Chemical synthesis of human protein S thrombin-sensitive module and first epidermal growth factor module

Human plasma protein S is a nonenzymatic cofactor for activated protein C (APC) in the inactivation of coagulation factors Va and VIIIa, and helps to provide an essential negative feedback on blood coagulation. Previous indirect evidence suggested that the thrombin-sensitive region (TSR:residues 47–75, 1 disulfide) and the first epidermal growth factorlike region (EGF1: residues 76–116, 3 disulfides) of protein S may be functionally important for expression of its APC cofactor activity. To study the functional importance of these modules directly, access to the isolated TSR and EGF1 modules would be preferred. Recombinant expression of protein S intact TSR and correctly folded EGF1 has not been possible. Here we describe the synthesis of both TSR and EGF1 modules by stepwise solid phase peptide synthesis using the in situ neutralization/2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate activation procedure for tert-butoxycarbonyl chemistry. For the TSR, correct intramodular disulfide bonding was confirmed. To overcome folding difficulties with the EGF1, a two-step oxidation procedure was used in which the cysteines involved in the middle, crossing, disulfide bond (Cys⁸⁵-Cys¹⁰²) remained protected with acetamidomethyl (Acm) groups after hydrogen fluoride treatment of the peptide resin. Selective formation of the first two disulfide bonds (Cys⁸⁰-Cys⁹³ and Cys¹⁰⁴-Cys¹¹³) was followed by release of the Acm groups and subsequent formation of the third disulfide bond (Cys⁸⁵-Cys¹⁰²). CD studies revealed 54% of β-sheet/turn in the EGF1 that is characteristic for EGF modules. Deuterium exchange studies suggested a very tightly packed core in EGF1 that is not accessible to the bulk solvent, likely a result from the compact structure caused by its three disulfide bonds. The 30% β-sheet structure observed in the TSR involved amide protons that could be readily exchanged by deuterons, likely reflecting a more flexible structure of the TSR loop in contrast to the rigid structure of EGF1. The establishment of synthetic access to the TSR and EGF1 of protein S provides a versatile tool to study interactions of these modules with the blood coagulation components of the anticoagulant plasma protein C pathway. © 1998 John Wiley & Sons, Inc. Biopoly 46: 53–63, 1998     

Reductive cleavage and regeneration of the disulfide bonds inStreptomyces subtilisin inhibitor (SSI) as studied by the carbonyl13C NMR resonances of cysteinyl residues

Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-¹³C]Cys. The¹³C signals were assigned by the¹⁵N,¹³C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys⁷¹-Cys¹⁰¹ disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met⁷³-Val⁷⁴, is more accessible to solvent than the other disulfide bond. Cys³⁵-Cys⁵⁰, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys⁷¹ and Cys¹⁰¹ regenerates at a much faster rate than that between Cys³⁵ and Cys⁵⁰.Nomenclature of the various states of SSI that are observed in the present study Fully oxidized-folded native or intact (without GuHCl or DTT) - half reduced-folded (Cys⁷¹-Cys¹⁰¹ reduced; DTT without GuHCl) - inversely half reduced-folded (Cys³⁵-Cys⁵⁰ reduced; a reoxidation intermediate from fully reduced-folded state) - fully reduced-unfolded (reduced by DTT in the presence of GuHCl) - fully reduced-folded (an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture)     

Assignment of the three disulfide bonds in ShK toxin: A potent potassium channel inhibitor from the sea anemone Stichodactyla helianthus

Summary ShK toxin, a 35-residue peptide isolated from the Caribbean sea anemone Stichodactyla helianthus, is a potent inhibitor of the Kv 1.3 potassium channel in lymphocytes. The natural toxin contains three disulfide bonds. The disulfide pairings of the synthetic ShK toxin were elucidated as a prerequisite for studies on its structure-function relationships. The toxin was fragmented at pH 6.5 using either thermolysin or a mixture of trypsin and chymotrypsin followed by thermolysin. The fragments were isolated by RP-HPLC and were identified by sequence analysis and MALDI-TOF mass spectrometry. The three disulfides were unambiguously identified in either proteolytic digest: Cys³ to Cys³⁵, Cys¹² to Cys²⁸ and Cys¹⁷ to Cys³². The Cys³-Cys³⁵ disulfide, linking the amino- and carboxyl-termini, defines the characteristic cyclic structure of the molecule. A similar disulfide pairing motif is found in the snake venom-derived potassium channel blocker dendrotoxin and the mammalian antibiotic peptide defensins.     

Theoretical structural insights into the snakin/GASA family

Among the main classes of cysteine-stabilized antimicrobial peptides, the snakin/GASA family has not yet had any structural characterization. Through the combination of ab initio and comparative modeling with a disulfide bond predictor, the three-dimensional structure prediction of snakin-1 is reported here. The structure was composed of two long α-helices with a disulfide pattern of Cys^I-Cys^IX, Cys^II-Cys^VII, Cys^III-Cys^IV, Cys^V-Cys^XI, Cys^VI-Cys^XII and Cys^VIII-Cys^X. The overall structure was maintained throughout molecular dynamics simulation. Snakin-1 showed a small degree of structural similarity with thionins and α-helical hairpins. This is the first report of snakin-1 structural characterization, shedding some light on the snakin/GASA family.     

Sequential Comparison of Peptides Containing Half-cystine Residues from Ovalbumins of Six Avian Species

Hen ovalbumin contains one cystine disulfide (Cys⁷³-Cys¹²⁰) and four cysteine sulfhydryl groups (Cys¹¹,Cys³⁰,Cys³⁶⁷, and Cys³⁸²) in a single polypeptide chain of 385 amino acid residues. To investigate whether or not such a structure is shared by related avian species, the contents of disulfide-involved half-cystine residues and their positions in the primary structure of ovalbumins from five species were compared with those of hen ovalbumin. Ovalbumins were alkylated with a fluorescent dye, IAEDANS, under disulfide-reduced and disulfide-intact conditions and digested with a number of proteolytic enzymes. The sequences were deduced from peptides containing half-cystine residues labeled with the fluorescent dye. The results showed that the number of free cysteine sulfhydryl groups of ovalbumins was different among the species, three for guinea fowl and turkey (Cys¹¹, Cys³⁶⁷, and Cys³⁸²); and two for Pekin duck, mallard duck, and Emden goose (Cys¹¹ and Cys³³¹). On the other hand, a single intrachain disulfide bond could be identified from ovalbumins of five species using a combination of peptide mapping and N-terminal amino acid sequencing analysis under reduced and non-reduced conditions, in which the intrachain disulfide bond was like that of hen ovalbumin (Cys⁷³-Cys¹²⁰). The results also indicated that the variations in amino acid sequences on these peptides containing half-cystine residues bear a close relationship with the phylogeny of the six species.     

Characterization of a Reduced Form of Plasma Plasminogen as the Precursor for Angiostatin Formation

Plasma plasminogen is the precursor of the tumor angiogenesis inhibitor, angiostatin. Generation of angiostatin in blood involves activation of plasminogen to the serine protease plasmin and facilitated cleavage of two disulfide bonds and up to three peptide bonds in the kringle 5 domain of the protein. The mechanism of reduction of the two allosteric disulfides has been explored in this study. Using thiol-alkylating agents, mass spectrometry, and an assay for angiostatin formation, we show that the Cys⁴⁶²-Cys⁵⁴¹ disulfide bond is already cleaved in a fraction of plasma plasminogen and that this reduced plasminogen is the precursor for angiostatin formation. From the crystal structure of plasminogen, we propose that plasmin ligands such as phosphoglycerate kinase induce a conformational change in reduced kringle 5 that leads to attack by the Cys⁵⁴¹ thiolate anion on the Cys⁵³⁶ sulfur atom of the Cys⁵¹²-Cys⁵³⁶ disulfide bond, resulting in reduction of the bond by thiol/disulfide exchange. Cleavage of the Cys⁵¹²-Cys⁵³⁶ allosteric disulfide allows further conformational change and exposure of the peptide backbone to proteolysis and angiostatin release. The Cys⁴⁶²-Cys⁵⁴¹ and Cys⁵¹²-Cys⁵³⁶ disulfides have −/+RHHook and −LHHook configurations, respectively, which are two of the 20 different measures of the geometry of a disulfide bond. Analysis of the structures of the known allosteric disulfide bonds identified six other bonds that have these configurations, and they share some functional similarities with the plasminogen disulfides. This suggests that the −/+RHHook and −LHHook disulfides, along with the −RHStaple bond, are potential allosteric configurations.     

The disulfide structure of denatured epidermal growth factor: preparation of scrambled disulfide isomers

The conformational stability of human epidermal growth factor (EGF) and the structure of denatured EGF were investigated using the technique of disulfide scrambling. Under denaturing conditions and in the presence of a thiol catalyst, the native EGF denatures by shuffling its three native disulfide bonds and converts to a mixture of scrambled isomers. Analysis by HPLC reveals that the denatured EGF is composed of about 10 fractions of scrambled isomers. The heterogeneity varies under different denaturing conditions, with the heat-denatured samples exhibiting the highest degree of heterogeneity. The disulfide structures of eight major scrambled isomers of EGF were determined. The most predominant isomer adopts the bead-form structure with disulfide bonds bridged by three pairs of neighboring cysteines: Cys⁶-Cys¹⁴, Cys²⁰-Cys³¹, and Cys³³-Cys⁴². The denaturation curve of EGF is determined by the relative yield of the scrambled and native species of EGF. EGF is a highly stable molecule and can be effectively denatured only by guanidine chloride at a concentration of greater than 4–5 M. At 8 M urea, less than 16% of the native EGF was denatured. The unusual conformational stability of EGF was compared with that of eight different disulfide proteins that were similarly characterized by the method of disulfide scrambling.     

Microwave-assisted solid-phase peptide synthesis of the 60–110 domain of human pleiotrophin on 2-chlorotrityl resin

A fast and efficient microwave-assisted solid phase peptide synthesis (MW-SPPS) of a 51mer peptide, the main heparin-binding site (60–110) of human pleiotrophin (hPTN), using 2-chlorotrityl chloride resin (CLTR-Cl) following the 9-fluorenylmethyloxycarbonyl/tert-butyl (Fmoc/tBu) methodology and with the standard N,N′-diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) coupling reagents, is described. An MW-SPPS protocol was for the first time successfully applied to the acid labile CLTR-Cl for the faster synthesis of long peptides (51mer peptide) and with an enhanced purity in comparison to conventional SPPS protocols. The synthesis of such long peptides is not trivial and it is generally achieved by recombinant techniques. The desired linear peptide was obtained in only 30 h of total processing time and in 51% crude yield, in which 60% was the purified product obtained with 99.4% purity. The synthesized peptide was purified by reversed phase high performance liquid chromatography (RP-HPLC) and identified by electrospray ionization mass spectrometry (ESI-MS). Then, the regioselective formation of the two disulfide bridges of hPTN 60–110 was successfully achieved by a two-step procedure, involving an oxidative folding step in dimethylsulfoxide (DMSO) to form the Cys⁷⁷–Cys¹⁰⁹ bond, followed by iodine oxidation to form the Cys⁶⁷–Cys⁹⁹ bond.     

Assignment of the three disulfide Bridges of huwentoxin-I, a neurotoxin from the spiderSelenocosmia huwena

The positions of the disulfide bonds of huwentoxin-I, a neurotoxin from the spiderSelenocosmia huwena, have been determined. The existence of three disulfide bonds in the native toxin was demonstrated by mass spectroscopy and the lack of reactivity with a thiol reagent. The assignment procedure involved a combination of tryptic digestion of the native toxin and sequence analysis of both intact andin situ S-carboxymethylated toxin.In situ carboxymethylation is shown to be a useful procedure in sequencing of cysteine- and cystine-containing peptides. Sequence analysis of the intact, cross-linked toxin indicated that no amino acid phenylthiohydantoin (PTH) derivative is seen for the first half-cystine in a cross-linked pair, but that the PTH of dehydroalanine, which can be detected at 313 nm, is seen at the position of the second half-cystine. By sequencing disulfide cross-linked tryptic fragments, the three disulfide linkages in huwentoxin-I could be assigned as Cys₂-Cys₁₇, Cys₉-Cys₂₂, and Cys₁₆-Cys₂₉.     

Solid-phase synthesis of somatostatin and glucagon-selective analogs in gram quantities

Somatostatin (SS) and two glucagon selective analogs [D -Cys^l4]-SS and [D -Trp⁸, D -Cys¹⁴]-SS have been synthesized in gram quantities by the solid-phase procedure. A general modification of Monahan and Gilon's procedure for esterification of the first protected amino acid onto the chloromethylated resin as well as a general protocol for solid-phase peptide synthesis on Beckman 990 automatic synthesizer are described. A new general procedure for disulfide formation, which involves the adaptation of the “high-dilution” principle to the ferricyanide oxidation and the optimization of the sequence of purification steps as applied to somatostatin and its analogs, yields highly purified peptides (≥ 199% pure) as checked by reverse-phase high-pressure liquid chromatography—which is shown to be a highly sensitive, resolutive, and quantitative analytical tool for evaluation of the homogeneity of peptides.     

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl]_1/2 at 3.4-5 M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1 mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys³⁶-Cys⁴⁹ and two disulfide bonds formed by two pair of consecutive cysteines, Cys²²-Cys²³ and Cys⁵⁶-Cys⁵⁷, a unique disulfide structure of polypeptide that has not been documented previously.     

Three-dimensional structure of α-conotoxin EI determined by1H NMR spectroscopy

Summary α-conotoxin EI is an 18-residue peptide (RDOCCYHPTCNMSNPQIC; 4–10, 5–18) isolated from the venom ofConus ermineus, the only fish-hunting cone snail of the Atlantic Ocean. This peptide targets specifically the nicotinic acetylcholine receptor (nAChR) found in mammalian skeletal muscle and the electric organTorpedo, showing a novel selectivity profile when compared to other α-conotoxins. The 3D structure of EI has been determined by 2D-NMR methods in combination with dynamical simulated annealing protocols. A total of 133 NOE-derived distances were used to produce 13 structures with minimum energy that complied with the NOE restraints. The structure of EI is characterized by a helical loop between THr⁹ and Met¹² that is stabilized by the Cys⁴-Cys¹⁰ disulfide bond and turns involving Cys⁴-Cys⁵ and Asn¹⁴-Pro¹⁵. Other regions of the peptide appear to be flexible. The overall fold of EI is similar to that of other α4/7-conotoxins (PnIA/B, MII, EpI). However, unlike these other α4/7-conotoxins, EI targets the muscular type nAChR. The differences in selectivity can be attributed to differences in the surface charge distribution among these α4/7-conotoxins. The implications for binding of EI to the muscular nAChR are discussed with respect to the current NMR structure of EI. Supplementary material available:¹H resonance assignments of α-conotoxin EI.     

Position of the sulfhydryl group and the disulfide bonds of human glucocerebrosidase

Purified human glucocerebrosidase isolated from placenta was modified with [¹⁴C]-iodoacetic acid without reduction and digested with both protease-V8 at pH 4.0 followed by-chymotrypsin at pH 7.5. The majority of radioactivity was found in a peptide that contained the [¹⁴C]-carboxymethylated-cysteine identified as CM-Cys₁₈. Direct sequencing of the N-terminus of the intact labeled protein confirmed the modification of Cys₁₈. For identification of disulfide bond-containing peptides, another portion of glucocerebrosidase was alkylated with nonlabeled iodoacetic acid and then digested with protease V8 and-chymotrypsin as before. Twenty-eight HPLC fragments were collected. These purified peaks were then reduced with-mercaptoethanol followed by S-carboxymethylation with [¹⁴C]-iodoacetic acid. Three peptides among these 28 peptides generated two radioactive daughter peptides. These peptides were sequenced and the position of the radioactive CM-cysteines identified. The locations of these disulfides are Cys₄-Cys₁₆, Cys₂₃-Cys₃₄₂, and Cys₁₂₆-Cys₂₄₈. Attempts to reproduce the free sulfhydryl labeling experiments using the glucocerebrosidase isolated from Ceredase proved unsuccessful. No label was incorporated by this enzyme prior to reduction. This result suggests that the form of the protein used in the clinic differs from the native protein.     

Assignment of the three disulfide Bridges of huwentoxin-I,a neurotoxin from the spiderSelenocosmia huwena

The positions of the disulfide bonds of huwentoxin-I, a neurotoxin from the spiderSelenocosmia huwena, have been determined. The existence of three disulfide bonds in the native toxin was demonstrated by mass spectroscopy and the lack of reactivity with a thiol reagent. The assignment procedure involved a combination of tryptic digestion of the native toxin and sequence analysis of both intact andin situ S-carboxymethylated toxin.In situ carboxymethylation is shown to be a useful procedure in sequencing of cysteine- and cystine-containing peptides. Sequence analysis of the intact, cross-linked toxin indicated that no amino acid phenylthiohydantoin (PTH) derivative is seen for the first half-cystine in a cross-linked pair, but that the PTH of dehydroalanine, which can be detected at 313 nm, is seen at the position of the second half-cystine. By sequencing disulfide cross-linked tryptic fragments, the three disulfide linkages in huwentoxin-I could be assigned as Cys₂-Cys₁₇, Cys₉-Cys₂₂, and Cys₁₆-Cys₂₉.     

Autocatalytic activation of a thermostable glutamyl endopeptidase capable of hydrolyzing proteins at high temperatures

Glutamyl endopeptidases (GSEs) specifically hydrolyze peptide bonds formed by α-carboxyl groups of Glu and Asp residues. We cloned the gene for a thermophilic GSE (designated TS-GSE) from Thermoactinomyces sp. CDF. A proform of TS-GSE that contained a 61-amino acid N-terminal propeptide and a 218-amino acid mature domain was produced in Escherichia coli. We found that the proform possessed two processing sites and was capable of autocatalytic activation via multiple pathways. The N-terminal propeptide could be autoprocessed at the Glu^?1-Ser¹ bond to directly generate the mature enzyme. It could also be autoprocessed at the Glu^?12-Lys^?11 bond to yield an intermediate, which was then converted into the mature form after removal of the remaining part of the propeptide. The segment surrounding the two processing sites was flexible, which allowed the proform and the intermediate form to be trans-processed into the mature form by either active TS-GSE or heterogeneous proteases. Deletion analysis revealed that the N-terminal propeptide is important for the correct folding and maturation of TS-GSE. The propeptide, even its last 11-amino acid peptide segment, could inhibit the activity of its cognate mature domain. The mature TS-GSE displayed a temperature optimum of 85 °C and retained approximately 90 % of its original activity after incubation at 70 °C for 6 h, representing the most thermostable GSE reported to date. Mutational analysis suggested that the disulfide bonds Cys³²-Cys⁴⁸ and Cys¹⁸⁰-Cys¹⁸³ cumulatively contributed to the thermostability of TS-GSE.     

Redox Regulation of Methionine Aminopeptidase 2 Activity

Protein translation is initiated with methionine in eukaryotes, and the majority of proteins have their N-terminal methionine removed by methionine aminopeptidases (MetAP1 and MetAP2) prior to action. Methionine removal can be important for protein function, localization, or stability. No mechanism of regulation of MetAP activity has been identified. MetAP2, but not MetAP1, contains a single Cys²²⁸-Cys⁴⁴⁸ disulfide bond that has an −RHStaple configuration and links two β-loop structures, which are hallmarks of allosteric disulfide bonds. From analysis of crystal structures and using mass spectrometry and activity assays, we found that the disulfide bond exists in oxidized and reduced states in the recombinant enzyme. The disulfide has a standard redox potential of −261 mV and is efficiently reduced by the protein reductant, thioredoxin, with a rate constant of 16,180 m⁻¹ s⁻¹. The MetAP2 disulfide bond also exists in oxidized and reduced states in glioblastoma tumor cells, and stressing the cells by oxygen or glucose deprivation results in more oxidized enzyme. The Cys²²⁸-Cys⁴⁴⁸ disulfide is at the rim of the active site and is only three residues distant from the catalytic His²³¹, which suggested that cleavage of the bond would influence substrate hydrolysis. Indeed, oxidized and reduced isoforms have different catalytic efficiencies for hydrolysis of MetAP2 peptide substrates. These findings indicate that MetAP2 is post-translationally regulated by an allosteric disulfide bond, which controls substrate specificity and catalytic efficiency.     

Three-dimensional structure of α-conotoxin EI determined by 1H NMR spectroscopy H resonance assignments of α-conotoxin EI

α-Conotoxin EI is an 18-residue peptide (RDOCCYHPTCNMSNPQIC; 4–10, 5–18) isolated from the venom of Conus ermineus, the only fish-hunting cone snail of the Atlantic Ocean. This peptide targets specifically the nicotinic acetylcholine receptor (nAChR) found in mammalian skeletal muscle and the electric organ Torpedo, showing a novel selectivity profile when compared to other α-conotoxins. The 3D structure of EI has been determined by 2D-NMR methods in combination with dynamical simulated annealing protocols. A total of 133 NOE-derived distances were used to produce 13 structures with minimum energy that complied with the NOE restraints. The structure of EI is characterized by a helical loop between Thr⁹ and Met¹² that is stabilized by the Cys⁴-Cys¹⁰ disulfide bond and turns involving Cys⁴-Cys⁵ and Asn¹⁴-Pro¹⁵. Other regions of the peptide appear to be flexible. The overall fold of EI is similar to that of other α4/7-conotoxins (PnIA/B, MII, EpI). However, unlike these other α4/7-conotoxins, EI targets the muscular type nAChR. The differences in selectivity can be attributed to differences in the surface charge distribution among these α4/7-conotoxins. The implications for binding of EI to the muscular nAChR are discussed with respect to the current NMR structure of EI.     

Conformational analysis of endothelin-1: Effects of solvation free energy

In order to investigate conformational preferences of the 21-residue peptide hormone endothelin-1 (ET-1), an extensive conformational search was carried out in vacuo using a combination of high temperature molecular dynamics / annealing and a Monte Carlo / minimization search in torsion angle space. Fully minimized conformations from the search were grouped into families using a clustering technique based on rms fitting over the Cartesian coordinates of the atoms of the peptide backbone of the ring region. A wide range of local energy minima were identified even though two disulfide bridges (Cys¹-Cys¹⁵ and Cys³-Cys¹¹) constrain the structure of the peptide. Low energy conformers of ET-1 as a nonionized species in vacuo arestabilized by intramolecular interaction of the ring region (residues 1-15) with the tail (residues 16–21). Strained conformations for individual residues are observed. Conformational similarity to protein loops is established by matching to protein crystal structures In order to assess the influence of aqueous environment on conformational preference, the electrostatic contribution to the solvation energy was calculated for ET-1 as a fully ionized species (Asp⁸, Lys⁹, Glu¹⁰, Asp¹⁸, N- and C-terminus) using a continuum electrostatics model (DelPhi) for each of the conformed generated in vacuo, and the total solvation free energy was estimated by adding a hydrophobic contribution proportional to solvent accessible surface area. Solvation dramatically alters the relative energetics of ET-1 conformers from that calculated in vacuo. Conformers of ET-1 favored by the electrostatic salvation energy in water include conformers with helical secondary structure in the region of residues 9–15. Perhaps of most importance, it was demonstrated that the contribution tosolvation by an individual charge depends not only on its solvent accessibility but on the proximity of other charges, i.e., it is a cooperative effect. This was shown by the calculation of electrostatic solvation energy as afunction of conformation with individual charges systematically turned “on” and “off”. The cooperative effect of multiple charges on solvation demonstrated in this manner calls into question models that relate solvation energysimply to solvent accessibility by atom or residue alone. © 1995 John Wiley & Sons, Inc.     

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.