GDF15 linked to maternal risk of nausea and vomiting during pregnancy.

GDF15, a hormone acting on the brainstem, has been implicated in the nausea and vomiting of pregnancy, including its most severe form, hyperemesis gravidarum (HG), but a full mechanistic understanding is lacking1-4. Here we report that fetal production of GDF15 and maternal sensitivity to it both contribute substantially to the risk of HG. We confirmed that higher GDF15 levels in maternal blood are associated with vomiting in pregnancy and HG. Using mass spectrometry to detect a naturally labelled GDF15 variant, we demonstrate that the vast majority of GDF15 in the maternal plasma is derived from the feto-placental unit. By studying carriers of rare and common genetic variants, we found that low levels of GDF15 in the non-pregnant state increase the risk of developing HG. Conversely, women with ß-thalassaemia, a condition in which GDF15 levels are chronically high5, report very low levels of nausea and vomiting of pregnancy. In mice, the acute food intake response to a bolus of GDF15 is influenced bi-directionally by prior levels of circulating GDF15 in a manner suggesting that this system is susceptible to desensitization. Our findings support a putative causal role for fetally derived GDF15 in the nausea and vomiting of human pregnancy, with maternal sensitivity, at least partly determined by prepregnancy exposure to the hormone, being a major influence on its severity. They also suggest mechanism-based approaches to the treatment and prevention of HG.

Fetally-encoded GDF15 and maternal GDF15 sensitivity are major determinants of nausea and vomiting in human pregnancy.

Human pregnancy is frequently accompanied by nausea and vomiting that may become severe and life-threatening, as in hyperemesis gravidarum (HG), the cause of which is unknown. Growth Differentiation Factor-15 (GDF15), a hormone known to act on the hindbrain to cause emesis, is highly expressed in the placenta and its levels in maternal blood rise rapidly in pregnancy. Variants in the maternal GDF15 gene are associated with HG. Here we report that fetal production of GDF15, and maternal sensitivity to it, both contribute substantially to the risk of HG. We found that the great majority of GDF15 in maternal circulation is derived from the feto-placental unit and that higher GDF15 levels in maternal blood are associated with vomiting and are further elevated in patients with HG. Conversely, we found that lower levels of GDF15 in the non-pregnant state predispose women to HG. A rare C211G variant in GDF15 which strongly predisposes mothers to HG, particularly when the fetus is wild-type, was found to markedly impair cellular secretion of GDF15 and associate with low circulating levels of GDF15 in the non-pregnant state. Consistent with this, two common GDF15 haplotypes which predispose to HG were associated with lower circulating levels outside pregnancy. The administration of a long-acting form of GDF15 to wild-type mice markedly reduced subsequent responses to an acute dose, establishing that desensitisation is a feature of this system. GDF15 levels are known to be highly and chronically elevated in patients with beta thalassemia. In women with this disorder, reports of symptoms of nausea or vomiting in pregnancy were strikingly diminished. Our findings support a causal role for fetal derived GDF15 in the nausea and vomiting of human pregnancy, with maternal sensitivity, at least partly determined by pre-pregnancy exposure to GDF15, being a major influence on its severity. They also suggest mechanism-based approaches to the treatment and prevention of HG.

Novel Mutation (T273R) in Thyroid Hormone Receptor ß Gene Provides Further Insight into Cryptic Negative Regulation by Thyroid Hormone.

Production of thyroid hormone is precisely regulated in a negative feed-back mechanism that depends critically on thyroid hormone receptor ß (TRß). This mechanism decreases production of thyrotropin- releasing hormone (TRH) and thyrotropin (TSH) in the hypothalamus and pituitary gland in response to high levels of circulating thyroid hormones (TH). Despite the wealth of accumulated knowledge, it is still not clear how exactly this negative regulation is executed. The syndrome of resistance to thyroid hormone (RTH), in which the levels of TH are not properly sensed, represents naturally occurring situations in which molecular components of this regulation are displayed and may be uncovered. TRß, which is central to this regulation, is in the majority of RTH cases mutated in a way that preserves some functions of the receptor. Approximately 150 different mutations in TRß have been identified to date. Here, we hypothesized that additional pathogenic mutations in TRß are likely to exist in human population and analysed clinical cases with suspected RTH. In keeping with our prediction, analysis of 17 patients from nine families led to identification of four presumed pathogenic mutations of TRß, including a previously unknown mutation, T273R. This suggests that threonine 273 is likely to be critical for the normal function of TRß, possibly due to its role in helix 12 mobility and interaction with coactivators, and thus supports the concept that TRß-dependent trans-activating function is necessary for the inhibition of TRH and TSH expression in response to elevated levels of TH.

Molecular Diagnostics of Copper-Transporting Protein Mutations Allows Early Onset Individual Therapy of Menkes Disease.

Menkes disease is a severe X-linked recessive disorder caused by a defect in the ATP7A gene, which encodes a membrane copper-transporting ATPase. Deficient activity of the ATP7A protein results in decreased intestinal absorption of copper, low copper level in serum and defective distribution of copper in tissues. The clinical symptoms are caused by decreased activities of copper-dependent enzymes and include neurodegeneration, connective tissue disorders, arterial changes and hair abnormalities. Without therapy, the disease is fatal in early infancy. Rapid diagnosis of Menkes disease and early start of copper therapy is critical for the effectiveness of treatment. We report a molecular biology-based strategy that allows early diagnosis of copper transport defects and implementation of individual therapies before the full development of pathological symptoms. Low serum copper and decreased activity of copperdependent mitochondrial cytochrome c oxidase in isolated platelets found in three patients indicated a possibility of functional defects in copper-transporting proteins, especially in the ATPA7 protein, a copper- transporting P-type ATPase. Rapid mutational screening of the ATP7A gene using high-resolution melting analysis of DNA indicated presence of mutations in the patients. Molecular investigation for mutations in the ATP7A gene revealed three nonsense mutations: c.2170C>T (p.Gln724Ter); c.3745G>T (p.Glu1249Ter); and c.3862C>T (p.Gln1288Ter). The mutation c.3745G>T (p.Glu1249Ter) has not been identified previously. Molecular analysis of the ATOX1 gene as a possible modulating factor of Menkes disease did not reveal presence of pathogenic mutations. Molecular diagnostics allowed early onset of individual therapies, adequate genetic counselling and prenatal diagnosis in the affected families.

Prorenin Receptor Homologue VHA-20 is Critical for Intestinal pH Regulation, Ion and Water Management and Larval Development in C. elegans.

The prorenin receptor (ATP6AP2) is a multifunctional transmembrane protein; it is a constituent of proton-translocating V-ATPase, a non-proteolytic activator of renin and an adaptor in the Wnt/ß-catenin pathway. Here, we studied vha-20, one of the two prorenin receptor homologues that are identified by sequence similarity in the C. elegans genome. We show that vha-20 (R03E1.2) is prominently expressed in the intestine, in the excretory cell and in amphid neurons, tissues critical for regulation of ion and water management. The expression of vha-20 in the intestine is dependent on NHR-31, a nuclear receptor related to HNF4. VHA-20 is indispensable for normal larval development, acidification of the intestine, and is required for nutrient uptake. Inhibition of vha-20 by RNAi leads to complex deterioration of water and pH gradients at the level of the whole organism including distention of pseudocoelome cavity. This suggests new roles of prorenin receptor in the regulation of body ion and water management and in acidification of intestinal lumen in nematodes.

Clinical and molecular characterization of a novel PLIN1 frameshift mutation identified in patients with familial partial lipodystrophy.

Perilipin 1 is a lipid droplet coat protein predominantly expressed in adipocytes, where it inhibits basal and facilitates stimulated lipolysis. Loss-of-function mutations in the PLIN1 gene were recently reported in patients with a novel subtype of familial partial lipodystrophy, designated as FPLD4. We now report the identification and characterization of a novel heterozygous frameshift mutation affecting the carboxy-terminus (439fs) of perilipin 1 in two unrelated families. The mutation cosegregated with a similar phenotype including partial lipodystrophy, severe insulin resistance and type 2 diabetes, extreme hypertriglyceridemia, and nonalcoholic fatty liver disease in both families. Poor metabolic control despite maximal medical therapy prompted two patients to undergo bariatric surgery, with remarkably beneficial consequences. Functional studies indicated that expression levels of the mutant protein were lower than wild-type protein, and in stably transfected preadipocytes the mutant protein was associated with smaller lipid droplets. Interestingly, unlike the previously reported 398 and 404 frameshift mutants, this variant binds and stabilizes ABHD5 expression but still fails to inhibit basal lipolysis as effectively as wild-type perilipin 1. Collectively, these findings highlight the physiological need for exquisite regulation of neutral lipid storage within adipocyte lipid droplets, as well as the possible metabolic benefits of bariatric surgery in this serious disease.

Hepatoerythropoietic Porphyria Caused by a Novel Homoallelic Mutation in Uroporphyrinogen Decarboxylase Gene in Egyptian Patients.

Porphyrias are metabolic disorders resulting from mutations in haem biosynthetic pathway genes. Hepatoerythropoietic porphyria (HEP) is a rare type of porphyria caused by the deficiency of the fifth enzyme (uroporphyrinogen decarboxylase, UROD) in this pathway. The defect in the enzymatic activity is due to biallelic mutations in the UROD gene. Currently, 109 UROD mutations are known. The human disease has an early onset, manifesting in infancy or early childhood with red urine, skin photosensitivity in sun-exposed areas, and hypertrichosis. Similar defects and links to photosensitivity and hepatopathy exist in several animal models, including zebrafish and mice. In the present study, we report a new mutation in the UROD gene in Egyptian patients with HEP. We show that the homozygous c.T163A missense mutation leads to a substitution of a conserved phenylalanine (amino acid 55) for isoleucine in the enzyme active site, causing a dramatic decrease in the enzyme activity (19 % of activity of wild-type enzyme). Inspection of the UROD crystal structure shows that Phe-55 contacts the substrate and is located in the loop that connects helices 2 and 3. Phe-55 is strictly conserved in both prokaryotic and eukaryotic UROD. The F55I substitution likely interferes with the enzyme-substrate interaction.

SMED-TLX-1 (NR2E1) is critical for tissue and body plan maintenance in Schmidtea mediterranea in fasting/feeding cycles.

Nuclear receptors (NRs), or nuclear hormone receptors (NHRs), are transcription factors that regulate development and metabolism of most if not all animal species. Their regulatory networks include conserved mechanisms that are shared in-between species as well as mechanisms that are restricted to certain phyla or even species. In search for conserved members of the NHR family in Schmidtea mediterranea, we identified a molecular signature of a class of NRs, NR2E1, in the S. mediterranea genome and cloned its complete cDNA coding sequence. The derived amino acid sequence shows a high degree of conservation of both DNA-binding domain and ligand- binding domain and a remarkably high homology to vertebrate NR2E1 and C. elegans NHR-67. Quantitative PCR detected approximately ten-fold higher expression of Smed-tlx-1 in the proximal part of the head compared to the tail region. The expression of Smed-tlx-1 is higher during fed state than during fasting. Smed-tlx-1 down-regulation by RNA interference affects the ability of the animals to maintain body plan and induces defects of brain, eyes and body shape during fasting and re-growing cycles. These results suggest that SMED-TLX-1 is critical for tissue and body plan maintenance in planaria.

A new mutation within the porphobilinogen deaminase gene leading to a truncated protein as a cause of acute intermittent porphyria in an extended Indian family.

Based on Internet search, we were contacted by a 50-year-old man suffering from severe abdominal pain. Acute hepatic porphyria was considered from positive Watson-Schwartz test. He, not being a health professional, searched for centres with ability to do molecular diagnosis and for information about therapeutic possibilities. He asked his physician for haem-arginate (Normosang, Orphan Europe, Paris) treatment, arranged sending his blood to our laboratory and mediated genetic counselling for him and his family. Molecular analyses of the PBGD gene revealed a novel mutation in exon 15, the 973insG. Subsequently, genetic analysis was performed in 18 members of the proband's extensive family. In 12 members of the family, the same mutation was found. The mutation, which consisted of one nucleotide insertion, resulted in addition of four different amino acids leading to a protein that is prematurely truncated by the stop codon. The effect of this mutation was investigated by expression of the wildtype and mutated PBGD in a prokaryotic expression system. The mutation resulted in instability of the protein and loss of enzymatic function. The increasing access to a number of disease- and symptom-oriented web pages presents a new and unusual venue for gaining knowledge and enabling self-diagnosis and self-help. It is, therefore, important that diseaseoriented Internet pages for public use should be designed with clarity and accurate current knowledge based background.

Deletion of V335 from the L2 domain of the insulin receptor results in a conformationally abnormal receptor that is unable to bind insulin and causes Donohue's syndrome in a human subject.

An infant with Donohue's syndrome (leprechaunism) was found to be homozygous for an in-frame trinucleotide deletion within the insulin receptor gene resulting in the deletion of valine 335. When transiently transfected into Chinese hamster ovary cells, mutant receptor was produced in a mature form, but at significantly lower levels compared with wild-type receptor. Cell surface biotinylation experiments revealed that significant amounts of the DeltaV335 receptor were expressed on the cell surface. Despite this, cells expressing this receptor showed no significant insulin binding or ligand-induced receptor autophosphorylation. Although the DeltaV335 receptor was capable of being immunoprecipitated with antibodies directed against the beta-subunit of the receptor, the mutant receptor could not be recognized by a panel of antibodies directed against different epitopes of the alpha-subunit, suggesting that the loss of V335 results in a major conformational alteration in the receptor alpha-subunit. This would be predicted by the positioning of V335 at a critical location within a strand that provides the main rigid scaffold for the two beta-sheet faces of the L2 domain of the receptor. The severe biochemical and clinical consequences of this novel mutation, which occur despite substantial expression on the cell surface, emphasize the crucial role of the L2 domain in ligand binding by the insulin receptor.

Solution structure of a neurotrophic ligand bound to FKBP12 and its effects on protein dynamics.

The structure of a recently reported neurotrophic ligand, 3-(3-pyridyl)-1-propyl(2S)-1-(3,3-dimethyl-1, 2-dioxopentyl)-2-pyrrolidinecarboxylate, in complex with FKBP12 was determined using heteronuclear NMR spectroscopy. The inhibitor exhibits a binding mode analogous to that observed for the macrocycle FK506, used widely as an immunosuppressant, with the prolyl ring replacing the pipecolyl moiety and the amide bond in a trans conformation. However, fewer favourable protein-ligand interactions are detected in the structure of the complex, suggesting weaker binding compared with the immunosuppressant drug. Indeed, a micromolar dissociation constant was estimated from the NMR ligand titration profile, in contrast to the previously published nanomolar inhibition activity. Although the inhibitor possesses a remarkable structural simplicity with respect to FK506, 15N relaxation studies show that it induces similar effects on the protein dynamics, stabilizing the conformation of solvent-exposed residues which are important for mediating the interaction of immunophilin/ligand complexes with molecular targets and potentially for the transmission of the neurotrophic action of FKBP12 inhibitors.

Mapping the active site of factor Xa by selective inhibitors: an NMR and MD study.

The structure of two selective inhibitors, Ac-Tyr-Ile-Arg-Ile-Pro-NH2 and Ac-(4-Amino-Phe)-(Cyclohexyl-Gly)-Arg-NH2, in the active site of the blood clotting enzyme factor Xa was determined by using transferred nuclear Overhauser effect nuclear magnetic resonance (NMR) spectroscopy. They represent a family of peptidic inhibitors obtained by the screening of a vast combinatorial library. Each structure was first calculated by using standard computational procedures (distance geometry, simulated annealing, energy minimization) and then further refined by systematic search of the conformation of the inhibitor docked in the active site and repeating the simulated annealing and energy minimization. The final structure was optimized by molecular dynamics simulations of the inhibitor-complex in water. The NMR restraints were kept throughout the refinement. The inhibitors assume a compact, very well defined conformation, embedded into the substrate binding site not in the same way as a substrate, blocking thus the catalysis. The model allows to explain the mode of action, affinity, and specificity of the peptides and to map the active site.

7Li nuclear-magnetic-resonance study of lithium binding to myo-inositolmonophosphatase.

The interaction of Li+ with myo-inositol monophosphatase was studied by 7Li-NMR spectroscopy. Li+ binding to the enzyme induces a downfield shift and broadening of the 7Li-NMR signal. Changes of the chemical shift were used to follow the titration of the enzyme with lithium and to determine a dissociation constant, Kd = (1.0 +/- 0.1) mM. Only one major binding site/enzyme subunit was inferred. The complex forms independently of the presence of inorganic phosphate. Metals from the group IIa of the periodic table compete with Li+ binding with the affinity increasing in the order Mg2+ < Ca2+ < Be2+. In contrast to lithium, their binding is enhanced by phosphate.

Echistatin: the refined structure of a disintegrin in solution by 1H NMR and restrained molecular dynamics.

The structure of the disintegrin echistatin has been determined by 1H NMR, distance geometry calculations and restrained molecular dynamics simulations. The structure has been refined from the preliminary distance geometry calculations with the inclusion of additional 1H NMR data and hydrogen bonds identified in early stages of the molecular dynamics calculations. The calculations reported here allow a distinction to be made between the two possible disulfide bridging patterns-echistatin is crosslinked as follows: Cys2-Cys11, Cys7-Cys32, Cys8-Cys37, Cys20-Cys39. The final set of structures gives an average pairwise root mean square distance of 0.100 nm (calculated over the backbone atoms of residues Ser4-Cys20 and Asp30-Pro40). The core of echistatin is a well defined though irregular structure, composed of a series of non-classical turns crosslinked by the disulfide bridges and stabilised by hydrogen bonds. The RGD sequence is located in a protruding loop whose stem is formed by two rigid, hydrogen-bonded strands (Thr18-Cys20, Asp30-Cys32). The RGD sequence is connected to this structure by short, flexible segments. High (but not unlimited) mobility is probably necessary for fast recognition and fitting to the integrin receptors. Sequence variability among the disintegrins is found in the segments flanking the RGD sequence, suggesting that these may be important in conferring specificity for the receptors.

Stimulation of cGMP-dependent protein kinase I alpha by a peptide from its own sequence. An investigation by enzymology, circular dichroism and 1H NMR of the activity and structure of cGMP-dependent protein kinase I alpha-(546-576)-peptide amide.

The structure of cGMP-dependent protein kinase I alpha-(546-576)-peptide amide (peptide-546) and its effects on cGMP-dependent protein kinase I alpha (G-kinase) have been studied. By primary sequence analysis and analogy to a peptide that stimulates protein kinase C, peptide-546 was predicted to form part of the protein/peptide binding site of G-kinase, and it was proposed that it would stimulate the enzyme by interaction with an autoinhibitory site. The portion of cAMP-dependent protein kinase analogous to peptide-546 forms part of the peptide substrate binding site, interacting with the peptide inhibitor residues Argp-2 and Phep-11 (where p is the pseudophosphorylation site), through residues at positions corresponding to Glu4, Pro10 and Ser13 in peptide-546. Peptide-546 is a reasonably potent G-kinase activator, increasing the turnover number with the peptide substrate Arg-Lys-Arg-Ser-Arg-Lys-Glu by about threefold with an activation constant that is about fivefold lower than the Km value of this peptide substrate. Peptide-546 does not appear to change the affinity of the enzyme for the above substrate, ATP or cGMP and does not affect the binding of [3H]cGMP to G-kinase. The activation does not seem to result from an interaction between peptide-546 and peptide substrates, and a kinetic scheme is proposed which is compatible with an action of peptide-546 on G-kinase independent of substrates. The activation is additive with that given by cGMP and causes the enzyme to enter a hitherto unrecognised superactive state. Peptide conformation has been monitored in mixed 2,2,2-trifluoroethanol/H2O solvents by circular dichroism: helical structure is observed in these mixtures when the 2,2,2-trifluoroethanol content is above 25%. The structure is lost only gradually on raising the temperature to 80 degrees C with no clear melting transition. Assignment of the resonances in the 1H-NMR spectrum has allowed the identification of elements of secondary structure from detected nuclear Overhauser effects. In particular, a helical segment from Met18 to Arg26 is observed. The four proline residues (Pro10, Pro11, Pro15 and Pro17) are all seen to be in the trans conformation, although additional, weaker peaks in the spectra may correspond to a minor conformer in which one or more of the prolines is in a cis conformation. The N-terminal residues are less structured but show some helical character.(ABSTRACT TRUNCATED AT 400 WORDS)

An echistatin C-terminal peptide activates GPIIbIIIa binding to fibrinogen, fibronectin, vitronectin and collagen type I and type IV.

Integrin binding to proteins often involves recognition of domains containing the arginine-glycine-aspartate (RGD) motif. Different binding affinities and specificities of the integrin-ligand protein interactions involve additional protein domains. The n.m.r. structure of the snake-venom protein echistatin suggested that the C-terminal portion of the molecule might be important, in addition to the RGD domain, in binding to the integrin glycoprotein IIbIIIa (GPIIbIIIa) [Saudek, Atkinson and Pelton (1991) Biochem. 30, 7369-7372]. The synthetic C-terminal peptide, echistatin-(40-49), PRNPHKGPAT, (1) inhibited binding of GPIIbIIIa to immobilized echistatin (IC50 3-6 mM), but did not inhibit binding of GPIIbIIIa to immobilized fibrinogen (up to 5 mM peptide), (2) activated GPIIbIIIa binding to fibronectin and vitronectin, usual ligands for the activated integrin, (3) activated binding of GPIIbIIIa to collagen type I and type IV, proteins not usually regarded as ligands for the integrin, and (4) stimulated 125I-fibrinogen binding by human platelets. These findings argue for an interaction of this non-RGD domain in echistatin with GPIIbIIIa, leading to activation of the integrin and extension of the ligand specificity to include immobilized collagen.

Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions.

A novel approach to tailored selective excitation for the measurement of NMR spectra in non-deuterated aqueous solutions (WATERGATE, WATER suppression by GrAdient-Tailored Excitation) is described. The gradient echo sequence, which effectively combines one selective 180 degrees radiofrequency pulse and two field gradient pulses, achieves highly selective and effective water suppression. This technique is ideally suited for the rapid collection of multi-dimensional data since a single-scan acquisition produces a pure phase NMR spectrum with a perfectly flat baseline, at the highest possible sensitivity. Application to the fast measurement of 2D NOE data of a 2.2 mM solution of a double-stranded DNA fragment in 90% H2O at 5 degrees C is presented.

Structure of neuropeptide Y dimer in solution.

The structure of porcine neuropeptide Y in 0.05 M CD3COOD/D2O was determined by nuclear magnetic resonance spectroscopy. Nuclear Overhauser spectra yielded 377 distances which define a helical segment formed by residues 11-36. An additional set of 24 distances were interpreted as intermolecular distances within a dimer. A combination of distance geometry calculations, energy minimization and molecular dynamics yielded a model of the dimer having antiparallel packing of two curved helical units whose hydrophobic sides form a well defined core. The N-terminus (residues 1-9) appears as an unstructured mobile segment. Large changes in the intrinsic fluorescence intensity of neuropeptide Y tyrosine residues allowed the determination of the dimer dissociation constant as 1.6 +/- 0.6 microM at pH 2-8 in aqueous buffers and also indicated the enclosure of several tyrosine residues in the hydrophobic environment of the interface region in the dimeric species. Fluorescence anisotropy data reveals the slow rotation of such shielded residues.

Three-dimensional structure of acylphosphatase. Refinement and structure analysis.

We report here the complete determination of the solution structure of acylphosphatase, a small enzyme that catalyses the hydrolysis of organic acylphosphates, as determined by distance geometry methods based on nuclear magnetic resonance information. A non-standard strategy for the distance geometry calculations was used and is described here some detail. The five best structures were then refined by restrained energy minimization and molecular dynamics in order to explore the conformational space consistent with the experimental data. We address the question of whether the solution structure of acylphosphatase follows the general principles of protein structure, i.e. those learned from analysing crystal structures. Static and dynamic features are discussed in detail. An uncommon beta-alpha-beta motif, so far found only in procarboxypeptidase B and in an RNA-binding protein, is present in acylphosphatase.

The secondary structure of echistatin from 1H-NMR, circular-dichroism and Raman spectroscopy.

Detailed biophysical studies have been carried out on echistatin, a member of the disintegrin family of small, cysteine-rich, RGD-containing proteins, isolated from the venom of the saw-scaled viper Echis carinatus. Analysis of circular-dichroism spectra indicates that, at 20 degrees C, echistatin contains no alpha-helix but contains mostly beta-turns and beta-sheet. Two isobestic points are observed as the temperature is raised, the conformational changes associated with that observed between 40 degrees C and 72 degrees C being irreversible. Raman spectra also indicate considerable beta-turn and beta-sheet (20%) structure and an absence of alpha-helical structure. Three of the four disulphide bridges are shown to be in an all-gauche conformation, while the fourth adopts a trans-gauche-gauche conformation. The 1H-NMR spectrum of echistatin has been almost fully assigned. A single conformation was observed at 27 degrees C with the four proline residues adopting only the trans conformation. A large number of backbone amide protons were found to exchange slowly, but no segments of the backbone were found to be in either alpha-helical or beta-sheet conformation. A number of turns could be characterised. An irregular beta-hairpin contains the RGD sequence in a mobile loop at its tip. Two of the four disulphide cross-links have been identified from the NMR spectra. The data presented in this paper will serve to define the structure of echistatin more closely in subsequent studies.