Structural analysis of the decanucleotide duplex d(GGTAATTACC)2 using 1H NMR spectroscopy.

The conformation of the decanucleotide duplex d(GGTAATTACC)2 has been investigated in solution by one- and two-dimensional proton NMR spectroscopy. Intra- and inter-nucleotide two-dimensional nuclear Overhauser enhancement data, recorded at mixing times between 15 and 250 ms, reveal a right-handed B-DNA structure. The data also show that the A-T basepairs of the TAATTA tract are highly propeller twisted and the minor groove is particularly narrow.     

Headspace-solid-phase microextraction fast GC in combination with principal component analysis as a tool to classify different chemotypes of chamomile flower-heads (Matricaria recutita l.)

Headspace-solid-phase microextraction gas chromatography-principal component analysis (HS-SPME GC-PCA) is proposed as a complementary or alternative method to essential oil (EO) GC-PCA in order to discriminate between flower-heads of chamomile of different chemotypes. Ninety-two EOs and the headspaces sampled by HS-SPME of the corresponding chamomile flower-heads were examined by conventional GC and fast GC (F-GC) and the results submitted to statistical analysis by PCA. HS-SPME F-GC-PCA showed itself to be a rapid technique by which to distinguish chamomile flower-head chemotypes a produced results in agreement with the accepted EO classification. Using this method, the analysis time was reduced from at least 4.5 h with EO conventional GC to less than 1 h with HS-SPME F-GC. This approach can thus successfully be used as an analytical decision maker in order to reduce the number of time-consuming EO conventional GC analyses by limiting them to those samples that cannot unequivocally be classified. The EO conventional GC and HS-SPME F-GC results of PCA were very uniform, but they did not provide quantitative correlations between the components as determined by the two methods. A different statistical approach and a larger number of samples will be needed in order to correlate components in the headspace sampled by SPME and those in the corresponding EO quantitatively through a function.     

Application of progress curve analysis to in situ enzyme kinetics using 1H NMR spectroscopy

The steady-state kinetics of enzymes in tissues, cells, and concentrated lysates can be characterized using high-resolution nuclear magnetic resonance spectroscopy; this is possible because almost invariably there are differences in the spectra of substrates and products of a reaction and these spectra are obtainable even from optically opaque samples. We used 1H spin-echo NMR spectroscopy to study the hydrolysis of alpha-L-glutamyl-L-alanine by cytosolic peptidases of lysed human erythrocytes. Nonlinear regression of the integrated Michaelis-Menten expression onto the progress-curve data yielded, directly, estimates of Vmax and Km for the hydrolase; a procedure for analyzing progress curves in this manner was adapted and compared with a commonly used procedure which employs the Newton-Raphson algorithm. We also performed a sensitivity analysis of the integrated Michaelis-Menten expression; this yielded equations that indicate under what conditions estimates of Km and Vmax are most sensitive to variations in experimental observables. Specifically, we showed that the most accurate estimates of the steady-state parameters from analysis of progress curves are obtained when the initial substrate concentration is much greater than Km. Furthermore, estimates of these parameters obtained by such an analysis are most sensitive to data obtained when the reaction is 60-80% complete, having started with the highest practicable initial substrate concentration.     

Visualizing the principal component of 1H,15N-HSQC NMR spectral changes that reflect protein structural or functional properties: application to troponin C

Laboratories often repeatedly determine the structure of a given protein under a variety of conditions, mutations, modifications, or in a number of states. This approach can be cumbersome and tedious. Given then a database of structures, identifiers, and corresponding (1)H,(15)N-HSQC NMR spectra for homologous proteins, we investigated whether structural information could be ascertained for a new homolog solely from its (1)H,(15)N-HSQC NMR spectrum. We addressed this question with two different approaches. First, we used a semi-automated approach with the program, ORBplus. ORBplus looks for patterns in the chemical shifts and correlates these commonalities to the explicit property of interest. ORBplus ranks resonances based on consistency of the magnitude and direction of the chemical shifts within the database, and the chemical shift correlation of the unknown protein with the database. ORBplus visualizes the results by a histogram and a vector diagram, and provides residue specific predictions on structural similarities with the database. The second method we used was partial least squares (PLS), which is a multivariate statistical technique used to correlate response and predictor variables. We investigated the ability of these methods to predict the tertiary structure of the contractile regulatory protein troponin C. Troponin C undergoes a closed-to-open conformational change, which is coupled to its function in muscle. We found that both ORBplus and PLS were able to identify patterns in the (1)H,(15)N-HSQC NMR data from different states of troponin C that correlated to its conformation.     

Determination of the illicit drug gamma-hydroxybutyrate (GHB) in human saliva and beverages by 1H NMR analysis

High resolution 1H NMR spectroscopy has been employed to investigate the detection and quantification of the illicit "date-rape" drug gamma-hydroxybutyrate (GHB) in both human saliva and a commonly-consumed low-alcohol beer product. Data acquired revealed that this multicomponent analytical technique provided unequivocal evidence for the detection of this agent by this technique in both of these matrices, i.e., all three of its resonances [those ascribable to the alpha-CH2 (t, delta=2.25 ppm), beta-CH2 (tt, delta=1.81 ppm) and gamma-CH2 (t, delta=3.61 ppm) group protons] were present in spectra acquired on human saliva, and two of these (the alpha- and beta-CH2 group signals) in the beverage product examined, the latter observation attributable to overlap of the gamma-CH2 1H resonance with those of carbohydrates. Since good linear calibration relationships between the intensities of each of the NMR-visible signals and added GHB concentration (the former normalised to that of an external 3-trimethylsilyl [2,2,3,3-2H4]- propionate standard present in a coaxial NMR tube insert) were observed, this illicit drug is also readily quantifiable in such multicomponent samples. Our data demonstrate the advantages offered by this technique when applied to the analysis of illicit drugs in multicomponent sample matrices such as human biofluids and beverage products.     

Lipid profiling of developing Jatropha curcas L. seeds using (1)H NMR spectroscopy

Seed development in Jatropha curcas L. was studied with respect to phenology, oil content, lipid profile and concentration of sterols. Seeds were collected at various stages of development starting from one week after fertilization and in an interval of five days thereafter till maturity. These were classified as stage I to stage VII. Moisture content of the seeds ranged from 8.8 to 90.3%; the lowest in mature seeds in stage VII and highest in stage I. The seed area increased as the seed grew from stage I to stage VI (0.2-10.2mm(2) per seed), however, the seed area shrunk at stage VII. Increase in seed area corresponded to increase in fresh weight of the seeds. (1)H NMR spectroscopy of hexane extracts made at different stages of seed development revealed the presence of free fatty acids (FFA), methyl esters of fatty acids (FAME) and triglycerol esters (TAG), along with small quantity of sterols. The young seeds synthesized predominantly polar lipids. Lipid synthesis was noticed nearly three weeks after fertilization. From the fourth week the seeds actively synthesized TAG. Stage III is a turning point in seed development since at this stage, the concentration of sterols decreased to negligible, there was very little FAME formation, accumulation of TAG increased substantially, and there was a sudden decrease in FFA concentration. The findings can be helpful in understanding the biosynthesis and in efforts to improve biosynthesis of TAG and reduce FFA content in the mature seeds.     

Metabolomic analysis of the plasma of patients with high-altitude pulmonary edema (HAPE) using 1H NMR

Upon rapid ascent to a high altitude, non-acclimatized individuals, although healthy, are highly prone to contracting high-altitude pulmonary edema (HAPE). Early diagnosis is difficult and there is no reliable biomarker available. We used proton ((1)H) NMR metabolomics to profile the altered metabolic patterns of blood plasma from HAPE patients. The plasmas of ten patients with HAPE and ten individuals without HAPE were collected and compared using (1)H NMR spectroscopy. Data were evaluated with several multivariate statistical analyses, including the principal components, the orthogonal partial least-squares discriminant, and the orthogonal signal correction partial least-squares discriminant. Multivariate statistical analyses revealed a significant disparity between subjects with HAPE and those in the control group. Compared to the plasma of the controls, the HAPE patients had significant increases in valine, lysine, leucine, isoleucine, glycerol phosphoryl choline, glycine, glutamine, glutamic acid, creatinine, citrate, and methyl histidine. These were accompanied by decreases in α- and β-glucose, trimethylamine, and the metabolic products of lipids. The data demonstrate that metabolomics may be effective for the diagnosis of HAPE in the future, and can be used for further understanding HAPE pathogenesis.     

Structural and functional analysis of the complement component factor H with the use of different enzymes and monoclonal antibodies to factor H.

The action of six different enzymes on the function and structure of Factor H was investigated by use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, haemagglutination, two enzyme-linked immunosorbent assay systems and an assay for Factor I cofactor activity. Six monoclonal antibodies directed against the 38 kDa tryptic fragment of Factor H [which contains the binding site for C3b (a 180 kDa fragment of the third component of complement) and the cofactor activity] were also used to detect cleavage products derived from the same fragment. Elastase, chymotrypsin A4 or trypsin first cleaved Factor H to 36-38 kDa fragments carrying all six monoclonal anti-(Factor H)-binding sites. In parallel, the interaction of Factor H with surface-bound C3b was lost, whereas the cofactor function was preserved. Further cleavage of the 36-38 kDa fragments into two 13-19 kDa fragments (one carrying the MAH4 and MRC OX 24 epitopes, the other the MAH1, MAH2, MAH3 and MRC OX 23 epitopes) destroyed cofactor activity. Pepsin, bromelain or papain rapidly split off a 13-15 kDa fragment of Factor H carrying the MAH1, MAH2, MAH3 and MRC OX 23 epitopes and destroyed all tested functions of Factor H. Ficin cleaved Factor H into disulphide-linked fragments smaller than 25 kDa, but did not affect the functions of the Factor H molecule. The 38 kDa tryptic fragment of Factor H is the N-terminal end of the Factor H molecule, as determined by N-terminal sequence analysis. A model is presented of the substructure of Factor H.     

1H NMR structural analysis of human ghrelin and its six truncated analogs.

Human ghrelin, the first recognized natural ligand of growth hormone secretagogue growth hormone secretagogue receptors (GHS-Rs) (M. Kojima, H. Hosada, Y. Date, M. Nakazato, H. Matsuo, and K. Kangawa, Nature, 1999, Vol. 402, pp. 656-660), consists of 28 amino acids of which Ser3 is modified by n-octanoylation. This new peptide hormone has been implicated not only in regulation of the GH secretion but also in regulation of food intake. The discovery of ghrelin opens up more opportunities to study the relationship of ghrelin with metabolic diseases. Until now, only mass spectometry analysis has been reported on the structure of ghrelin. NMR analysis is a suitable way to study if any tertiary structure of unbound ghrelin is present in solution. NMR studies were carried out on human ghrelin and its five truncated analogs. The full-length ghrelin and its fragments exhibited random coil behavior in aqueous solution. Additional studies were carried out on the shortest active segment of human ghrelin, which consists of the first five amino acids of the ghrelin sequence (M. A. Bednarek, S. D. Feighner, S.-S. Pong, K. K. McKee, D. L. Hreniuk, M. V. Silva, V. A. Warrem, A. D. Howard, L. H. Y. Van der Ploeg, and J. V. Heck, Journal of Medical Chemistry, 2000, Vol. 43, pp. 4370-4376), to compare the spectral features with their counterparts in the full-length ghrelin. The NMR data showed behavior similar to ghrelin except for two additional nuclear Overhauser effects (NOEs) between the Phe4 NH and the protons of the beta-methylene of Ser3. CD on human ghrelin and its short active analog in water were indicative of random coil peptides. Molecular modeling based on NMR data was carried out to probe which structural features were similar to growth hormone-releasing peptide-6 (GHRP-6), a hexapeptide that binds to GHS-R releasing GH and stimulating food intake. Modeling suggested some similarities, but they were not of a nature to account for binding properties of these compounds.     

An application of common principal component analysis to cranial morphometry of Microtus californicus and M. ochrogaster(Mammalia, Rodentia)

Principal component analysis (PCA) is a one-group method. Its purpose is to transform correlated variables into uncorrelated ones and to find linear combinations accounting for a relatively large amount of the total variability, thus reducing the number of original variables to a few components only.
In the simultaneous analysis of different groups, similarities between the principal component structures can often be modelled by the methods of common principal components (CPCs) or partial CPCs. These methods assume that either all components or only some of them are common to all groups, the discrepancies being due mainly to sampling error.
Previous authors have dealt with the k-group situation either by pooling the data of all groups, or by pooling the within-group variance-covariance matrices before performing a PCA. The latter technique is known as multiple group principal component analysis or MGPCA (Thorpe, 1983a). We argue that CPC- or partial CPC-analysis is often more appropriate than these previous methods.
A morphometrical example using males and females of Microtus californicus and M. ochrogaster is presented, comparing PCA, CPC and partial CPC analyses. It is shown that the new methods yield estimated components having smaller standard errors than when groupwise analyses are performed. Formulas are given for estimating standard errors of the eigenvalues and eigenvectors, as well as for computing the likelihood ratio statistic used to test the appropriateness of the CPC- or partial CPC-model.     

A comparison of human serum and plasma metabolites using untargeted <Superscript>1</Superscript>H NMR spectroscopy and UPLC-MS

Introduction

Differences in the metabolite profiles between serum and plasma are incompletely understood.

Objectives

To evaluate metabolic profile differences between serum and plasma and among plasma sample subtypes.

Methods

We analyzed serum, platelet rich plasma (PRP), platelet poor plasma (PPP), and platelet free plasma (PFP), collected from 8 non-fasting apparently healthy women, using untargeted standard 1D and CPMG ¹H NMR and reverse phase and hydrophilic (HILIC) UPLC-MS. Differences between metabolic profiles were evaluated using validated principal component and orthogonal partial least squares discriminant analysis.

Results

Explorative analysis showed the main source of variation among samples was due to inter-individual differences with no grouping by sample type. After correcting for inter-individual differences, lipoproteins, lipids in VLDL/LDL, lactate, glutamine, and glucose were found to discriminate serum from plasma in NMR analyses. In UPLC-MS analyses, lysophosphatidylethanolamine (lysoPE)(18:0) and lysophosphatidic acid(20:0) were higher in serum, and phosphatidylcholines (PC)(16:1/18:2, 20:3/18:0, O-20:0/22:4), lysoPC(16:0), PE(O-18:2/20:4), sphingomyelin(18:0/22:0), and linoleic acid were lower. In plasma subtype analyses, isoleucine, leucine, valine, phenylalanine, glutamate, and pyruvate were higher among PRP samples compared with PPP and PFP by NMR while lipids in VLDL/LDL, citrate, and glutamine were lower. By UPLC-MS, PE(18:0/18:2) and PC(P-16:0/20:4) were higher in PRP compared with PFP samples.

Conclusions

Correction for inter-individual variation was required to detect metabolite differences between serum and plasma. Our results suggest the potential importance of inter-individual effects and sample type on the results from serum and plasma metabolic phenotyping studies.

     

Phenotypic covariance structure in tamarins (genus Saguinus): a comparison of variation patterns using matrix correlation and common principal component analysis

Constancy of variation/covariation structure among populations is frequently assumed in order to measure the differential selective forces which have caused population differentiation through evolutionary time. Following Steppan ([1997] Evolution 51:571-594), this assumption is examined among closely related tamarin species (genus Saguinus), using two distinct approaches applied to the task of evaluating similarity in patterns of morphological variation: common principal component analysis and matrix correlations. While the results of these analyses may appear contradictory, closer examination reveals them as complementary, highlighting the wisdom of combined methodologies. Overall, the results reveal a close relationship among the morphologically based variance structures of the tamarin species a relationship whose pattern is consistent with the pattern of phylogenetic relatedness as found via a molecular genetic study. More specifically, both methodological approaches provide some support for divergence of S. geoffroyi and S. oedipus (with regards to their patterns of morphological variation) from other tamarin species. This suggests that variance/covariance structure may have diverged through evolutionary time in the tamarin lineage, placing assumptions of constancy in doubt.     

Characterization of human fetal cord blood steroid profiles in relation to fetal sex and mode of delivery using temperature-dependent inclusion chromatography and principal component analysis (PCA)

In the present work, human male and female fetal cord blood samples were purified, selectively extracted and separated to examine a fraction of steroids ranging from polar estetrol to relatively non-polar progesterone using solid phase extraction based on C-18 tubes and beta-cyclodextrin driven temperature dependent inclusion chromatography. Resulting UV diode array chromatographic patterns revealed the presence of 27 peaks. Chromatographic patterns of UV detected steroids were analyzed using principal components analysis which revealed differences between male/female and labour/not-in-labour clusters. Quantitative analysis of nine identified steroids including: estetrol, 17beta-estradiol, estrone, estriol, cortisol, cortisone, progesterone, 20 alpha-hydroxyprogesterone and 17 alpha-hydroxyprogesterone were not significantly different between males and females. Significant differences between male and female fetuses were related to as yet unidentified compounds. Four peaks were significantly different with labour which corresponded with cortisol, cortisone and two unidentified compounds. This protocol may distinguish significant differences between clinical groups that are not readily identifiable using univariate measurements of single steroids or different low molecular mass biomarkers. Moreover, we have provided new evidence that despite the absence of testosterone there are number of steroids and low molecular mass compounds that differ between male and female fetuses.     

Site-specific N-glycosylation of human chorionic gonadotrophin--structural analysis of glycopeptides by one- and two-dimensional 1H NMR spectroscopy.

Glycopeptides representing individual N-glycosylation sites of the heterodimeric glycoprotein hormone human chorionic gonadotrophin (hCG) were obtained from subunits hCG alpha (N-glycosylated at Asn-52 and Asn-78) and hCG beta (N-glycosylated at Asn-13 and Asn-30) by digestion with trypsin and chymotrypsin, respectively. Following purification by reverse-phase HPLC and identification by amino acid sequencing, the glycopeptides were analysed by one- and two-dimensional 1H NMR spectroscopy. The results are summarized as follows: (i) oligosaccharides attached to Asn-52 of hCG alpha comprised monosialylated 'monoantenary' NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-4'), disialylated diantennary NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[NeuAc alpha 2-3-Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N2), and the monosialylated hybrid-type structures NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-3Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-A) and NeuAc alpha 2-3Gal-beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-3(Man alpha 1-6)Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-AB) in a ratio approaching 5:2:2:1; (ii) Asn-78 of hCG alpha carried N2 and N1-4' almost exclusively (ratio approximately 3:2); (iii) both N-glycosylation sites of hCG beta contained predominantly component N2, partially (approximately 25%) and completely alpha 1-6-fucosylated at the N-acetylglucosamine linked to Asn-13 and Asn-30, respectively. The distinct site-specific distribution of the oligosaccharide structures among individual N-glycosylation sites of hCG appears to reflect primarily the influence of the surrounding protein structure on the substrate accessibility of the Golgi processing enzymes alpha-mannosidase II, GlcNAc transferase II and alpha 1,6-fucosyltransferase.     

Comparative analysis of diversification and population structure of kenaf (Hibiscus cannabinus L.) and roselle (H. sabdariffa L.) using SSR and RGA (resistance gene analogue) markers

Multiple DNA marker systems and complementary analytical approaches are often useful in population genetic analysis and speciation of plants. We investigated population structure of kenaf (Hibiscus cannabinus) and roselle (H. sabdariffa) for gaining insight in evolution and geographic separation of these crop species using SSR and RGA (resistance gene analogues) markers through Bayesian clustering and principal coordinate analysis (PCoA) methods. Genotyping by 12 SSR and 16 RGA markers amplified a total of 172 loci in the study population. The RGA markers generated higher number of alleles per marker (8.2) as compared to SSR (3.4), but exhibited lower heterozygosity in the population. Genetic variance and heterozygosity in roselle population for both marker systems were lower than in kenaf. RGA markers revealed higher variation among populations. Bayesian structure as well as PCoA analysis using RGA marker revealed distinct cluster for roselle, while SSR-based classification revealed high admixture. Results indicate geographic isolation and natural selection for adaptive RGA alleles in kenaf. The Indian kenaf landraces were distinct from the exotic kenaf accessions, suggesting separate lineage formation by geographic separation. Possible origin and domestication of roselle in South India is proposed.     

An application of common principal component analysis to cranial morphometry of Microtus californicus and M. ochrogaster (Mammalia, Rodentia): further remarks

We welcome Dr Thorpe's interesting discussion (Thorpe, 1988), and we would like to take this opportunity to clarify some points.
Both MGPCA (multiple group principal component analysis) and CPCA (common principal component analysis) serve essentially the same purpose, namely estimation of principal components simultaneously in several groups, based on the assumption of equality of principal component directions across groups, while eigenvalues may differ between groups. However, CPCA has the distinct advantage that this assumption can actually be tested, using the (CPC) statistic. In analyses involving more than two variables, it is usually difficult to decide, without a formal test, whether or not the assumption of common directions of principal components is reasonable.
There is also a conceptual difficulty with MGPCA. In statistical terms, both methods assume that:
(a) a certain set of parameters (namely those determining the eigenvectors) are common to all groups
(b) there are sets of parameters (namely p eigenvalues per group) which are specific to each group.
CPCA sets up a model that reflects this structure and estimates the parameters accordingly. MGPCA, on the other hand, ignores part (b), at least temporarily, by pooling the variance-covariance matrices and extracting eigenvectors from the single pooled matrix. This may lead to reasonable results, but there is no guarantee that it will indeed do so. The reader may find a more familiar analog in the fitting of regression lines when data are in groups. If it is assumed that all regression lines are parallel, one should set up an appropriate model based on a single slope parameter common to all groups, and groupwise intercepts. One should then estimate the parameters of this model, and not simply apply a technique which is appropriate in the one-group case only.     

Phylogenetic studies of the subgenus Nialoe (s. lat.) (Coleoptera, Carabidae, Genus Pterostichus), part 1: Homology of the component parts of male genitalia and higher phylogeny

A phylogenetic hypothesis of the subgenus Nialoe (s. lat.) of genus Pterostichus is proposed based on a cladistic analysis of seventeen morphological characters. Through comparative studies of the sclerites on the endophallus of male genitalia, it became apparent that the arrangement of these sclerites reflects seven different origins. The results show that Nialoe (s. lat.) is monophyletic and composed of four clades, while some traditional taxa are para- or polyphyletic, and are defined only by symplesiomorphies. Pterostichus (Nialoe) mosaicus sp. nov., which is indispensable for tracing the homology of sclerites in this subgenus, is described.     

Determination of the enantiomeric composition of chiral delta-2-oxazolines-1,3 by 1H and 19F NMR spectroscopy using chiral solvating agents

Studies of the perturbing effect of chiral solvating agents (CSAs) 5a and mostly of 5c upon the NMR spectra of chiral Delta(2)-oxazoline 1 demonstrated the ability of these fluoroalcohols to afford diastereomeric solvates from these solutes. Thus, for all tested Delta(2)-oxazolines 1Aa-d, 1Ba, and 1e there is at least one possibility to proceed to their enantiomeric discrimination either by (1)H or (19)F NMR using these CSAs (see Fig. 1). NMR results are discussed from substrate and CSA structure standpoints and a solvation model is proposed on the basis of the inequivalence senses generally observed. Then the method was applied to extracts of incubated locust tissues obtained by solid phase extraction (SPE) after a partial unmasking of the substrate 1.     

Conformational analysis of biologically active truncated linear analogs of endothelin-1 using NMR and molecular modeling.

Some linear truncated analogs of endothelin-1 display potent agonistic activity at the ET(B) receptor, especially when the side chain of Trp21 is N-formylated. Then, the three-dimensional arrangements of six structurally reduced linear analogs, three formylated and three nonformylated, have been investigated by high resolution NMR spectroscopy and molecular modeling, in order to pinpoint the conformational features related to the biological activity. Two-dimensional double-quantum-filtered correlation spectroscopy (DQFCOSY), total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY) were recorded and analyzed for each molecule. Interspatial distance constraints were derived from the intensity of the NOESY connectivities. The formation of hydrogen bonding was monitored from the temperature dependence of the NH chemical shifts. Molecular models calculated by means of distance geometry, simulated annealing and energy minimization, using the NMR constraints, strongly suggested a global elongated structure for the formylated analogs exhibiting biological activity, and a folded arrangement for the unformylated derivatives. Homology comparisons allowed the identification of a beta-turn-like folding of the C-terminal segment Asp18-Trp21 as a probable key-factor for activity.