gram-negative bacteria but does not lyse Jurkat cells or liposomes. To explore a new approach for high expression of the G13 domain, we fused the sequence encoding G13 to thioredoxin (Trx) gene to construct the recombinant expression vector (pThioHisA-G13). A cyanogen bromide (CNBr) cleavage site was introduced between the Trx and G13 to facilitate final release of the recombinant G13. The recombinant expression vector, pThioHisA-G13, was transformed into E. coli BL21 (DE3). Upon induction by IPTG Trx-G13 fusion protein was expressed and took the form of inclusion bodies counting 58% (W/W) of total cellular proteins. The inclusion body was solved by urea (8 mol/L) and then cleaved by CNBr. We purified the recombinant peptide G13 by one-step cation exchange chromatography. Results of agarose diffuse assay analysis indicated that the recombinant G13 exhibited antibacterial activity. The procedure described in this study will provide a reliable and simple method for highly efficient production of some cationic antimicrobial peptides.

Modification of the composition of articular cartilage collagen fibrils with increasing age.

Recent studies have identified a range of interactions between type IX collagen and other cartilage matrix components. To determine the extent to which these interactions are important in maintaining the integrity of ageing articular cartilage, we analyzed an age range of normal healthy articular cartilage samples by Western blotting, immunohistochemical, and PCR analyses. Reduced levels of type IX collagen were detected in post adolescence cartilage. Type IX collagen epitopes were evident throughout the matrix in all cartilage samples up to 19 years of age. Post adolescence, however, the pattern of immunoreactivity revealed territorial staining only. Type IX collagen expression at the transcriptional level is maintained at all ages. Type IX collagen fragments were extracted from young tissue, supporting the hypothesis that young cartilage is continually remodelled, while mature cartilage maintains relatively low levels of collagen turnover. Clearly the age changes we observed may have significant effects on the integrity of the tissue as the chondrocytes in ageing articular cartilage have limited capacity to turnover the interterritorial matrix. However, this study provides evidence that even in old age, the chondrocyte attempts to maintain its pericellular environment and hence its mechanical role. Therefore, the potential of type IX collagen to interact with other matrix components continues to be of importance in the territorial environment, and these interactions may have significant roles in mechanotransduction.

Hydrophobic interaction chromatography of soluble interleukin I receptor type II to reveal chemical degradations resulting in loss of potency.

A hydrophobic interaction chromatography method was developed to analyze recombinant soluble Interleukin 1 receptor type II (sIL-1R type II) drug substance and assess the stability of the drug under accelerated degradation studies. HIC resolved the degraded molecules into three peaks. A combination of several analytical techniques, including cyanogen bromide cleavage, reversed-phase chromatography, mass spectrometry, and N-terminal sequencing, were used to identify the origins of these peaks. We found that accelerated degradation resulted from three different events, deamidation and isomerization at asparagine 317 (Asn317), C-terminal cleavage, and aggregation. The iso-aspartate 317 (iso-Asp317)-containing species were shown to elute in HIC peak I and the Asp317-containing species in HIC peak II, respectively. Deamidation-isomerization to iso-Asp317, but not deamidation to Asp317, resulted in altered retention time on HIC companied by loss of potency, presumably by introducing a significant conformational change. CNBr C-terminal analysis showed that the inactive HIC peak I consisted of sIL-1R type II with "large" C-terminal truncations of 13 or 14 amino acids, whereas the active HIC peak II contained C-terminally full length and "small" C-terminal clips of two amino acids. Molecular modeling indicates that the short loop D317-S320, in the third domain of IL-1R type II, has a crucial impact on the stability of the molecule.

Effect of oversulfated chondroitin-6-sulfate or oversulfated fucoidan in the activation of glutamic plasminogen by tissue plasminogen activator: role of lysine and cyanogen bromide-fibrinogen.

Fucoidan and chondroitin-6-sulfate were oversulfated using chlorosulfonic acid-pyridine complex and were isolated as the sodium salt. Infrared analysis of oversulfated compounds showed introduction of sulfate groups in new positions, and in-vitro studies of the compounds showed a significant increase in the anticoagulant property. Addition of 28.6 microg/ml oversulfated compound gave a two-fold to four-fold increase in the rate of enhancement of activation of glutamic plasminogen by tissue plasminogen activator using 0.05 mol/l Tris buffer (pH 7.35) containing physiological concentrations of NaCl (0.9%). Under these conditions, unfractionated heparin was not active and the native compounds gave less than 30% enhancement. In the present study, the effect of lysine or cyanogen bromide-treated fibrinogen, alone or in combination with the oversulfated compounds, on the activation of glutamic plasminogen by tissue plasminogen activator was investigated. Addition of 16.2 mmol/l L-lysine gave three-fold to four-fold enhancement of activation, which was further enhanced to five-fold to six-fold by addition of 2.86 microg/ml oversulfated chondroitin-6-sulfate or oversulfated fucoidan. Cyanogen bromide-treated fibrinogen (50 microg/ml) gave a 10-fold enhancement of activation by itself, and addition of 2.86 microg/ml oversulfated compounds amplified this to 15-fold. A 25-fold to 35-fold enhancement of activation of glutamic plasminogen was obtained when 2.86 microg/ml oversulfated compounds were combined with 16.2 mmol/l lysine and 50 microg/ml cyanogen bromide-treated fibrinogen. Dilution studies showed that the amplification of the enhancement of lysine by 2.86 microg/ml oversulfated compound was related to interaction of the cofactors with both glutamic plasminogen and tissue plasminogen activator.

Cyanogen bromide peptides of the fibrillar collagens I, III, and V and their mass spectrometric characterization: detection of linear peptides, peptide glycosylation, and cross-linking peptides involved in formation of homo- and heterotypic fibrils.

The network of the fibrillar collagens I, III, and V, extracted from fetal calf skin and cleaved with cyanogen bromide, was studied by means of ultraviolet matrix-assisted laser desorption ionization time-of-flight mass spectrometry (UV-MALDI MS). Nearly all of the expected cyanogen bromide peptides of the different alpha chains were detected. Distinct peptides are identified that can serve as a reference signal for the individual alpha-chains. Homo- and heterotypic cross-linking patterns, some of which have not been described before for bovine collagen, are indicated by comparison of the mass spectrometric data with documented amino acid sequences. Potential cross-linking mechanisms are discussed. For example, the mass spectrometric data suggest that the formation of heterotypic I/III and I/V fibrils is substantially determined by the telo-regions of type I collagen, which are covalently connected to the corresponding helical and nonhelical cross-linking domains of adjacent molecules either by 4D or 0D-stagger bonds. The chemical nature of the cross-links can be concluded. The data also indicate a disturbed formation of heterotypic fibrils. Finally, collagen glycosylation can also be identified.

Overexpression and purification of single zinc finger peptides of human zinc finger protein ZNF191.

ZNF191, a new human zinc finger protein, probably relates to some hereditary diseases and cancers. To obtain structural information of zinc finger domain a convenient method for obtaining milligram quantities of each zinc finger peptide of ZNF191 is necessary. Here, we report an Escherichia coli expression system for rapid and high-level expression of zinc finger 3 and zinc finger 4 of ZNF191. The gene of zinc finger 3 or zinc finger 4 was cloned into pET31b vector to allow expression of single zinc finger peptide as a ketosteroid isomerase (KSI) fusion protein. The KSI-single zinc finger fusion protein was overexpressed in the form of inclusion body, which can be purified by washing several times using buffer solutions, and then be cleaved directly by cyanogen bromide to release single zinc finger peptide. The more than 20mg/L yield of single zinc finger peptide was achieved with more than 95% purity by using YM ultrafiltration membranes. Circular dichroism spectra of these two single zinc finger peptides titrated with Zn(2+) ions demonstrate that they have different secondary structures.

Localization of cocaine analog [125I]RTI 82 irreversible binding to transmembrane domain 6 of the dopamine transporter.

The site of cocaine binding on the dopamine transporter (DAT) was investigated using the photoactivatable irreversible cocaine analog [125I]3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([125I]RTI 82). The incorporation site of this compound was mapped to transmembrane domains (TMs) 4-6 using epitope-specific immunoprecipitation of trypsin fragments and further localized using cyanogen bromide (CNBr), which hydrolyzes proteins on the C-terminal side of methionine residues. CNBr hydrolysis of [125I]RTI 82-labeled rat striatal and expressed human DATs produced fragments of approximately 5-10 kDa consistent with labeling between Met(271/272) or Met(290) in TM5 to Met(370/371) in TM7. To further define the incorporation site, substitution mutations were made that removed endogenous methionines and inserted exogenous methionines in combinations that would generate labeled CNBr fragments of distinct masses depending on the labeling site. The results obtained were consistent with the presence of TM6 but not TMs 4, 5, or 7 in the labeled fragments, with additional support for these conclusions obtained by epitope-specific immunoprecipitation and secondary digestion of CNBr fragments with endoproteinase Lys-C. The final localization of [125I]RTI 82 incorporation to rat DAT Met(290)-Lys(336) and human DAT I291M to R344M provides positive evidence for the proximity of cocaine binding to TM6. Residues in and near DAT TM6 regulate transport and transport-dependent conformational states, and TM6 forms part of the substrate permeation pathway in the homologous Aquifex aeolicus leucine transporter. Cocaine binding near TM6 may thus overlap the dopamine translocation pathway and function to inhibit TM6 structural rearrangements necessary for transport.

Disulfide linkages and a three-dimensional structure model of the extracellular ligand-binding domain of guanylyl cyclase C.

Guanylyl cyclase C (GC-C) is a single-transmembrane receptor that is specifically activated by endogenous ligands, including guanylin, and the exogenous ligand, heat-stable enterotoxin. Using combined HPLC separation and MS analysis techniques the positions of the disulfide linkages in the extracellular ligand-binding domain (ECD) of GC-C were determined to be between Cys7-Cys94, Cys72-Cys77, Cys101-Cys128 and Cys179-Cys226. Furthermore, a three-dimensional structural model of the ECD was constructed by homology modeling, using the structure of the ECD of GC-A as a template (van den Akker et al., 2000, Nature, 406: 101-104) and the information of the disulfide linkages. Although the GC-C model was similar to the known structure of GC-A, importantly its ligand-binding site appears to be located on the quite different region from that in GC-A.

Paired cysteine mutagenesis to establish the pattern of disulfide bonds in the functional intact secretin receptor.

The amino-terminal domain of class B G protein-coupled receptors contains six conserved cysteine residues involved in structurally and functionally critical disulfide bonds. The mapping of these bonds has been unclear, with one pattern based on biochemical and NMR structural characterizations of refolded, nonglycosylated amino-terminal fragments, and another pattern derived from functional characterizations of intact receptors having paired cysteine mutations. In the present study, we determined the disulfide bonding pattern of the prototypic class B secretin receptor by applying the same paired cysteine mutagenesis approach and confirming the predicted bonding pattern with proteolytic cleavage of intact functional receptor. As expected, systematic mutation to serine of the six conserved cysteine residues within this region of the secretin receptor singly and in pairs resulted in loss of function of most constructs. Notable exceptions were single mutations of the 4th and 6th cysteine residues and paired mutations involving the 1st and 3rd, 2nd and 5th, and 4th and 6th conserved cysteines, with secretin eliciting statistically significant cAMP responses above basal levels of activation for each of these constructs. Immunofluorescence microscopy confirmed similar levels of plasma membrane expression for each of the mutated receptors. Furthermore, cyanogen bromide cleaved a series of wild type and mutant secretin receptors, yielding patterns that agreed with our paired cysteine mutagenesis results. In conclusion, these data suggest the same pattern of disulfide bonding as that predicted previously by NMR and thus support a consistent pattern of amino-terminal disulfide bonds in class B G protein-coupled receptors.

Reformable intramolecular cross-linking of the N-terminal domain of heparin cofactor II: effects on enzyme inhibition.

The crystal structure of a heparin cofactor II (HCII)-thrombin Michaelis complex has revealed extensive contacts encompassing the N-terminal domain of HCII and exosite I of the proteinase. In contrast, the location of the N-terminal extension in the uncomplexed inhibitor was unclear. Using a disulfide cross-linking strategy, we demonstrate that at least three different sites (positions 52, 54 and 68) within the N terminus may be tethered in a reformable manner to position 195 in the loop region between helix D and strand s2A of the HCII molecule, suggesting that the N-terminal domain may interact with the inhibitor scaffold in a permissive manner. Cross-linking of the N terminus to the HCII body does not strongly affect the inhibition of alpha-chymotrypsin, indicating that the reactive site loop sequences of the engineered inhibitor variants, required for interaction with one of the HCII target enzymes, are normally accessible. In contrast, intramolecular tethering of the N-terminal extension results in a drastic decrease of alpha-thrombin inhibitory activity, both in the presence and in the absence of glycosaminoglycans. Treatment with dithiothreitol and iodoacetamide restores activity towards alpha-thrombin, suggesting that release of the N terminus of HCII is an important component of the multistep interaction between the inhibitor and alpha-thrombin.

Sequence-independent acylation of the vaccinia virus A-type inclusion protein.

N-Terminal myristoylation of proteins typically occurs cotranslationally via an amide bond to the penultimate glycine residue within the canonical motif (M)GXXX(S/T/A) in a reaction catalyzed by N-myristoyltransferase. A second, less common myristoylation reaction occurs internally at dibasic amino acid doublets of proteins such as alpha-TNF. In this case, myristoylation occurs within a portion of the preprotein, which is subsequently removed by N-terminal proteolysis. The identity of the enzyme catalyzing internal myristoylation is unknown. Considering this information, the vaccinia virus (VV) A-type inclusion protein (ATI) presents a conundrum. Although this cytosolic protein is clearly myristoylated, the protein does not have the N-terminal myristoylation motif nor is it subject to proteolytic maturation. In the experiments reported here, we cleaved VV ATI with cyanogen bromide and determined that the myristoyl moiety was present in the C-terminal half of the protein. We also subjected a tryptic digest of VV ATI to liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry analyses, which indicated that ATI is randomly myristoylated at six different lysines or arginines. Analysis of the modification sites reveals no obvious conserved acceptor motifs or dibasic doublets. Mutation of these residues alone or in combination does not abrogate myristoylation of the protein, suggesting utilization of alternative modification sites. This information implies that the VV ATI protein is myristoylated in a sequence-independent manner. Because viral acylproteins typically utilize the host cell modification apparatus, this result suggests there may be an alternative type of myristoylation pathway in mammalian cells.

Histidine 167 is the phosphate acceptor in glucose-6-phosphatase-beta forming a phosphohistidine enzyme intermediate during catalysis.

The glucose-6-phosphatase (Glc-6-Pase) family comprises two active endoplasmic reticulum (ER)-associated isozymes: the liver/kidney/intestine Glc-6-Pase-alpha and the ubiquitous Glc-6-Pase-beta. Both share similar kinetic properties. Sequence alignments predict the two proteins are structurally similar. During glucose 6-phosphate (Glc-6-P) hydrolysis, Glc-6-Pase-alpha, a nine-transmembrane domain protein, forms a covalently bound phosphoryl enzyme intermediate through His(176), which lies on the lumenal side of the ER membrane. We showed that Glc-6-Pase-beta is also a nine-transmembrane domain protein that forms a covalently bound phosphoryl enzyme intermediate during Glc-6-P hydrolysis. However, the intermediate was not detectable in Glc-6-Pase-beta active site mutants R79A, H114A, and H167A. Using [(32)P]Glc-6-P coupled with cyanogen bromide mapping, we demonstrated that the phosphate acceptor in Glc-6-Pase-beta is His(167) and that it lies inside the ER lumen with the active site residues, Arg(79) and His(114). Therefore Glc-6-Pase-alpha and Glc-6-Pase-beta share a similar active site structure, topology, and mechanism of action.

Spatial proximity between a photolabile residue in position 19 of salmon calcitonin and the amino terminus of the human calcitonin receptor.

Calcitonins are 32-amino acid peptide hormones with both peripheral and central actions mediated via specific cell surface receptors, which belong to the class II subfamily of G protein-coupled receptors. Understanding receptor function, particularly in terms of ligand recognition by calcitonin receptors, may aid in the rational design of calcitonin analogs with increased potency and improved selectivity. To directly identify sites of proximity between calcitonin and its receptor, we carried out photoaffinity labeling studies followed by protein digestion and mapping of the radiolabeled photoconjugated receptor. A fully active salmon calcitonin analog [Arg(11,18),Bpa19]sCT, incorporating a photolabile p-benzoyl-L-phenylalanine into position 19 of the ligand, has been used to demonstrate spatial proximity between residue 19 of the peptide and the amino-terminal extracellular domain of the receptor. Cyanogen bromide cleavage together with endoproteinase Asp-N digestion indicated that binding was predominantly to the region delimited by receptor residues Cys134 and Met187. Binding to this fragment was supported further by cyanogen bromide-digestion of receptors that were mutated to remove the predicted cleavage site at Met133 (M133A, M133L). Binding within the 54-amino acid fragment was refined further by digestion with endoproteinase Lys-C to the 8-amino acid region corresponding to Cys134-Lys141. These results provide the first direct demonstration of a contact domain between salmon calcitonin and its receptor and will contribute toward modeling of the calcitonin-receptor interface.

Identification of a hexapeptide binding region in the nociceptin (ORL1) receptor by photo-affinity labelling with Ac-Arg-Bpa-Tyr-Arg-Trp-Arg-NH2.

The interaction of Ac-Arg-Tyr-Tyr-Arg-Trp-Arg-NH(2) (HP1), a high-affinity partial agonist of the opioid receptor like (ORL1) receptor, has been investigated using the photo-labile analogue [p-benzoyl-L-Phe (Bpa)(2)]-HP1. In recombinant CHO cells expressing the human ORL1 receptor, [Bpa(2)]-HP1 binds the receptor with high affinity (K; approximately 3nM) and is as potent as HP1 in stimulating GTPgammaS binding (50-60% of nociceptin maximal effect). UV irradiation at 365nm of the complex formed by the ORL1 receptor and radio-iodinated [Bpa(2)]-HP1 results in the irreversible labelling of a glycoprotein of M(r) approximately 66kDa, as determined by SDS-PAGE. Cyanogen bromide (CNBr) and enzymatic footprints of the photo-labelled receptor and an engineered receptor mutant (L113M), containing an additional CNBR cleavage site, allowed the photoreactive region to be identified as ORL1[107-113] at the C-terminal of TM helix II. In addition the presence of a disulphide bridge between Cysl23 and Cys200 has been confirmed biochemically.

Spatial approximation between two residues in the mid-region of secretin and the amino terminus of its receptor. Incorporation of seven sets of such constraints into a three-dimensional model of the agonist-bound secretin receptor.

Photoaffinity labeling of receptors by bound agonists can provide important spatial constraints for molecular modeling of activated receptor complexes. Secretin is a 27-residue peptide hormone with a diffuse pharmacophoric domain that binds to the secretin receptor, a prototypic member of the Class B family of G protein-coupled receptors. In this work, we have developed, characterized, and applied two new photolabile probes for this receptor, with sites for covalent attachment in peptide positions 12 and 14, surrounding the previously most informative site of affinity labeling of this receptor. The [Tyr10,(BzBz)Lys12]rat secretin-27 probe covalently labeled receptor residue Val6, whereas the [Tyr10,(BzBz)Lys14]rat secretin-27 probe labeled receptor residue Pro38. When combined with previous photoaffinity labeling data, there are now seven independent sets of constraints distributed throughout the peptide and receptor amino-terminal domain that can be used together to generate a new molecular model of the ligand-occupied secretin receptor. The amino-terminal domain of this receptor presented a stable platform for peptide ligand interaction, with the amino terminus of the peptide hormone extended toward the transmembrane helix domain of the receptor. This provides clear insights into the molecular basis of natural ligand binding and supplies testable hypotheses regarding the molecular basis of activation of this receptor.

Role of elastin in pathologic calcification of xenograft heart valves.

Bioprosthetic heart valves fabricated from glutaraldehyde crosslinked porcine aortic valves often fail because of calcific degeneration. Calcification occurs in both cusp and aortic wall portions of bioprosthetic heart valves. The purpose of this study was to discern the role of different aortic wall components in the calcification process. Thus, we selectively extracted cells and other extracellular matrix proteins from porcine aorta using trypsin/DNase/RNase, cyanogen bromide (CNBr), and sodium hydroxide (NaOH) treatments and subdermally implanted these pretreated aortas in young rats. Total DNA and phospholipid data showed complete removal of cells by CNBr and NaOH treatments, whereas trypsin/DNase/RNase treatment was effective in removing DNA but not phospholipids. As shown by amino acid data and Masson's trichrome staining, collagen was removed in CNBr and NaOH treatments. Control fresh porcine aorta calcified significantly after 21 days of implantation (Ca 26.4 +/- 2.4 microg/mg). Removal of cells and collagen from the aorta by CNBr treatment did not lead to a statistically significant reduction in aortic calcification (Ca 20.8 +/- 3.0 microg/mg). Moreover, partial degradation of elastin fibers caused by NaOH (during extraction) and trypsin treatment (after implantation) of the aorta significantly increased elastin-oriented calcification (Ca 94.4 +/- 9.3 and 58.4 +/- 4.6 microg/mg, respectively). Our results indicate that the elastin component of the aorta may undergo independent calcification irrespective of devitalized cell-mediated calcification observed in glutaraldehyde crosslinked aortas. Our results also demonstrate the importance of studying elastin-oriented calcification in decellularized elastin-rich aortic matrices currently used in tissue-engineering applications.

Met174 side chain is the site of photoinsertion of a substance P competitive peptide antagonist photoreactive in position 8.

Numerous photoaffinity studies of the NK-1 receptor have been carried out with peptide agonist analogues of substance P (SP). However, no information is available with regard to the domain interaction of peptide antagonists within this receptor. We describe herein the photoaffinity labelling of the SP receptor with a peptide antagonist analogue, Bapa(0)[(pBzl)Phe(8),DPro(9),MePhe(10),Trp(CHO)(11)]SP. Photolabelling, enzymatic or chemical cleavage of the covalent complex, purification via streptavidin-coated beads and matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis led us to show that the methyl of Met174 side chain, within the receptor's second extracellular loop, is covalently linked to the antagonist photoreactive at position 8.

Efficient expression of isotopically labeled peptides for high resolution NMR studies: application to the Cdc42/Rac binding domains of virulent kinases in Candida albicans.

The production of bioactive peptides and small protein fragments is commonly achieved via solid-phase chemical synthesis. However, such techniques become unviable and prohibitively expensive when the peptides are large (e.g., >30 amino acids) or when isotope labeling is required for NMR studies. Expression and purification of large quantities of unfolded peptides in E. coli have also proved to be difficult even when the desired peptides are carried by fusion proteins such as GST. We have developed a peptide expression system that utilizes a novel fusion protein (SFC120) which is highly expressed and directs the peptides to inclusion bodies, thereby minimizing in-cell proteolysis whilst maintaining high yields of peptide expression. The expressed peptides can be liberated from the carrier protein by CNBr cleavage at engineered methionine sites or through proteolysis by specific proteases for peptides containing methionine residues. In the present systems, we use CNBr, due to the absence of methionine residues in the target peptides, although other cleavage sites can be easily inserted. We report the production of six unfolded protein fragments of different composition and lengths (19 to 48 residues) derived from the virulent effector kinases, Cla4 and Ste20 of Candida albicans. All six peptides were produced with high yields of purified material (30-40 mg/l in LB, 15-20 mg/l in M9 medium), pointing to the general applicability of this expression system for peptide production. The enrichment of these peptides with (15)N, (15)N/(13)C and even (15)N/(13)C/(2)H isotopes is presented allowing speedy assignment of poorly-resolved resonances of flexible peptides.

Photoaffinity labeling demonstrates physical contact between vasoactive intestinal peptide and the N-terminal ectodomain of the human VPAC1 receptor.

Vasoactive intestinal peptide (VIP) is a prominent neuropeptide whose actions are mediated by VPAC receptors belonging to class II G protein-coupled receptors. To identify contact sites between VIP and its VPAC1 receptor, an analog of VIP substituted with a photoreactive para-benzoyl-l-Phe (Bpa) at position 22 has been synthesized and evaluated in Chinese hamster ovary cells stably expressing the recombinant human receptor. Bpa22-VIP and native VIP are equipotent in stimulating adenylyl cyclase activity in cell membranes. Cyanogen bromide cleavage of the covalent 125I-[Bpa22-VIP]-hVPAC1R complex yielded a single labeled fragment of 30 kDa that shifted to 11 after deglycosylation, most consistent with the 67-137 fragment of the receptor N-terminal ectodomain. Further cleavage of this fragment with V8 endoproteinase and creation of receptor mutants with new CNBr cleavage sites (XàMet), demonstrated that 125I-[Bpa22-VIP] was covalently attached to the short receptor 109-120 fragment (GWTHLEPGPYPI). In a three-dimensional model of the receptor N-terminal ectodomain, this fragment is located on one edge of the putative VIP binding groove and encompasses several amino acids previously shown to be crucial for VIP binding (reviewed in Laburthe, M., Couvineau, A., and Marie, J. C. (2002) Receptors Channels 8, 137-153). Our data provide the first direct evidence for a physical contact between VIP and the N-terminal ectodomain of the hVPAC1 receptor.