1H-NMR assignment and secondary structure of a herpes simplex virus glycoprotein D-1 antigenic domain

The peptide alpha Ahx-Met-Ala-Asp-Pro-Asn-Arg-Phe-Arg-Gly-Lys-Asp-Leu-Pro-Val-Leu- Asp-Gln-Leu-Thr-Asp-Pro-Pro-alpha Ahx (epsilon Ahx = 6-aminohexanoyl), the antigenic sequence 11-32 from Herpes simplex virus glycoprotein D-1, has been synthesised. Its 1H-NMR spectrum has been assigned by a combination of two-dimensional techniques in H2O and 2H2O. Its secondary structure has been defined by nuclear Overhauser effects and amide proton exchange rates, and also to some extent chemical shifts, coupling constants and amide proton temperature coefficients. These latter parameters are shown to be less reliable as guides to secondary structure. The peptide has a helical (type I/III) turn at residues Pro-14-Asn-15 and helical structure at residues Lys-20-Val-24, in rapid equilibrium with random-coil structure. A beta-turn at residues Arg-18-Gly-19 may be present as a minor component. These locations of secondary structure correspond with previously determined regions of antigenic activity.     

Peptide Subunits of γ-Aminobutyric AcidA/Benzodiazepine Receptors from Bovine Cerebral Cortex

The gamma-aminobutyric acidA/benzodiazepine receptor complexes from bovine cerebral cortex were purified by immunoaffinity chromatography, and the main component peptide subunits were characterized. The peptide band originally thought to be a single beta subunit [57,000 Mr band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] is composed of at least four different peptides of 54,000-57,000 Mr. Two peptides of 55,000 and 57,000 Mr were recognized by the beta subunit-specific monoclonal antibody 62-3G1. Peptides in the range of 54,000-57,000 Mr were photoaffinity-labeled with [3H]muscimol. A different 57,000 Mr peptide was photoaffinity-labeled by [3H]flunitrazepam, but neither was recognized by the monoclonal antibody 62-3G1 nor photoaffinity-labeled with [3H]muscimol. Some peptides could be identified by their differential mobility shift in SDS-PAGE after treatment with endoglycosidase H. Two additional subunit peptides of 51,000 and 53,000 Mr were also photoaffinity-labeled by [3H]flunitrazepam and reacted with antiserum A. However, the 57,000 Mr peptide that also was photoaffinity-labeled by [3H]flunitrazepam did not react with antiserum A.     

The structure of an antigenic determinant in a protein

The immunogenic and antigenic determinants of a synthetic peptide and the corresponding antigenic determinants in the parent protein have been elucidated. Four determinants have been defined by reactivity of a large panel of antipeptide monoclonal antibodies with short, overlapping peptides (7-28 amino acids), the immunizing peptide (36 amino acids), and the intact parent protein (the influenza virus hemagglutinin, HA). The majority of the antipeptide antibodies that also react strongly with the intact protein recognize one specific nine amino acid sequence. This immunodominant peptide determinant is located in the subunit interface in the HA trimeric structure. The relative inaccessibility of this site implies that antibody binding to the protein is to a more unfolded HA conformation. This antigenic determinant differs from those previously described for the hemagglutinin and clearly demonstrates the ability of synthetic peptides to generate antibodies that interact with regions of the protein not immunogenic or generally accessible when the protein is the immunogen.     

Subunit location and sequences of the cysteinyl peptides of pig heart NAD-dependent isocitrate dehydrogenase

Pig heart NAD-dependent isocitrate dehydrogenase has a subunit structure consisting of alpha 2 beta gamma, with the alpha subunit exhibiting a molecular weight of 39,000 and the beta and gamma each having molecular weights of 41,000. The amino-terminal sequences (33-35 residues) and the cysteinyl peptide sequences have now been determined by using subunits separated by chromatofocusing or isoelectric focusing and electroblotting. Displacement of the N-terminal sequence of the alpha subunit by 11-12 amino acids relative to that of the larger beta and gamma subunits reveals a 17 amino acid region of great similarity in which 10 residues are identical in all three subunits. The complete enzyme has 6.0 free SH groups per average subunit of 40,000 daltons, but yields 15 distinguishable cysteines in isolated tryptic peptides. Six distinct cysteines in sequenced peptides have been located in the alpha subunit. The beta and gamma subunits contain seven and five cysteines, respectively, with tryptic peptides containing three cysteines being common to the beta and gamma subunits. The three subunits appear to be closely related, but beta and gamma are more similar to each other than either is to the alpha subunit. The NAD-specific isocitrate dehydrogenase from pig heart has been shown to have 2 binding sites/enzyme tetramer for isocitrate, manganous ion, NAD+, and the allosteric activator ADP [Colman, R. F. (1983) Pept. Protein Rev. 1, 41-69]. It is proposed that the catalytically active tetrameric enzyme is organized as a dimer of dimers in which the alpha beta and alpha gamma dimers are nonidentical but functionally similar.     

MSP-1 malaria pseudopeptide analogs: biological and immunological significance and three-dimensional structure

Merozoite Surface Protein-1 (MSP-1) has been considered as a malaria vaccine candidate. It is processed during the Plasmodium falciparum invasion process of red blood cells (RBCs). A conserved MSP-1 C-terminal peptide was identified as a high-activity erythrocyte-binding peptide (HAEBP) termed 1585. Since conserved HAEBPs are neither antigenic nor immunogenic we decided to assess the significance of a single peptide bond replacement in 1585. Thus, two pseudopeptides were obtained by introducing a Y[CH2-NH] reduced amide isoster into the 1585 critical binding motif. The pseudopeptides bound to different HLA-DR alleles, suggesting that backbone modifications affect MHC-II binding patterns. Pseudopeptide-antibodies inhibit in vitro parasite RBC invasion by recognizing MSP-1. Each pseudopeptide-induced antibody shows distinct recognition patterns. 1H-NMR studies demonstrated that isoster bonds modulate the pseudopeptides' structure and thus their immunological properties, therefore representing a possible subunit malaria vaccine component.     

Multiple very late antigen (VLA) heterodimers on platelets. Evidence for distinct VLA-2, VLA-5 (fibronectin receptor), and VLA-6 structures

After removal of very late antigen (VLA) 2 material from a radiolabeled detergent lysate of platelets, another VLA heterodimer was precipitated using antibody to the common VLA beta subunit. This structure was identified as VLA-5 because it contained VLA beta plus an alpha subunit that was (i) recognized by anti-alpha 5 antibodies and (ii) cleaved by V8 protease to yield a characteristic alpha 5-like pattern of peptide fragments. Besides VLA-2 and VLA-5, a third heterodimer, here named VLA-6, was also present on platelets. VLA-6 (an alpha 6 beta complex) was defined using the monoclonal antibody GoH3 (Sonnenberg, A., Janssen, H., Hogervorst, F., Calafat, J., and Hilgers, J. (1987) J. Biol. Chem. 262, 10376-10383). Although it resembled VLA-5 in size, VLA-6 was different from VLA-5 because (i) removal of the alpha 5 subunit did not remove alpha 6, (ii) removal of alpha 6 by the GoH3 antibody did not remove alpha 5, (iii) the alpha 5 and alpha 6 subunits had very distinct one-dimensional V8 peptide maps, and (iv) the alpha 6 and alpha 5 subunits had distinct migration patterns on two-dimensional O'Farrell gels. The beta subunit of VLA-6 was identified as the common VLA beta subunit because (i) it was recognized by anti-VLA beta antibody and (ii) it yielded a V8 protease cleavage map characteristic of beta. VLA-6 was not readily seen in anti-VLA beta immunoprecipitations, apparently because the alpha 6 subunit is only loosely or partially associated with the VLA beta subunit. Because VLA-5 and VLA-6 both closely resemble the previously defined Ic-IIa platelet protein complex, it is likely that there is more than one platelet "Ic" protein complexed with IIa.     

Topographic analysis of antigenic determinants recognized by monoclonal antibodies to the photoreceptor guanyl nucleotide-binding protein, transducin

Antigenic sites for six monoclonal antibodies that bind to the alpha subunit (G alpha) of the photoreceptor guanyl nucleotide-binding protein (G-protein or transducin) have been determined. Five of these antibodies (4A, 7A, 7B, 7C, and 7D) were shown in the preceding paper (Hamm, H. E., Deretic, D., Hofmann, K. P., Schleicher, A., and Kohl, B. (1987) J. Biol. Chem. 262, 10831-10838) to block G-protein-rhodopsin interaction. We have blotted tryptic and chymotryptic peptides of G-protein to nitrocellulose paper and found that these antibodies bind to peptides that contain the COOH-terminal end of the protein assessed by 32P-ADP-ribosylation of the COOH-terminus by pertussis toxin. The antigenic site is not exactly at the COOH-terminus since the antibodies also bind two peptides which lack a 2-kDa piece from the COOH-terminus. Antigenic sites are therefore on the 7-kDa chymotryptic peptide and 5-kDa tryptic peptide more than 2 kDa away from the COOH-terminus. Further evidence for this antigenic site comes from the ability of these antibodies to block pertussis toxin-mediated ADP-ribosylation while still binding to the previously ADP-ribosylated protein both on nitrocellulose blots and in immunoprecipitations. Antibody 4H, which was shown not to interrupt any of the functions studied, binds to the 11-kDa major tryptic fragment. To aid in the mapping of these sites onto the surface of G alpha, a model of the three-dimensional structure of G alpha has been generated using the G alpha primary sequence, predicted secondary structure, hydropathy plot, and the constraints of the GDP-binding site of the GTP-binding protein elongation factor Tu solved by Jurnak (Jurnak, F. (1985) Science 230, 32-36).     

Molecular basis of ligand recognition by integrin alpha5beta 1. II. Specificity of arg-gly-Asp binding is determined by Trp157 OF THE alpha subunit

Different beta(1) integrins bind Arg-Gly-Asp (RGD) peptides with differing specificities, suggesting a role for residues in the alpha subunit in determining ligand specificity. Integrin alpha(5)beta(1) has been shown to bind with high affinity to peptides containing an Arg-Gly-Asp-Gly-Trp (RGDGW) sequence but with relatively low affinity to other RGD peptides. The residues within the ligand-binding pocket that determine this specificity are currently unknown. A cyclic peptide containing the RGDGW sequence was found to strongly perturb the binding of the anti-alpha(5) monoclonal antibody (mAb) 16 to alpha(5)beta(1). In contrast, RGD peptides lacking the tryptophan residue acted as weak inhibitors of mAb 16 binding. The epitope of mAb 16 has previously been localized to a region of the alpha(5) subunit that contains Ser(156)-Trp(157). Mutation of Trp(157) (but not of Ser(156) or surrounding residues) to alanine blocked recognition of mAb 16 and perturbed the high affinity binding of RGDGW-containing peptides to alpha(5)beta(1). The same mutation also abrogated recognition of the alpha(5)beta(1)-specific ligand peptide Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA). Based on these findings, we propose that Trp(157) of alpha(5) participates in a hydrophobic interaction with the tryptophan residue in RGDGW, and that this interaction determines the specificity of alpha(5)beta(1) for RGDGW-containing peptides. Since the RGD sequence is recognized predominantly by amino acid residues on the beta(1) subunit, our results suggest that Trp(157) of alpha(5) must lie very close to these residues. Our findings therefore provide new insights into the structure of the ligand-binding pocket of alpha(5)beta(1).     

Structure of the pore-forming transmembrane domain of a ligand-gated ion channel

The structure of the pore-forming transmembrane domain of the nicotinic acetylcholine receptor from Torpedo has been investigated by infrared spectroscopy. Treatment of affinity-purified receptor with either Pronase or proteinase K digests the extramembranous domains (roughly 75% of the protein mass), leaving hydrophobic membrane-imbedded peptides 3-6 kDa in size that are resistant to peptide (1)H/(2)H exchange. Infrared spectra of the transmembrane domain preparations exhibit relatively sharp and symmetric amide I and amide II band contours centered near 1655 and 1545 cm(-)1, respectively, in both (1)H(2)O and (2)H(2)O. The amide I band is very similar to the amide I bands observed in the spectra of alpha-helical proteins, such as myoglobin and bacteriorhodopsin, that lack beta structure and exhibit much less beta-sheet character than is observed in proteins with as little as 20% beta sheet. Curve-fitting estimates 75-80% alpha-helical character, with the remaining peptides likely adopting extended and/or turn structures at the bilayer surface. Infrared dichroism spectra are consistent with transmembrane alpha-helices oriented perpendicular to the bilayer surface. The evidence strongly suggests that the transmembrane domain of the nicotinic receptor, the most intensively studied ligand-gated ion channel, is composed of five bundles of four transmembrane alpha-helices.     

Gαs protein C‐terminal α‐helix at the interface: does the plasma membrane play a critical role in the Gαs protein functionality?

The heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins, Galphabetagamma) mediate the signalling process of a large number of receptors, known as G protein-coupled receptors. The C-terminal domain of the heterotrimeric G protein alpha-subunit plays a key role in the selective activation of G proteins by their cognate receptors. The interaction of this domain can take place at the end of a cascade including several successive conformational modifications. Galpha(s)(350-394) is the 45-mer peptide corresponding to the C-terminal region of the Galpha(s) subunit. In the crystal structure of the Galpha(s) subunit it encompasses the alpha4/beta6 loop, the beta6 beta-sheet segment and the alpha5 helix region. Following a previous study based on the synthesis, biological activity and conformational analysis of shorter peptides belonging to the same Galpha(s) region, Galpha(s)(350-394) was synthesized and investigated. The present study outlines the central role played by the residues involved in the alpha4/beta6 loop and beta6/alpha5 loops in the stabilization of the C-terminal Galpha(s)alpha-helix. H(2)O/(2)H(2)O exchange experiments, and NMR diffusion experiments show interesting evidence concerning the interaction between the SDS micelles and the polypeptide. These data prompt intriguing speculations on the role of the intracellular environment/cellular membrane interface in the stabilization and functionality of the C-terminal Galpha(s) region.     

Conformations and pharmacophores of cyclic RGD containing peptides which selectively bind integrin alpha(v)beta3.

This paper reports a detailed conformational characterization in solution by 1H-NMR in H2O and DMSO-d6 and molecular modeling simulations of cyclic peptides containing the RGDDV pharmacophore and the RGDY(Me)R pharmacophore. These two pentapeptide sequences when properly constrained in cyclic peptides are low to sub-nanomolar inhibitors of integrin alpha(v)beta3. The peptides containing the RGDDY(Me)R sequence bind potently to integrin alphaIIb3 as well. The conformations found in H2O and in DMSO-d6 solutions are valuable for the design of peptidomimetics of these two pharmacophores. The structure-activity relationships of the RGDDV and RGDY(Me)R pharmacophores within cyclic peptides are discussed. Specifically, the orientation of surface-accessible chemical features on the ligand, such as hydrophobic, positive and negative ionizable groups, which are considered to be responsible for the desired biological activity, is focused on.     

Determination of the disulfide bond arrangement of dengue virus NS1 protein

The 12 half-cystines of NS1 proteins are absolutely conserved among flaviviruses, suggesting their importance to the structure and function of these proteins. In the present study, peptides from recombinant Dengue-2 virus NS1 were produced by tryptic digestion in 100% H(2)(16)O, peptic digestion in 50% H(2)(18)O, thermolytic digestion in 50% H(2)(18)O, or combinations of these digestion conditions. Peptides were separated by size exclusion and/or reverse phase high performance liquid chromatography and examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry, matrix-assisted laser desorption ionization post-source decay, and matrix-assisted laser desorption ionization tandem mass spectrometry. Where digests were performed in 50% H(2)(18)O, isotope profiles of peptide ions aided in the identification and characterization of disulfide-linked peptides. It was possible to produce two-chain peptides containing C1/C2, C3/C4, C5/C6, and C7/C12 linkages as revealed by comparison of the peptide masses before and after reduction and by post-source decay analysis. However, the remaining four half-cystines (C8, C9, C10, and C11) were located in a three-chain peptide of which one chain contained adjacent half-cystines (C9 and C10). The linkages of C8/C10 and C9/C11 were determined by tandem mass spectrometry of an in-source decay fragment ion containing C9, C10, and C11. This disulfide bond arrangement provides the basis for further refinement of flavivirus NS1 protein structural models.     

Mammalian beta-adrenergic receptors. Structural differences in beta 1 and beta 2 subtypes revealed by peptide maps

Photoaffinity labeling techniques using p-azido-m-[125I]iodobenzylcarazolol have recently demonstrated that both the beta 1- and beta 2-adrenergic receptor-binding subunits from mammalian tissues including heart, lung, and erythrocytes reside on peptides of Mr approximately equal to 62,000-64,000. In this study, a two-dimensional gel electrophoresis method for peptide mapping was used to investigate and compare the structure of beta 1 - and beta 2-adrenergic receptor subtypes. When the photoaffinity labeled Mr approximately equal to 62,000 peptides from the beta 2-adrenergic receptors of rat lung and erythrocyte are subjected to simultaneous proteolysis using Staphylococcus aureus V8 proteinase or papain, exactly the same peptide fragments are generated from each subunit. In contrast, when the Mr approximately equal to 62,000 peptide containing the beta 1-adrenergic receptor-binding subunit derived from the rat heart is proteolyzed simultaneously with the Mr approximately equal to 62,000 peptide containing the beta 2-adrenergic receptors from either lung or erythrocyte, the peptide fragments generated are distinctly different. Peptide maps of beta 1-adrenergic receptors from the myocardial tissue of different species (pig versus rat) yield slightly different maps while the maps derived from the beta 2-adrenergic receptors of hamster lung and rat lung or erythrocytes reveal no interspecies differences. These data suggest: 1) alterations in the primary structure of the beta-adrenergic receptor may be responsible for the pharmacological specificities characteristic of beta 1- and beta 2-adrenergic receptor subtypes; and 2) alterations in the primary structure of similar beta-adrenergic receptor subtypes across different species may relate to the magnitude of their phylogenetic differences.     

The peptide-substrate-binding domain of human collagen prolyl 4-hydroxylases. Backbone assignments,secondary structure,and binding of proline-rich peptides

The collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of 4-hydroxyproline by the hydroxylation of proline residues in -Xaa-Pro-Gly-sequences. The vertebrate enzymes are alpha 2 beta 2 tetramers in which protein-disulfide isomerase serves as the beta subunit. Two isoforms of the catalytic alpha subunit have been identified and shown to form [alpha(I)]2 beta 2 and [alpha(II)]2 beta 2 tetramers, the type I and type II C-P4Hs, respectively. The peptide-substrate-binding domain of type I C-P4H has been shown to be located between residues 138 and 244 in the 517-residue alpha(I) subunit and to be distinct from the catalytic domain that is located in the C-terminal region. We report here that a recombinant human C-P4H alpha(I) polypeptide Phe144-Ser244 forms a folded domain consisting of five alpha helices and one short beta strand. This structure is quite different from those of other proline-rich peptide-binding modules, which consist mainly of beta strands. Binding of the peptide (Pro-Pro-Gly)2 to this domain caused major chemical shifts in many backbone amide resonances, the residues showing the largest shifts being mainly hydrophobic, including three tyrosines. The Kd values determined by surface plasmon resonance and isothermal titration calorimetry for the binding of several synthetic peptides to the alpha(I) and the corresponding alpha(II) domain were very similar to the Km and Ki values for these peptides as substrates and inhibitors of the type I and type II C-P4H tetramers. The Kd values of the alpha(I) and alpha(II) domains for (Gly-Pro-4Hyp)5 were much higher than those for (Pro-Pro-Gly)5, indicating a marked decrease in the affinity of hydroxylated peptides for the domain. Many characteristic features of the binding of peptides to the type I and type II C-P4H tetramers can thus be explained by the properties of binding to this domain rather than the catalytic domain.     

Specific macromolecular interactions between tau and the microtubule system

The microtubule-associated protein Tau, a major component of brain microtubules, shares common repeated C-terminal sequences with the high molecular-weight protein MAP-2. It has been shown that tau peptides V¹⁸⁷-G²⁰⁴ and V²¹⁸-G²³⁵ representing two main repeats, induced brain tubulin assembly in a concentration-dependent fashion. The specific roles of these repeats in the interaction of tau with microtubules, and its antigenic nature were investigated using synthetic tau peptides and site-directed monoclonal antibodies. Tau peptides appeared to compete with MAP-2 incorporation into assembled microtubules. The interactions of the tau fragments with -tubulin peptides bearing the tau binding domain on tubulin were analyzed by fluorescence spectroscopy. The specificity of the binding was further demonstrated by the reactivity of tau and the tau peptides with a monoclonal anti-idiotypic antibody produced after immunization with the -11(422–434) tubulin peptide, as assessed by enzyme-linked immunoassay. Western blots confirmed the interaction of tau with the monoclonal antibody. In addition, immunoassays revealed a competition between the MAP-reacting monoclonal antibody and the tubulin peptide -11(422–434) for their interaction with the tau molecule.     

The beta 2-microglobulin dissociation rate is an accurate measure of the stability of MHC class I heterotrimers and depends on which peptide is bound.

Stable, recombinant, water-soluble complexes of HLA-A2 and HLA-B27 were reconstituted from 125I-labeled beta 2-microglobulin (beta 2m), a synthetic peptide, and HLA H chain fragments expressed as inclusion bodies in the Escherichia coli cytoplasm. Using this system, we were able to show: 1) the t1/2 of beta 2m dissociation from HLA complexes at 37 degrees C varied from approximately 40 h to less than 1 h, depending on the peptide employed for reconstitution. Peptide length and composition were found to be critical factors in determining the beta 2m dissociation rate. Endogenous peptides form complexes that are about as stable as those formed with typical antigenic peptides. 2) Peptide exchange reactions, in which an exogenous peptide replaces the peptide that is already bound by the class I molecule, proceed readily for complexes that have rapid beta 2m dissociation rates. Thus, difficulties in demonstrating peptide binding to complexes that contain endogenous peptides can be attributed to the stability of the endogenous peptide/class I molecule complex. 3) The peptide exchange reaction does not require concomitant beta 2m dissociation. 4) Distal parts of the class I molecule, which are not directly involved in peptide binding or beta 2m binding, have a major impact on the stability of class I molecules. Thus, these studies show that the dissociation rate of beta 2m is an excellent measure of how tightly a given peptide binds to class I MHC molecules, that the ability to bind peptide is tightly coupled to the binding of beta 2m and vice versa, and that regions of the molecule distal from the binding site influence the stability of peptide binding.     

The design and production of semisynthetic ribonucleases with increased thermostability by incorporation of S-peptide analogues with enhanced helical stability

Recent work has shown that, with synthetic analogues of C-peptide (residues 1-13 of ribonuclease A), the stability of the peptide helix in H2O depends strongly on the charge on the N-terminal residue. We have asked whether, in semisynthetic ribonuclease S reconstituted from S-protein plus an analogue of S-peptide (1-15), the stability of the peptide helix is correlated with the Tm of the reconstituted ribonuclease S. Six peptides have been made, which contain Glu9----Leu, a blocked alpha-COO- group (-CONH2), and either Gln11 or Glu11. The N-terminal residue has been varied; its charge varies from +2 (Lys) to -1 (succinyl-Ala). We have measured the stability of the peptide helix, the affinity of the peptide for S-protein (by C.D. titration), and the thermal stability of the reconstituted ribonuclease S. All six peptide analogues show strongly enhanced helix formation compared to either S-peptide (1-15) or (1-19), and the helix content increases as the charge on the N-terminal residue changes from +2 to -1. All six peptides show increased affinity for S-protein compared to S-peptide (1-19), and all six reconstituted ribonucleases S show an increase in Tm compared to the protein with S-peptide (1-19). The Tm increases as the charge on residue 1 changes from +2 to -1. The largest increment in Tm is 6 degrees. The results suggest that the stability of a protein can be increased by enhancing the stability of its secondary structure.     

Interpretation of 1H-NMR spectra of the cyclic analog of kallidin in solutions

1H-NMR resonances of [cyclo (10-1 epsilon)]kallidin (cyclo-KL) in (CD3)2SO and H2O have been assigned by combined analysis of two-dimensional phase-sensitive COSY and NOESY spectra. The presence of three slowly interchangeable conformers cyclo-KL I, cyclo-KL II, and cyclo-KL III, has been established, their population in (CD3)2SO being 25, 35 and 40%, respectively. Cyclo-KL I conformer possesses trans-configuration of all peptide bonds, but in the cyclo-KL II and cyclo-KL III conformers the Pro3-Pro4 and Arg2-Pro3 peptide bonds, respectively, have cis-configuration. In----solution, the following exchange occurs between the conformers: cyclo-KL II----cyclo-KL I----cyclo-KL III. The assignment of 1H-NMR signals of all the three cyclo-KL conformers has been carried out in H2O by gradual titration with (CO3)2SO. The conformer populations in H2O are 45, 25 and 30%, respectively.     

Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptides spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex.     

Monoclonal antibodies as structural probes of surface residues in the regulatory subunit of cAMP-dependent protein kinase II from porcine heart

Two murine monoclonal antibodies (H5 and B6) generated against bovine heart type II regulatory subunit of cAMP-dependent protein kinase were shown to cross-react equally well with the homologous subunit from porcine heart. The antibodies demonstrated specificity for only the type II regulatory subunit and showed negligible cross-reactivity with the type I regulatory subunit, the catalytic subunit, and cGMP-dependent protein kinase. Following limited proteolysis of type II regulatory subunit with chymotrypsin, the H5 monoclonal antibody was shown to cross-react with the Mr = 37,000 cAMP-binding domain corresponding to the COOH-terminal region of the polypeptide chain. To more specifically localize the antigenic sites, the porcine type II regulatory subunit was carboxymethylated and cleaved with cyanogen bromide. Both monoclonal antibodies cross-reacted with the NH2-terminal CNBr peptide, and this peptide demonstrated affinities similar to native bovine type II regulatory subunit in competitive displacement radioimmunoassays. Tryptic cleavage of this CNBr fragment destroyed all antigenicity for both monoclonal antibodies, whereas antigenicity was retained following chymotryptic digestion. A single major immunoreactive chymotryptic fragment that cross-reacted with H5 was isolated by gel filtration and reverse phase high performance liquid chromatography. this peptide retained the complete antigenic site and had the following sequence: Asn-Pro-Asp-Glu-Glu-Glu-Glu-Asp-Thr-Asp-Pro-Arg-Val-Ile-His-Pro-Lys-Thr-Asp-Gl n. This antigenic site was localized just beyond the major site of autophosphorylation, approximately a third of the distance from the NH2-terminal end of the polypeptide chain.