Mapping and characterization of the entomocidal domain of the Bacillus thuringiensis CrylA(b) protoxin

The amino acid sequences necessary for entomocidal activity of the CryIA(b) protoxin of Bacillus thuringiensis were determined. Introduction of stop codons behind codons Arg601, Phe604 or Ala607 showed that amino acid residues C-terminal to Ala607 are not required for insecticidal activity and that activation by midgut proteases takes place distal to Ala607. The two shortest polypeptides, deleted for part of the highly conserved β-strand, were prone to proteolytic degradation, explaining their lack of toxicity. Apparently, this β-strand is essential for folding of the molecule into a stable conformation. Proteolytic activation at the N-terminus was investigated by removing the first 28 codons, resulting in a translation product extending from amino acid 29 to 607. This protein appeared to be toxic not only to susceptible insect larvae such as Manduca sexta and Heliothis virescens, but also to Escherichia coli cells. An additional mutant, encoding only amino acid residues 29–429, encompassing the complete putative pore forming domain, but lacking a large part of the receptor-binding domain, was similarly toxic to E. coli cells. This suggests a role for the N-terminal 28 amino acids in rendering the toxin inactive in Bacillus thuringiensis, and indicates that the cytolytic potential of the pore forming domain is only realized after proteolytic removal of these residues by proteases in the insect gut. In line with this hypothesis are results obtained with a mutant protein in which Arg28 at the cleavage site was replaced by Asp. This substitution prevented the protein from being cleaved by trypsin in vitro, and reduced its toxicity to M. sexta larvae.     

Dissection of functional domains in Escherichia coli DNA photolyase by linker-insertion mutagenesis.

Summary The phr gene, which encodes protein of 472 amino acid residues, is required for light-dependent photoreactivation and enhances light-independent excision repair of ultraviolet light (UV)-induced DNA damage. In this study, dodecamer HindIII linker insertions were introduced into the cloned phr gene and the functional effects of the resulting mutations on photoreactivation and light-independent dark repair in vivo were studied. Among 22 mutants obtained, 7 showed no photoreactivation as well as no enhancement of light-independent repair. Four of these were located in amino acid residues between Gln333 and Leu371 near the 3 end of the gene, two were located in a small region at Glu275 to Glu280 near the middle of the gene and the remaining one was between Pro49 and Arg50. Three mutants that had insertions located in the 42 by segment from 399 to 441 by of the phr coding sequence (corresponding to amino acid residues Ile134 to Lys149) lost the light-independent repair effect but retained photoreactivation. These results suggest that (i) Escherichia coli DNA photolyase contains several critical sites that are distributed over much of the enzyme molecule, and (ii) a functional domain required for the effect on light-independent repair is at least in part distinct from that necessary for light-dependent photoreactivation.     

Chemical modification of xylanase from Trichosporon cutaneum shows the presence of carboxyl groups and cysteine residues essential for enzyme activity

The endo--1,4-xylanase (EC 3.2.1.8) from Trichosporon cutaneum was chemically modified using amino acid-specific reagents. The enzyme does not bear arginines essential for activity, since 1,2-cyclohexanedione and 2,3-butanedione, although they modify the enzyme (after chromatographic analysis), have no effect on its activity. Reaction of the enzyme with tetranitromethane and N-acetylimidazole did not result in a significant activity loss as a result of modification of tyrosine residues. The water-soluble carbodiimide 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide inactivated the xylanase rapidly and completely in a pseudo-first-order process, and kinetic analysis indicated that at least one molecule of carbodiimide binds to the enzyme for inactivation. A mixture of neutral xylooligomers provided significant protection of the enzyme against this carbodiimide inactivation. Reaction of the xylanase with 2,4,6-trinitrobenzene sulfonic acid did not result in a significant activity loss as a result of modification of lysine residues. Titration of the enzyme with 5,5-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetamide and p-chloromercuribenzoate indicated the presence of a free/active thiol group. Xylan completely protected the enzyme from inactivation by p-hydroxymercuribenzoate, suggesting the presence of cysteine at the substrate-binding site. Inactivation of xylanase by p-hydroxymercuribenzoate could be restored by cysteine.     

Differential effect of amino acid residues on the stability of double helices formed from polyribonucleotides and its possible relation to the evolution of the genetic code

Summary The interaction of amino acid residues with polyribonucleotides was characterized by measurements of melting temperatures (t_m) for poly(A) poly(U) and poly(I)poly(C) as functions of the concentrations of various amino acid amides. The amides of hydrophilic amino acids lead to a continuous increase of tm with increasing concentration, whereas amides of hydrophobic amino acids induce a decrease of t_m at low concentrations (1 mM) followed by an increase at higher concentrations. Analysis of the data by a simple site model provides the affinity of each ligand for the double helix relative to that for the single strands. This parameter decreases in the order Ala>Gly>Ser>Asn>Pro>Met, Val>Ile, Leu for poly(A) poly(U) and Ala, Gly, Ser>Asn>Pro>Val>Ile, Met, Leu for poly(I)poly(C). The special effects of hydrophobic amino acids may be related to the similarity of the codons for these amino acids. A simple model for assignment of codons to amino acids is proposed.     

Phage display of a double-headed proteinase inhibitor: Analysis of the binding domains of potato proteinase inhibitor II

Potato proteinase inhibitor II (PI2) is a serine proteinase inhibitor composed of two domains that are thought to bind independently to proteinases. To determine the activities of each domain separately, various inactive and active domain combinations were constructed by substituting amino acid residues in the active domains by alanines. These derivatives were expressed as soluble protein inEscherichia coli and exposed on M13 phage as fusions to gene 3 in a phagemid system for monovalent phage display. Inactivation of both active domains by Ala residues reduced binding of phage to trypsin and chymotrypsin by 95%. Ten times more phage were bound to proteinases by domain II compared to domain I, while a point mutation (Leu⁵ Arg) altered the binding specificity of domain I of PI2 phage from chymotrypsin to trypsin. The mutants were used to show that functional PI2 phage mixed with nonfunctional PI2 phage could be enriched 323 000-fold after three rounds of panning. Thus, these results open up the possibility to use phage display for the selection of engineered PI2 derivatives with improved binding characteristics towards digestive proteinases of plants pests.The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number L37519 (p303.51).     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

A comparison between the amino acid composition of an egg parasitoid wasp and some of its hosts

Amino acid compositions of the eggs of five lepidopteran hosts for Trichogramma minutum were compared with each other and with a non-host species, Rhodnius prolixus, in which T. minutum oviposits but does not develop. Host eggs are quite homogeneous, particularly when compared according to groupings of potentially interconvertible amino acids. Combined mole percent values for glycine, serine and alanine were higher in hosts (27.5–29.2 mole%) than in R. prolixus eggs (21.5 mole%), in bovine serum albumin (14.9%), which has been used as a protein source in artificial diets for T. minutum, or in many of the mixtures used in published diets for this species. Since these three amino acids make up 26.3 mole% of the adult amino acid content of T. minutum, their deficiency in diets could require metabolic compensation detrimental to development.Adult T. minutum arising from eggs of Manduca sexta, Choristoneura fumiferana, and Sitotroga cerealella are similar in amino acid composition to each other and, in general, to their hosts. Variability appears greater in hosts than in adult wasp composition, suggesting some interconversion of host amino acids to accommodate inflexible nutritional requirements of T. minutum.In the three host species tested, free amino acids constituted 15.8–19.3% by weight of the amino acid in egg contents. In M. sexta eggs, glycine, serine and alanine together make up 28.4% by weight of the total free amino acid, a much higher proportion than in many published diets. The four free amino acids (isoleucine, leucine, phenylalanine and histidine) reported to be oviposition stimulants in experiments on encapsulated diets are present in sufficient concentrations to induce oviposition in the host species tested and in R. prolixus. S. cerealella egg contents having approximately 1.8 g amino acid, yield one or rarely two adult T. minutum (1g amino acid/insect). In contrast, M. sexta eggs with 94 g amino acid each yield an average of 10–12 adults (8.2g amino acid/insect). This suggests that small hosts are allocated few eggs which can only develop into small adults because of nutrient supply (parasitoid size in metabolically restricted), whereas much larger hosts are allocated proportionately fewer eggs than the former resulting in larger, and presumably more viable and fecund, adults (parasitoid size is established behaviourally).     

Proteome Composition in Plasmodium falciparum: Higher Usage of GC-Rich Nonsynonymous Codons in Highly Expressed Genes

The parasite Plasmodium falciparum, responsible for the most deadly form of human malaria, is one of the extremely AT-rich genomes sequenced so far and known to possess many atypical characteristics. Using multivariate statistical approaches, the present study analyzes the amino acid usage pattern in 5038 annotated protein-coding sequences in P. falciparum clone 3D7. The amino acid composition of individual proteins, though dominated by the directional mutational pressure, exhibits wide variation across the proteome. The Asn content, expression level, mean molecular weight, hydropathy, and aromaticity are found to be the major sources of variation in amino acid usage. At all stages of development, frequencies of residues encoded by GC-rich codons such as Gly, Ala, Arg, and Pro increase significantly in the products of the highly expressed genes. Investigation of nucleotide substitution patterns in P. falciparum and other Plasmodium species reveals that the nonsynonymous sites of highly expressed genes are more conserved than those of the lowly expressed ones, though for synonymous sites, the reverse is true. The highly expressed genes are, therefore, expected to be closer to their putative ancestral state in amino acid composition, and a plausible reason for their sequences being GC-rich at nonsynonymous codon positions could be that their ancestral state was less AT-biased. Negative correlation of the expression level of proteins with respective molecular weights supports the notion that P. falciparum, in spite of its intracellular parasitic lifestyle, follows the principle of cost minimization. [Reviewing Editor : Dr. Richard Kliman]     

Dihaploids of Elymus from the interspecific crosses E. dolichatherus x E. tibeticus and E. brevipes x E. panormitanus

Summary Dihaploids (n=2x=14, SY) of two Elymus species, i.e., E. dolichatherus (Keng) Löve (2n=4x=28, SSYY) and E. brevipes (Keng) Löve (2n=4x=28, SSYY), were obtained from the interspecific hybrid combinations E. dolichatherus () x E. tibeticus (Meld.) G. Singh () and E. brevipes () x E. panormitanus (Parl.) Tzvelev (). The dihaploids were probably formed through selective elimination of male parental chromosomes in early embryo development. Meiotic chromosome behavior was studied in E. dolichatherus, E. brevipes, and their dihaploids. The two parental Elymus species had regular meioses with predominantly ring bivalent formation. A low frequency of homoeologous chromosome pairing was observed, with an average of 0.81 bivalents and 0.03 trivalents in the dihaploid of E. dolichatherus, and 0.26 bivalents in the dihaploid of E. brevipes. Up to two chromatid bridges accompanied by small fragments were present at anaphase I of the E. dolichatherus dihaploid. It is concluded from this study that: (i) both E. dolichatherus and E. brevipes are allotetraploid species; (ii) a low affinity exists between the S and Y genomes of the two Elymus species.     

Biophysical characterization of the MerP-like amino-terminal extension of the mercuric reductase from<Emphasis Type="Italic"> Ralstonia metallidurans</Emphasis> CH34

The purified native mercuric reductase (MerA) from Ralstonia metallidurans CH34 contains an N-terminal sequence of 68 amino acids predicted to be homologous to MerP, the periplasmic mercury-binding protein. This MerP-like protein has now been expressed independently. The protein was named MerAa by homology with Ccc2a, the first soluble domain of the copper-transporting ATPase from yeast. a has been characterized using a set of biophysical techniques. The binding of mercury was followed using circular dichroism spectroscopy and electrospray mass spectrometry. The two cysteine residues contained in the consensus sequence GMTCXXC are involved in the binding of one mercury atom, with an apparent affinity comparable to that of MerP for the same metal. The metal-binding site is confirmed by NMR chemical shift changes observed between apo- and metal-bound MerAa in solution. NMR shift and NOE data also indicate that only minor structural changes occur upon metal binding. Further NMR investigation of the fold of MerAa using long-range methyl–methyl NOE and backbone residual dipolar coupling data confirm the expected close structural homology with MerP. ¹⁵N relaxation data show that MerAa is a globally rigid molecule. An increased backbone mobility was observed for the loop region connecting the first -strand and the first -helix and comprising the metal-binding domain. Although significantly reduced, this loop region keeps some conformational flexibility upon metal binding. Altogether, our data suggest a role of MerAa in mercury trafficking.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-003-0495-yAbbreviations CCA -cyano-4-hydroxy-trans-cinnamic acid - CSI chemical shift index - HSQC ¹H-detected heteronuclear single-quantum coherence - MerAa the 68 amino acid N-terminal extension of the mercuric reductase - NOE nuclear Overhauser effect - RDCs residual dipolar couplings - TCEP-HCl tris(2-carboxyethyl)phosphine hydrochloride     

Primary structure characterization of Bothrops jararacussu snake venom lectin

The complete amino acid sequence of the lectin from Bothrops jararacussu snake venom (BJcuL) is reported. The sequence was determined by Edman degradation and amino acid analysis of the S-carboxymethylated BJcuL derivative (RC-BJcuL) and from its peptides originated from enzymatic digestion. The sequence of amino acid residues showed that this lectin displays the invariant amino acid residues characterized in C-type lectins. Amino acids analysis revealed a high content of acidic amino acids and leucine. These findings suggest that BJcuL, like other snake venom lectins, possesses structural similarities to the carbohydrate recognition domain (CRD) of calcium-dependent animal lectins belonging to the C-type -galactoside binding lectin family.     

Covalent Structure of Botulinum Neurotoxin Type B; Location of Sulfhydryl Groups and Disulfide Bridge and Identification of C-Termini of Light and Heavy Chains

Botulinum neurotoxin (NT) serotype B, produced by Clostridium botulinum (proteolytic strain), is a 150-kDa single-chain polypeptide of 1291 amino acids, of which 10 are Cys residues [Whelan et al. (1992), Appl. Environ. Microbiol. 58, 2345–2354] The posttranslational modifications of the gene product were found to consist of excision of only the initiating Met residue, limited proteolysis (nicking) of the 1290-residue-long protein between Lys 440 and Ala 441, and formation of at least one disulflde bridge. The dichain (nicked) protein, in a mixture with the precursor single-chain (unnicked) molecules, was found to have a 50-kDa light chain (Pro 1 through Lys 440) and a 100-kDa heavy chain (Ala 441 through Glu 1290). The limited in vivo nicking of the single-chain NT to the dichain form, by protease endogenous to the bacteria, and the nonfacile in vitro cleavage by trypsin of the Lys 440–Ala 441 bond appear to be due to the adjacent Ala 441–Pro 442 imide bond's probable cis configuration in a mixed population of molecules with cis and trans configurations. The two chains were found connected by an interchain disulfide formed by Cys 436 and Cys 445. Six other Cys residues, at positions 70, 195, 308, 777, 954, and 1277, were found in sulfhydryl form. In addition, a Cys at position 1220 or 1257 appeared to be in sulfhydryl form, hence our experimental results could not unambiguously identify presence of an intrachain disulfide bridge near the C-terminus of the NT. A total of 384 amino acid residues, including the 6 Cys residues at positions 70, 195, 308, 436, 445, and 1277, were identified by direct protein-chemical analysis; thus 29.7% of the protein's entire amino acid sequence predicted from the nucleotide sequence was confirmed. The 6 amino acids, residues 945–950, did not match with the sequence predicted in 1992, but did match with a later report of 1995. The above determinations were made by a combination of chemical (CNBr and acidic cleavage at Asp–Pro) and enzymatic (trypsin, clostripain, and pepsin) cleavages of the NT, and NT carboxymethylated with iodoacetamide (with or without ¹⁴C label), separation and isolation of the fragments by SDS–PAGE (followed by electroblotting onto PVDF membrane), and/or reversed-phase HPLC, and analyses of the fragments for the N-terminal amino acid sequences by Edman degradation and amino acid compositions.     

Kinetic characterization of the Escherichia coli oligopeptidase A (OpdA) and the role of the Tyr residue

Oligopeptidase A (OpdA) belongs to the M3A subfamily of bacterial peptidases with catalytic and structural properties similar to mammalian thimet-oligopeptidase (TOP) and neurolysin (NEL). The three enzymes have four conserved Tyr residues on a flexible loop in close proximity to the catalytic site. In OpdA, the flexible loop is formed by residues 600-614 (⁶⁰⁰SHIFAGGYAAGYYSY⁶¹⁴). Modeling studies indicated that in OpdA the Tyr⁶⁰⁷ residue might be involved in the recognition of the substrate with a key role in catalysis. Two mutants were constructed replacing Tyr⁶⁰⁷ by Phe (Y607F) or Ala (Y607A) and the influence of the site-directed mutagenesis in the catalytic process was examined. The hydrolysis of Abz-GXSPFRQ-EDDnp derivatives (Abz = ortho-aminobenzoic acid; EDDnp N-[2,4-dinitrophenyl]-ethylenediamine; X = different amino acids) was studied to compare the activities of wild-type OpdA (OpdA WT) and those of Y607F and Y607A mutants The results indicated that OpdA WT cleaved all the peptides only on the X-S bond whereas the Y607F and Y607A mutants were able to hydrolyze both the X-S and the P-F bonds. The kinetic parameters showed the importance of Tyr⁶⁰⁷ in OpdA catalytic activity as its substitution promoted a decrease in the k_cat/K_m value of about 100-fold with Y607F mutant and 1000-fold with Y607A. Both mutations, however, did not affect protein folding as indicated by CD and intrinsic fluorescence analysis. Our results indicate that the OpdA Tyr⁶⁰⁷ residue plays an important role in the enzyme-substrate interaction and in the hydrolytic activity.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Covalent structure of botulinum neurotoxin type A: Location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains

Botulinum neurotoxin Type A is synthesized byClostridium botulinum as a 150 kD single chain polypeptide. The posttranslational processing of the 1296 amino acid residue long gene product involves removal of the initiating methionine, formation of disulfide bridges, and limited proteolysis (nicking) by the bacterial protease(s). The mature dichain neurotoxin is made of a 50-kD light chain and a 100-kD heavy chain connected by a disulfide bridge. DNA derived amino acid sequencepredicted a total of 9 Cys residues (Binzet al., 1990,J. Biol. Chem. 265, 9153–9158; Thompsonet al., 1990,Eur. J. Biochem. 189, 73–81). Treatment of the dichain neurotoxin, dissolved in 6 M guanidine. HCl, with 4-vinylpyridine converted 5 Cys residues into S-pyridylethyl cysteine residues; but alkylation after mercaptolysis converted all 9 Cys residues in the S-pyridylethylated form. After confirming the predicted number of Cys residues by amino acid analysis, the positions of the 5 Cys residues carrying sulfhydryl groups and the 4 involved in disulfide bridges were determined by comparing the elution patterns in reversed-phase HPLC of the cyanogen bromide mixtures of the exclusively alkylated and the mercaptolyzed-alkylated neurotoxin. The chromatographically isolated components were identified by N-terminal amino acid sequence analysis. The HPLC patterns showed characteristic differences. The Cys residuespredicted in positions 133, 164, 790, 966, and 1059 were found in the sulfhydryl form; Cys 429 and 453 were found disulfide-bridged connecting the light and heavy chains, and Cys 1234 and 1279 were found in an intrachain disulfide-bridge near the C-terminus in the heavy chain. Ten amino acid residues, Thr 438-Lys 447,predicted to be present in the single chain neurotoxin were not found in the dichain neurotoxin. Nicking of single-chain neurotoxin by the protease(s) endogenous to the bacteria therefore appears to excise these 10 amino acid residues from the nicking region which leaves Lys 437 as the C-terminus of the light chain and Ala 448 as the N-terminus of the heavy chain. The N-terminal Pro 1 and C-terminal Leu 1295,predicted from the nucleotide sequence, remain conserved after nicking. Residues Pro 1-Lys 437 and Ala 448-Leu 1295 constitute the light and heavy chains, respectively. The C-termini were determined by isolation of short C-terminal peptide fragments and subsequent sequence analysis by Edman degradation. About 20% of the amino acid sequence predicted from DNA analysis was confirmed in these studies by protein-chemical methods.     

Resonant recognition model of neuropeptide Y family: hot spot amino acid distribution in the sequences

The resonant recognition model is used to predict structurally and functionally important amino acid residues (so-called hot spots) in the neuropeptide Y (NPY) family. Thirty-three polypeptides belong to this family. All of them consist of 36 amino acids. The model predicts that residues 10 and 28 in the polypeptides are hot spots. In the 33 polypeptides, most of the amino acids at residue 10 are acidic amino acids, glutamic acid and aspartic acid. Other minor amino acids, serine, glycine, and proline, have high probabilities of -turn occurrence. Amino acids at residue 28 are all branched hydrophobic amino acids, isoleucine, leucine, and valine. The profile for predicting hot spots indicates repeating patterns of residues 1–18 and residues 19–36. Absolute values at residue i and residue i + 18 are the same, but these residues have opposite signs. Therefore the model of the NPY family predicts hot spots concerning a combination of residue i and residue i + 18.