INVESTIGATION OF THE ROLE OF TRYPTOPHAN IN α-MSH*:

Two analogues of α-MSH are described, in which the tryptophan residue occuring in position 9 of the natural hormone has been replaced by pentamethyl-phenylalanine and phenylalanine, respectively. The analogues were synthesized via a conventional procedure and the [Phe⁹]-analogue also by a semi-synthetic approach, which demonstrated thefavourable properties of the applied, new amino-protecting Msc function for this purpose. The widely different electron donor properties of the substituted residues were accompanied by a large difference in melanocyte stimulating activity of the analogues. The [Pmp⁹]-analogue, having donor properties comparable to those of the natural compound, was four to five times more active than the analogue containing the poorly donating Phe residue. The opposite effect was noted in in vivo lipolysis in rabbits.     

Improved solid‐phase synthesis of α,α‐dialkylated amino acid‐rich peptides with antimicrobial activity*

Abstract: A homologous series of nonapeptides and their acetylated versions were successfully prepared using solid‐phase synthetic techniques. Each nonapeptide was rich in α,α‐dialkylated amino acids [one 4‐aminopiperidine‐4‐carboxylic acid (Api) and six α‐aminoisobutyric acid (Aib) residues] and also included lysines or lysine analogs (two residues). The incorporation of the protected dipeptide 9‐fluorenylmethyloxycarbonyl (Fmoc)‐Aib‐Aib‐OH improved the purity and overall yields of these de novo designed peptides. The helix preference of each nonapeptide was investigated in six different solvent environments, and each peptide's antimicrobial activity and cytotoxicity were studied. The 3₁₀‐helical, amphipathic design of these peptides was born out most prominently in the N‐terminally acetylated peptides. Most of the peptides exhibited modest activity against Escherichia coli and no activity against Staphylococcus aureus. The nonacetylated peptides (concentrations ≤100 μm ) and the acetylated peptides (concentrations ≤200 μm ) did not exhibit any significant cytotoxicity with normal (nonactivated) murine macrophages.     

Effect of divalent cations on structure-function relationships of the antitumor protein α-sarcin

α-Sarcin binds one Zn(II) cation per protein molecule, with a K_d value of 0.9 mM, determined by equilibrium dialysis experiments. Ca(II), Mg(II), and Mn(II) do not bind to α-sarcin. Cd(II) and Co(II) also behave as Zn(II). The binding produces local modifications on the protein conformation affecting the microenvironment of tryptophan residues. The three cations modify the fluorescence emission of the protein. The near-u. v. circular dichroism spectrum of the protein is also altered. The binding of Zn(II) and related cations does not modify the secondary structure of the protein. The ribonucleolytic activity of a-sarcin is inhibited upon Zn(II) binding, but no alteration of the ability of the protein to aggregate phospholipid vesicles has been observed.     

Binding of Prazosin to α1-Acid Glycoprotein and Albumin: Effect of Protein Purity and Concentrations

Abstract: Prazosin is extensively bound in human serum/plasma. In the present study a bound fraction of 93–95% was observed at 37° for therapeutic drug concentrations. Both α₁-acid glycoprotein (AAG) and albumin (HSA) are established as transport proteins for prazosin, but their individual contribution to the extent and variability of protein binding in serum/plasma is unclear. The present study showed that AAG possesses one binding site per molecule with high affinity (Kd≈0.8 μM) for prazosin. HSA, essentially globulin-free, bound prazosin with lower affinity (Kd≈30 μM) with an average of 0.3 binding sites per molecule. However, less purified HSA, containing globulins, exhibited apparently higher affinity (Kd≈8 μM), but lower binding capacity (0.07 sites per molecule) for prazosin. In mixtures of highly purified proteins, the concentrations of AAG, and not HSA, determined the extent and variability of prazosin binding.     

SYNTHESIS,CHARACTERIZATION AND FLUORESCENCE STUDIES ON N-α-DANSYLALANYLLYSYL TRYPSINO (HIS-46)-METHANE

The 1-dimethylamino-5-naphthalene sulfonyl (dansyl) derivative of alanyllsyl chloromethane was synthesized and employed to introduce the fluorescent dansyl moiety specifically into the active site of trypsin via affinity labelling. The potential of dansylalanyllysyl chloromethane lies in its high degree of selectivity and markedly faster rate of enzyme inactivation when compared to previously synthesized, single residue affinity label chromophores. This permits the practical utilization of stoichiometric amounts of the inhibitor to achieve 100% inactivation of trypsin, even at high dilutions. The transfer of energy between the four tryptophan residues of trypsin and the bound dansyl group has been investigated in the fluorescent inhibitor-enzyme conjugate. From transfer efficiency measurements mean distances of 19.0 Å and 19.3 Å between the point of attachment of the dansyl group and the four tryptophan residues of trypsin have been calculated. These compare well with the mean value of 18.8 Å derived from calculations based on crystallo-graphic model studies.     

TRINITROPHENYLATION OF BOVINE GROWTH HORMONE

Bovine growth hormone was chemically modified with picryl sulfonic acid, at pH 8.4 during 2 and 5 min of reaction. The N-terminal residue provides the most reactive amino group followed by the amino groups of lysine 179 and lysines 143, 69, 111, 170 and 166 in decreasing order. These results agree with those obtained previously with equine growth hormone, except that residue 156 is not modified in bovine growth hormone, An important decrease in biological activity occurs between 2 and 5 min of reaction without sensible modification in the α-helix content of the molecule.     

Variation of amino acid properties in protein secondary structures, α-helices and β-strands

A study was made on the physical, chemical, energetic, conformational, geometric, and dynamic property potentials of amino acid residues in protein secondary structures: α-helix and β-strand. Property patterns were obtained by computing the average property values for specified residue units partitioned longitudinally and transversely about the chain. It was found that in r-helices with not more than 15 residues, there exist longitudinally opposing portions, one characteristically higher in average property potentials than the other. The helical chain, in general, acquires either an increasing or decreasing average potential in the N-terminal to C-terminal direction. The sequence-wise and surface-wise variations of property potentials in the elements of β-structure also revealed such general patterns. Possible wrong predictions in statistical methods of one secondary structural class over the other are pointed out.     

Effect of terminal arrangement of tryptophan on biological activity of symmetric α‐helix‐forming peptides

It is traditionally believed that the distribution of tryptophan (Trp) residues is critical for the novo design of antimicrobial peptides (AMPs). However, there is scarce knowledge regarding Trp residues arrangement at the head group level. Thus, a set of α‐helical AMPs containing different Trp residue arrangements at the N‐/C‐terminal of sequence were designed to increase the strategy database and analyze their biological activities. The arrangement of the N‐terminal Trp residue significantly improved the bacteriostatic activity of the peptides, but the C‐terminal Trp residue arrangement reduced the biocompatibility of them. WL and LW were effective against Gram‐negative microbes and had high selectivity for bacteria as compared to human erythrocytes and mammalian cells. They both maintained a relatively desirable activity in the presence of physiological salts and serum. It is observed through electron microscope, flow cytometry and fluorescence spectroscopy that target peptides can penetrate bacterial cell membrane and kill it by damaging cell membrane integrity. Collectively, we determined the structure–activity relationship of Trp residue distributions in a symmetric sequence structure and filled the gap in knowledge related to Trp arrangements at the head group level. The obtained results will be helpful in designing of artificial peptide‐based antimicrobials.     

Local structural differences between α and β-elicitins shown by circular dichroism and ultraviolet difference spectroscopy

In order to investigate differences in the conformation of elicitins exhibiting different levels of activity (toxicity to tobacco plants). the environment of the tyrosyl residues in four elicitins has been compared by different spectroscopic methods (difference absorption and circular dichroism). We compared two α-elicitins (capsicein and parasiticein) and two β-elicitins (β-cryptogein and β-cinnamomin), that are 50–100 times more toxic than the α-ones. Thermal difference UV spectroscopy and titration experiments clearly showed the exposure of Tyr-85 by comparison of parasiticein lacking Tyr-85 and the accessibility of its hydroxyl group to the solvent. The adjacent Ty-87 was also suggested to be located at the surface. In β-cryptogein, β-cinnamoniin and capsicein the pK was measured at between 10.5 and 10.8, while in parasiticein it is higher (11.5) owing to a difference in the local environment. Thermal difference UV spectroscopy showed one more exposed tyrosine in β-elicitins than in α-ones. This difference was attributed to Tyr-12, considering the more hydrophilic characteristic of the sequence around residue 13 in β-elicitins and the role of this region in the toxicity. However, no difference in titration behaviour was noted among elicitins concerning Tyr-12. The other two tyrosines also presented an abnormal pK of titration (> 12). In all elicitins Tyr-47 was probably exposed, while Tyr-33 was probably buried and not titrated, except in β-cinnamomin at very alkaline pH.     

SELECTIVE CLEAVAGE AT THE SINGLE TRYPTOPHAN RESIDUE IN BOVINE SOMATOTROPIN BY 2-(2-NITROPHENYLSULFENYL)-3-METHYL-3‘-BROMOINDOLEMINE

SELECTIVE CLEAVAGE AT THE SINGLE TRYPTOPHAN RESIDUE IN BOVINE SOMATOTROPIN BY 2-(2-NITROPHENYLSULFENYL)-3-METHYL-3′-BROMOINDOLEMINE The single tryptophan residue at position 86 in bovine somatotropin has been cleaved by the use of 2-(2-nitrophenylsulfenyl)-3-methyl-3′-bromoindolemine. This reaction was carried out with native bovine growth hormone as well as its reduced and alkylated derivative. When performed on the intact hormone, followed by the reduction and alkylation of the disulfides to liberate the two fragments, the cleavage reaction yielded the fragment containing residues 87–191 in a monomeric state. The other fragment, containing residues 1–86, could only be obtained in a highly aggregated form admixed with other material. When the cleavage reaction was carried out on the reduced and alkylated hormone, both the N-terminal fragment of reduced-carbamidomethylated bovine somatotropin as a monomer and the C-terminal fragment as a dimer were obtained in 35–40% yield. Since the cleavage reaction also oxidized methionine to methionine sulfoxide, the sulfoxides were reduced back to the methionine with N-methylmercapto acetamide. In this manner the following new derivatives and fragments of bovine somatotropin have been prepared: completely reduced and alkylated hormone, reduced and alkylated hormone with its methionines and tryptophan oxidized, reduced and alkylated hormone with its tryptophan oxidized, the N-terminal fragment of the hormone containing residues 1–86 with its methionine and tryptophan oxidized, the C-terminal fragment containing residues 87–191, both with its methionines oxidized and in their native state. The chemical and physical properties of these derivatives and fragments have been determined. Biological activity in the rat tibia was found to be negligible for all derivatives and fragments.     

Preparation of photoreactive derivatives of α-melanotropin by selective modification of the lysine or tryptophan residue

Photoreactive derivatives of α-melanotropin were prepared by selective modification of the single tryptophan residue in position 9 or the amino group of the lysine residue in position 11 by reaction with 2-nitro-4-azidophenylsulfenyl chloride. [2-Nitro-4-azidophenylsulfenyl-Trp⁹]-α-melanotropin was found to be five times as potent as α-melanotropin in stimulating lipolysis in isolated rabbit adipocytes. [N^ε-2-nitro-4-azidophenylsulfenyl-Lys¹¹]-α-melanotropin had only 11% of the lipolytic potency of α-melanotropin.     

Thiophilic interaction chromatography of Alzheimer's β‐amyloid peptides

Abstract: Alzheimer's disease is characterized by a progressive formation of senile plaques in the brain, the major constituent of which is β‐amyloid (Aβ) peptide, a proteolytic product of the transmembrane β‐amyloid precursor protein (APP). Prior to the measurement of levels of the Aβ peptide for diagnostic purposes, this peptide must be isolated from the myriad of proteins resident in the human serum. Thiophilic interaction chromatography is an effective method for the isolation of proteins and peptides containing clusters of aromatic residues such as tryptophan, phenylalanine and tyrosine. The purpose of the present study was to develop a protocol for binding and recovery of Aβ peptides (1–38), (1–40) and (1–42) to T‐gels by varying T‐gel type and elution conditions such as the salt concentration and type of eluent. We established the minimal salt concentration necessary for the binding of the Aβ(1–40) peptide to the 3S‐gel; binding at that concentration was subsequently compared with that of model proteins, lysozyme and α‐chymotrypsin and this methodology was extended to 2S‐gels and PyS. β‐Amyloid peptide (1–40) showed a remarkably strong affinity for all three types of T‐gels in comparison to lysozyme and α‐chymotrypsin and was found to bind best to 2S‐gel.     

CONFORMATIONAL ANALYSIS OF A CHAIN REVERSAL IN α-CHYMOTRYPSIN

A complete conformational analysis of the fold (Asp-Lys-Thr-Gly) (residues 35–38), and additional adjacent residues of α-chymotrypsin has been performed. A comparison of these findings with those of Lewis et al. (1) is made, and a discussion of the implications of protein-fold models is discussed. This particular residue sequence prefers to bend over maintaining a helical conformation. However, the bend conformation of the tetramer is different from that of the native bend. The native bend conformation is nearly realized when an additional residue of the native primary structure is added to each side of the tetramer. Early and late folding-sequence studies suggests that while the native fold is of low energy, there are fold-points along the primary structure which are more stable. The structural implications of this finding are discussed.     

The preferred solid-state conformation of (αMe)Trp peptides

The two Z-l -Ala-d l -(xMe)Trp-NH₂ diastereomeric dipeptides were synthesized from (Z-l -Ala)₂O and H-dl -(xMe)Trp-NH₂. The latter racemate, prepared by phase-transfer catalyzed alkylation of the N^α-benzylidene derivative of alanine amide followed by acidic hydrolysis of the resulting Schiff base, was characterized by X-ray diffraction. The molecular and crystal structure of Z-l -Ala-l -(αMe)Trp-NH², separated from its diastereomer by silica-gel column chromatography, was determined by X-ray diffraction analysis. Both independent molecules in the asymmetric unit of the dipeptide adopt a type-II β-bend conformation. However, only the more regularly folded conformation of molecule B is stabilized by a 1←4 C=O…H—N intramolecular H bond. The present results indicate that: (i) the Cα-methylated (αMe)Trp residue is a strong β-bend and helix former, and (ii) the relationship between (αMe)Trp chirality and helix screw sense tends to be opposite to that of protein amino acids. The implications for the use of the (αMe)Trp residue in designing conformationally restricted analogs of bioactive peptides are briefly discussed. ©Munksgaard 1995.     

Isolation and properties of two phospholipases A2 from the venom of an Australian elapid snake (Pseudechis australis)

Two phospholipases A2 (Pa-11 and Pa-13) were purified from the venom of an Australian elapid snake (subfamily Acanthophiinae) Pseudechis australis (king brown snake) by chromatography on CM-cellulose CM-52 followed by gel filtration on a Sephadex G-75 column. The apparent molecular weights of the two phospholipases A2 (Pa-11 and Pa-13) were 14,000 and 13,500, respectively, by gel filtration analysis on a Sephadex G-75 column. Each enzyme molecule consists of a single polypeptide chain of 118 amino acid residues. The isoelectric points of Pa-11 and Pa-13 were 10.5 and 10.0, respectively. The optimum pH values of Pa-11 and Pa-13 for hydrolysis of egg-yolk phosphatidylcholine were 7.8 and 7.5, respectively. Pa-11 was lethal to mice (LD50 0.23 micrograms/g body weight), whereas Pa-13 showed no lethal activity at a dose level of 7.4 micrograms/g mouse. Each enzyme was inactivated by reaction with p-bromophenacylbromide on the sole histidine residue (Pa-11) or on one of the two histidine residues (Pa-13). Oxidation of the tryptophan residues in Pa-11 and Pa-13 with N-bromosuccinimide led to a decrease in the phospholipase A activity. A complete loss of both enzymic and lethal activities of Pa-11 was observed upon oxidation of one of the two tryptophan residues of the molecule.     

The biological consequences of replacing hydrophobic amino acids in deltorphin I with amphiphilic α‐hydroxymethylamino acids

Abstract: New analogues of deltorphin I (DT I), in which the Phe residue in position 3, and the Val residue in position 5 or 6 are replaced with respective amphiphilic α‐hydroxymethylamino acid residues (HmAA), were synthesized and tested for receptor affinity and selectivity to μ and δ opioid receptors. The analogue with (R)‐HmPhe at position 3 lost receptor selectivity, as a result of a partial decrease of affinity to δ and a significant increase of affinity to μ receptors. In contrast, an analogue with (S)‐HmPhe in the same position, was very potent and more specific to δ receptors than parent DT I. The analogue with (R)‐HmVal at position 5 expressed higher δ affinity and selectivity than parent DT I. The analogue with other possible isomer (S)‐HmVal was less selective for δ opioid receptors, as a result of decreasing affinity to δ and increasing affinity to μ receptors. The analogues with (R)‐ or (S)‐HmVal in position 6 expressed equally low receptor affinity and selectivity. The data obtained support a previously proposed model of active conformation of deltorphins.     

Conformational investigation of α, β-dehydropeptides

The crystal structure of Ac-Pro-ΔVal-NHCH₃ was examined to determine the influence of the α,β-dehydrovaline residue on the nature of peptide conformation. The peptide crystallizes from methanol-diethyl ether solution at 4° in needle-shaped form in orthorhombic space group P2₁2₁2₁ with a= 11.384(2) Å, b = 13.277(2) Å, c = 9.942(1) Å. V = 1502.7(4) ų Z = 4, D_m= 1.17 g cm^?3 and D_c=1.18 g cm^?3 The structure was solved by direct methods using SHELXS-86 and refined to an R value of 0.057 for 1922 observed reflections. The peptide is found to adopt a β-bend between the type I and the type III conformation with φ₁=?68.3(4)°, ψ₁=? 20.1(4)°, φ₂=?73.5(4)°= and Ψ₂=?14.1(4)°=. An intramolecular hydrogen bond between the carbonyl oxygen of ith residue and the NH of (i+ 3)th residue stabilizes the β-bend. An additional intermolecular N.,.O hydrogen bond joins molecules into infinite chains. In the literature described crystal structures of peptides having a single α,β-dehydroamino acid residue in the (i+ 2) position and forming a β-bend reveal a type II conformation.     

SYNTHESIS AND GROWTH-PROMOTING ACTIVITY OF N α-ACETYL-HGH-(95–136)*

An N α-acetyl peptide with 42 amino acids corresponding to the amino acid sequence in residues 95–136 of the HGH molecule has been synthesized by the solid phase method. The synthetic product was found to possess some growth-promoting activity as revealed by the rat tibia test.     

Analysis of γβ, βγ, γγ, ββ continuous turns in proteins

Abstract: We report the observation of continuous turns in proteins which comprise individual γ‐turns or β‐turns or both that are situated immediately one after the other along the polypeptide chain. The continuous turns were identified from a representative data set of three‐dimensional protein crystal structures. The γβ/βγ, γγ and ββ continuous turns represent peptides of varying amino acid residue lengths and conformations. The continuous turns frequently observed in proteins were: γβ, between a coil and a strand; βγ, between a helix and a strand; γγ, between coils; and ββ, either between a strand and a coil or between strands or coils. We determined the statistically significant amino acid residue preferences at individual positions in the turn, calculated amino acid positional potentials and analyzed main chain hydrogen bonds and side‐chain interactions likely to stabilize the continuous turns. The data on continuous turns have been integrated in the database of structural motifs in proteins (DSMP) on our web server at ( http://www.cdfd.org.in/dsmp.html ). This is useful to make queries on sequences compatible with different continuous turns.     

DISULFIDE BONDS OF GLYCOPROTEIN HORMONES: Their Selective Reduction in the β Subunits of Bovine Lutropin and Thyrotropin

Treatment of the β subunit of either bovine lutropin (LH) or thyrotropin (TSH) at pH 8.2 by 5 mM dithioerythritol produces a set of partially reduced intermediates which, after alkylation with [¹⁴C] iodoacetate, can be separated by anion-exchange chromatography. Fractions from LH-β are obtained which represent molecules with 1, 2, 3 and 4 or 5 of the six disulfide bonds reduced. After full reduction, complete alkylation with non-radioactive iodoacetate and subsequent isolation of tryptic peptides, the radioactivity of each carboxymethyl cysteine residue was determined. With LH-β, the data show that disulfide bonds between half-cystines 93–100 and 26–110 open sequentially and that a third bond is between half-cystine 72 and either half-cystine 23 or 88; a specific disulfide interchange appears to have occurred between the two last positions. As reduction proceeds, extensive interchange occurs which prevents further assignment of disulfides; the half-cystines involved in disulfides 93–100 and 26–110, however, are not involved in the interchange. It is of interest that the assignments of bonds at 93–100 and 26–110 are in agreement with results of partial hydrolysis experiments from other laboratories, while the positions most involved in interchange are those where assignments from other laboratories differ. Under the same conditions of reduction, TSH-β yielded fractions containing one bond open (positions 88–95, analogous to 93–100 in LH-β) and small amounts of material in which reduction had occurred at positions 19–105 and 31–85 (analogous to 26–110 and 23–72 in LH-β). The data indicate that during the initial steps of reduction, the β subunits of LH and TSH open in a similar fashion, though disulfide interchange may begin sooner in the case of TSH. The results strongly support the assignments of two of the six disulfides in β subunits and show that unequivocal assignment of the four remaining bonds remains to be achieved.