Polypeptides. 53. Water-soluble copolypeptides of L-glutamic acid, L-lysine, and L-alanine

The synthesis and some of the physical-chemical properties of tricopolymers of L -glutamic acid, L -lysine, and L -alanine are reported here. The molar ratios of the glutamyl: lysyl: alanyl residues were 1:1:X or 3:2:X, where the alanyl content X was increased in regular steps. The α-helix content calculated from the optical rotatory dispersion of the polypeptides is compared with a predicted helix content estimated from the composition of the polymers and the known behavior of the homopolypeptides at pH 3, 8, and 12. At pH 3 copolypeptides containing 20 mole-% or more alanine exhibit a helix content equal to the sum of their alanyl and glutamyl residue contents. At pH 8 the helix content equals the alanyl content when the latter was 40 mole-% or higher; at lower alanyl contents the electrostatic interaction between charged glutamyl and lysyl residues makes some contribution. At pH 12 the amount of helix observed is proportional to the mole ratio of alanine residues present in the polymer. The helix content of a tricopolymer containing 1:1:3 mole ratios of glutamyl: lysyl: alanyl residues was determined in solutions of lithium bromide and in urea solutions. Both reagents led to a decrease in helix content at pH 3 and 8 to a minimum of approximately 20% helix in 8M urea or 5.5M LiBr. The helix–random chain transition curves at pH 3 and 8 are parallel when the urea concentration is varied, but differ in shape when the lithium bromide concentration is varied at pH 3 and 8. The mode of action of these two “denaturing” reagents may thus be different. Heating the same tricopolypeptide at pH 3 or 8 from 5 to 80°C. also led to a helix–random chain transition centered at approximately 45°C.     

Circular dichroism studies of α-aminoisobutyric acid-containing peptides: Chain length and solvent effects in alternating Aib-L-Ala and Aib-L-Val sequences

The CD spectra of the peptides Boc-X-(Aib-X)_n-OMe (n = 1, 2, 3) and Boc-(Aib-X)₅-OMe, where X = L -Ala or L -Val have been examined in several solvents. The X = Ala and Val peptides behave similarly in all solvents, suggesting that the Aib residues dominate the folding preferences of these peptides. The decapeptides adopt helical conformations in methanol and trifluoroethanol, with characteristic negative CD bands at 222 and 205 nm. In the heptapeptides, similar spectra with reduced intensities are observed. Comparison with nmr studies suggest that estimates of helical content in oligopeptides by CD methods may lead to erroneous conclusions. The pentapeptides yield solvent-dependent spectra indicative of conformational perturbations. Peptide association in dioxane results in an unusual spectrum with a single negative band at 210 nm for the decapeptides. Disaggregation is induced by the addition of methanol or water to dioxane solutions. Aggregation of the heptapeptides is less pronounced in dioxane, suggesting that a critical helix length may be necessary to promote association stabilized by helix dipole–dipole interactions.     

Detection by circular dichroism of conformational transitions in pH and thermosensitive copolymers based on N-isopropylacrylamide and N-methacryloyl-L-leucine

Circular dichroism (CD) spectra in the region of 210-250 nm allow visualization of intrachain phase transition of pH- and thermosensitive polyelectrolytes. Indeed, in 0.001 M citrate and acetate buffers, at pH 4.0-5.5, aqueous solutions of a poly(N-isopropylacrylamide-co-N-methacryloyl-L-leucine) (NIPAAm-MALEU) copolymer containing 90.9 mol% of NIPAAm residues exhibit a well-defined sigmoidal increase in the CD signal at 220 nm with increasing temperature. This phenomenon is suggestive of a highly cooperative transition which occurs at lower temperatures compared to that observed by cloud point measurements. The change in the CD signal is less sharp at higher pH, indicating varying cooperativity with pH. For pH 6.0 and higher, no such phenomena are observed.     

Synthesis and conformation of a polyoxyethylene-bound undecapeptide of the alamethicin helix and (2-methylalanyl-L-alanine)1–7

The stepwise synthesis and conformational studies of the N-terminal helical partial sequence of the membrane-modifying polypeptide antibiotic alamethicin are described. The polyoxyethylen esters of the fragments N-t-Boc-L -Pro-Aib-Ala-Gln-Aib-Val-Aib-Gly-OH and N-Ac-Aib-L -Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-OH are synthesized using polyoxyethylene (molecular mass 10,000) as solubilizing support. CD spectra of each intermediate in ethanol show α-helix formation of the N-protected peptide polymers beginning with the nonapeptide and of the N-protonated sequences beginning with the decapeptide. Compared to the helix of alamethicin, temperature- and solvent-dependent CD measurements indicate analogous conformational behavior. The results suggest that in lipophilic media the alamethicin helix can extend the full length of the partial sequence between the two proline residues and that aqueous media favor an increase of random-coil conformation. For model studies of the particular lipid interaction of alamethicin, the stepwise synthesis of peptides with the alternating (Aib-L -Ala)_n sequence (n = 1–7) was carried out on a polyoxyethylene support (molecular mass 6000). CD and ORD studies in ethanol showed a change from the random coil to a right-handed α-helix with increasing peptide length. This change is observed for the N-protected peptides at a chain length of 8 residues and for the N-protonated peptides at a length of 9 residues. The comparison of the CD data of free and polyoxyethylene-bound peptides revealed that the solubilizing polymeric support cannot induce conformational changes. The intensities of the CD bands of t-Boc-(Aib-L -Ala)_n-OPOE (n ≥ 6) are higher than those of alamethicin, and these model peptides show similar temperature and solvent inducible changes of their helix contents.     

Cu(II)–protamine interaction. II. The formation and structure of Cu(II) complexes of clupeine YII and of peptides mimicking clupeine N-terminals

The interaction of Cu(II) with the protamine clupeine YII (containing proline at the N-terminal) and with four peptides (H-Ala-Arg-OMe, H-Ala-Arg₂-OMe, H-Pro-Arg-OMe, and H-Arg₄-Tyr) has been studied by means of absorption, CD, and pH neasurements. The first two peptides mimic clupeine YI and Z N-terminals; the third, the clupeine YII N-terminal. At 1:1 molar ratio, clupeine YII yields two complexes: the first (I), at pH 6.6, through coordination via the N-terminal and the contiguous peptide nitrogen forming a five-membered chelate; the second (II), at pH 8.5, through the occupancy of the other two corners of the coordination square by amino nitrogens of the lateral chains. These complexes are strictly analogous and occur at the same pH as those formed with clupeine Z. Under the same conditions, all the peptides yield complex I in the first step, although the pH at which this complex is fully defined depends on the number of residues in the chain. It is 8.5 for dipeptides, decreases to 6.5 by the addition of a third residue to the chain, and remains constant when the number of residues is three or more. The amino nitrogens of lateral chains are unable to coordinate to the metal in a second step unless one additional peptide bond lies between the N-terminal residue and that containing the lateral chain bound to the metal. Thus, H-Ala-Arg-OMe and H-Pro-Arg-OMe form hydroxyl complexes in a second step (pH 11), by deprotonation of one of the water molecules coordinated to the metal; one of the lateral chains of H-Ala-Arg₂-OMe is able to coordinate in a second step (pH 8.5), but it is only with H-Arg₄-Tyr that a second complex (II) is obtained in which two amino nitrogens of lateral chains supersede the oxygens of water molecules in I, at pH 8.5.     

Thiolysis of O-2,4-dinitrophenyltyrosines. Spectrophotometric monitoring of the reaction and its use in peptide synthesis.

The thiolytic cleavage of O-2,4-dinitrophenyl (Dnp) derivatives of phenols was applied to the synthesis of tyrosine-containing peptides. This paper describes the preparation and properties of starting materials for such syntheses and illustrates their use in the synthesis of some peptides containing tyrosine at either the C- or N-terminus. A spectrophotometric method for following the thiolytic removal of Dnp groups from O-Dnp-tyrosines was developed and used to establish optimal conditions for quantitative deblockage in aqueous and nonaqueous solvents. The method is based on the fact that upon thiolysis, the colorless solution of O-Dnp-tyrosine (λ_max at 298 nm, pH 8.5) becomes yellow due to the formation of a dinitrophenylated thiol (for S-Dnp-2-mercaptoethanol, λmax at 340 nm, pH 8.5). This gives rise to a difference spectrum with a maximum at 354 nm (Δ?_M = + 8680 M^?1 cm^?1), a minimum at 298 nm (Δ?_M = ?5900 M^?1 cm^?1) and a crossover point at 318 nm, which is different (in the 290–320 nm range) from the difference spectrum obtained upon thiolysis of N^Im-Dnp-histidine. This method provides a useful analytical tool in peptide and polypeptide synthesis as well as in protein chemistry.     

Optical properties and viscosity of hyaluronic acid in mixed solvents: Evidence of conformational transition

Circular dichroism, optical rotatory dispersion, and viscosity of hyaluronic acid at various solvents compositions, concentrations, and pH values have been studied. The data show a large change in the molecular properties in organic/water solvents such as ethanol, p-dioxane, or acetonitrile/water at pH ? pK_a. At this pH range of aqueous solution, hyaluronic acid shows a CD minimum near 210 nm whereas in the presence of organic solvent it exhibits a strong negative dichroism (below 200 nm) and a positive band near 226 nm. It undergoes a sharp, cooperative transition with respect to pH and solvent. The observed CD features are assigned to the π-π* and n-π* transitions of the amide and carboxyl chromophores. The ORD results show a gradual blue shift of trough at 220 nm with increasing magnitude of rotation when the organic solvents and hydrogen ion concentrations are increased. A one-term Drude's equation was used to analyze the ORD data, and the result show a variation of dispersion parameters with different solvents in accordance with the observed CD changes. The intrinsic viscosity of hyaluronic acid in mixed solvent at pH 2.6 is lower than that of aqueous solution. All the observed property changes of hyaluronic acid are reversed on addition of foramide in mixed solvents indicating that the hydrogen bonds are involved in this transition. The observed spectroscopic and hydrodynamic features are attributed to a conformational change of hyaluronic acid in a mixed solvent involving intramolecular hydrogen bonding between the acetamido and carboxyl groups. The possible conformational state of hyaluronic acid in solution under various conditions is discussed in terms of the reported helical structure of hyaluronic acid from x-ray diffraction studies.     

Co-oligopeptides of aromatic amino acids and glycine with a variable distance between the aromatic residues. II. Ultraviolet absorption and circular dichroism properties of co-oligopeptides of tryptophan and glycine

The uv absorption and circular dichroism (CD) properties in water (pH 5.9) and trifluoroethanol of several co-oligopeptides of glycine and tryptophan have been investigated. These compounds contain one tryptophyl residue, such as H-Gly-Trp-OH, H-Trp-Gly-OH, and H-Gly-Trp-Gly-OH; or two, such as H-Trp-Trp-OH, H-Gly-Trp-Trp-OH, H-Trp-Trp-Gly-OH, and H-Gly-Trp-(Gly)_n-Trp-Gly-OH (I, n = 0,1,2). Furthermore, the case of some protected derivatives, such as Z-Trp-Gly-OMe, Z-Gly-Trp-Gly-OMe, and Z-Trp-Trp-OMe, Z-Trp-Trp-Gly-OMe, Z-Gly-Trp-Trp-Ome were investigated. The extinction coefficients in H₂O in the region of the L bands, referred to one mole of tryptophyl residue, are essentially the same for all compounds within the limit of experimental error, thus indicating that no strong chromophoric interaction takes place in the oligopeptides containing two aromatic residues. However, in the far uv region, anomalies in the absorption properties cannot be excluded. The investigated compounds show marked differences in their CD properties. Whereas in the case of lower molecular-weight peptides (e.g., H-Trp-Gly-OH and H-Gly-Trp-OH), these differences can be ascribed, at least in part, to the influence of the charge end groups on the indole chromophore, in the case of the compounds containing two tryptophyl residues, the differences in CD properties are assumed to reflect the different structures and conformations of the oligopeptides. In particular, we observed a negative dichroic band at ～225 nm in H-Gly-Trp-Trp-OH and I(n = 0) in water. In trifluoroethanol, this band has a much larger intensity, and it is present also in Z-Gly-Trp-Trp-OMe, Z-Trp-Trp-Gly-OMe, and Z-Trp-Trp-OMe. It is argued that such a negative band is characteristic of the sequence -CO-Trp-Trp-, and the possibility that it derives from intramolecular chromophoric interaction is discussed. It is also pointed out, particularly in view of the high ellipticity values found in some cases (up to 100,000 deg cm² dmol^?1), that this feature may reflect a high degree of conformational rigidity connected with the sequence -CO-Trp-Trp-.     

Block oligopeptides (L-lysyl)m-(L-alanyl)n-L-Tyrosyl-(L-Alanyl)n-(L-Lysyl)m. II. Circular dichroism and pulsefluorimetry conformational studies

The conformation of chromatographically pure block oligopeptides (L -lysyl)_m-(L -alanyl)_n- L -tyrosyl-(L -alanyl)_n-(L -lysyl)_m with n = 3 and m = 6 or 3 is investigated. By circular dichroism it is shown that these peptides may exhibit a partially α-helical structure depending upon pH, ionic strength, solvent, and temprerature. An attempt is made to describe the helical content of these small peptides by utilizing the data obtained on high-molecular-weight poly(L -lysine). By measurement of the quantum yield and the decays of the peptides fluorescence, it is shown that, in aqueous solution, at neutral pH, the fluorescence of the peptides is quenched by interactions with the peptide carbonyl groups. The decays are multiexponential, which shows the presence of several conformations of the phenolic chromophore relative to the peptide chain. The addition of methanol, which induced the helix formation, decreases the quenching of the fluorescence and the multiexponential character of the decays. In presence of sodium hydroxide, which further increases the helical content of the peptides, a dynamic quenching occured that can be attributed to interactions between the phenol hydroxyl group of tyrosine (ith residue) and the ε-amino groups of the (i+4)th and (i -4)th lysyl residues.     

Sequential polypeptides containing arginine as histone models: synthesis and conformational studies

The sequential polypeptides (L -Arg-X-Gly)_n, where X represents amino acid residues Ala, Val, and Leu, were prepared as models of arginine-rich histones to be used in studying their structure and their interactions with DNA. The polymerization was carried out on the pentachlorophenyl active esters of the appropriate tripeptides, while the toluene-4-sulfonyl group was used for protecting the arginine guanido group. CD was employed to investigate the conformation of (L -Arg-X-Gly)_n polymers in aqueous solutions, at different pH, as well as in trifluoroenthanol and hexafluoroisopropyl alcohol solutions. In aqueous solutions (at pH 7 and 12) the prepared sequential polymers behaved as a random coil. The CD spectra in various trifluoroethanol–water or hexafluoroisopropyl alcohol–water mixtures indicated that the degree of helical conformation of the studied polytripeptides increased in the order of Ala → Val → Leu. The opposite was true for the β-structure. Characteristics of β-turn are excluded from the poly(L -Arg-L -Leu-Gly), which assumed the most pronounced helical conformation. The poly(L -Arg-L -Val-Gly) exerts a significant preference to the β-turn structure compared to that of poly(L -Arg-L -Ala-Gly). Thus the probability for helical, β-structure or β-turn conformations of the polymers was analyzed in relation to the bulkiness and length, and to the special features of the X-residue side chain (β-branching). We concluded that the prepared sequential arginine-containing polypeptides are plausible models for histone fractions, f₃ and f₂α₁.     

Copper(II) binding to two novel histidine-containing model hexapeptides: evidence for a metal ion driven turn conformation

The solution conformation and the copper(II) binding properties have comparatively been investigated for the two novel hexapeptides Ac-HPSGHA-NH₂ (P2) and Ac-HGSPHA-NH₂ (P4). The study has been carried out by means of CD, NMR, EPR and UV-Vis spectroscopic techniques in addition to potentiometric measurements to determine the stability constants of the different copper(II) complex species formed in the pH range 3-11. The peptides contain two histidine residues as anchor sites for the metal ion and differ only for the exchanged position of the proline residue with glycine. CD and NMR results for the uncomplexed peptide ligands suggest a predominantly unstructured peptide chain in aqueous solution. Potentiometric and spectroscopic data (UV-Vis, CD and EPR) show that both peptides strongly interact with copper(II) ions by forming complexes with identical stoichiometries but different structures. Furthermore, Far-UV CD experiments indicate that the conformation of the peptides is dramatically affected following copper(II) complexation with the P4 peptide adopting a β-turn-like conformation.     

Conformational aspects of gastrin-related peptides: A circular dichroism study

The conformational properties of a series of gastrin-related peptides in aqueous solution and in 2,2,2-trifluoroethanol (TFE) have been investigated by CD measurements. In aqueous solution the peptides Leu³²-HG-34 (human big gastrin), Nle¹⁵-HG-17 (human little gastrin), and Nle¹¹-HG-13 assume a random-coil structure in the pH range 3–7. In TFE the three hormones fold into partially ordered structures, consisting of mixtures of α-helix, β-form and random coil. Comparison with the CD properties of the shorter gastrin peptides HG-4 (tetragastrin), N^α-Boc-HG-5 (pentagastrin), and HG-7 (heptagastrin) indicates that the biologically important C-terminal sequence Trp-Met-Asp-Phe-NH₂ in TFE does not maintain the same geometry upon elongation of the chain at the N-terminus from 4 to 34 residues. Thus, the various conformations in solution of the gastrin peptides examined do not provide a structural explanation for their very similar biological activity. Therefore, we hypothesize that the C-terminal tetrapeptide amide folds into an “active” structure only upon interaction with the receptor.     

Ribonuclease Activity of the Peptides with Alternating Arginine and Leucine Residues Conjugated to Tetrathymidilate

RNA cleaving conjugates have been prepared by attachment of oligodeoxyribonucleotide TTTT to peptides containing arginine, leucine, proline and serine residues. The highest activity was displayed by the conjugates containing peptides with alternating arginine and leucine residues (LR)₄G‐amide. Ribonuclease activity of the conjugates pep‐T₄ decreases in the order T₄‐(LR)₄G > T₄‐(LR)₂G > T₄‐(LLRR)2G > T₄‐(LR)₂PRLRG > S₂R₃‐Hmda‐T₄ ≥ R₅ ≠ (LR)₃. According to CD spectra, the free peptide (LR)₄G‐amide in water solution at neutral pH and physiological ionic strength has no pronounced secondary structure whereas conjugated to oligonucleotide it acquires a folding similar to α‐helix.     

Purification and characterization of a maltooligosaccharide-forming amylase that improves product selectivity in water-miscible organic solvents, from dimethylsulfoxide-tolerant Brachybacterium sp. strain LB25

A bacterium that secretes maltooligosaccharide-forming amylase in a medium containing 12.5% (vol/vol) dimethylsulfoxide (DMSO) was isolated and identified as Brachybacterium sp. strain LB25. The amylase of the strain was purified from the culture supernatant, and its molecular mass was 60 kDa. The enzyme was stable at pH 7.0–8.5 and active at pH 6.0–7.5. The optimum temperature at pH 7.0 was 35°C in the presence of 5 mM CaCl₂. The enzyme hydrolyzed starch to produce maltotriose primarily. The enzyme was active in the presence of various organic solvents. Its yield and product selectivity of maltooligosaccharides in the presence of DMSO or ethanol were compared with those of the industrial maltotriose-forming amylase from Microbacterium imperiale. Both enzymes improved the production selectivity of maltotriose by the addition of DMSO or ethanol. However, the total maltooligosaccharide yield in the presence of the solvents was higher for LB25 amylase than for M. imperiale amylase.     

Stereochemical studies on cyclic peptides. IX. Conformational studies on cyclic tetrapeptides containing alternating cis and trans peptide units

Conformational analyses of cyclic tetrapeptides consisting of alternating cis and trans peptide units have been made using contact criteria and energy calculations. This study has been restricted to those structures having a symmetry element in the backbone ring, such as a twofold axis (d) or a center of inversion (i). There are five main results. (1) There are two distinct types of conformations, which are stereochemically favorable corresponding to each of twofold and inversion-symmetrical structures, designated as d₁, d₂ (for twofold symmetrical) and i₁, i₂ (for inversion-symmetrical). Among these, the i₁ type has the lowest energy when glycyl residues occur at all four α-carbon atoms. (2) With the glycyl residue at all four α-carbon atoms, methyl substitution at the cis peptide nitrogen atoms is possible in all the four types, whereas the substitution at trans peptide nitrogen atoms is possible only for the i₁ type. Thus only in the i₁ type can all the nitrogen atoms be methylated simultaneously. The conformation of the molecule in the crystal structure of cyclotetrasarcosyl belongs to the i₁ type. (3) When alanyl residues occur at all four α-carbon atoms, the possible symmetrical type is dependent on the enantiomorphic form and the actual sequence of the alanyl residues. (4) The methyl substitution at peptide nitrogen atoms for cyclic tetrapeptides having alanyl residues causes more stereochemical restriction in the allowed conformations than with glycyl residues. (5) The prolyl residue can be incorporated favorably at the cis-trans junction of both d and i types of structures. The results of the present study are compared with the data on cyclic tetrapeptides available from the crystal structure and nmr studies. The results show an overall agreement both regarding the type of symmetry and the conformational parameters.     

Synthesis and conformational study of peptides possessing helically arranged ΔZPhe side chains

Z-Dehydrophenylalanine (Δ^zPhe) possessing four oligopeptides, Boc-(L -Ala-Δ^zPhe-Aib)_n-OCH₃ (n = 1–4: Boc, t-butoxycarbonyl; Aib, α-aminoisobutyric acid), were synthesized, and their solution conformations were investigated by ¹H-nmr, ir, uv, and CD spectroscopy and theoretical CD calculation. ¹H-nmr (the solvent accessibility of NH groups) and ir studies indicated that all the NH groups except for those belonging to the N-terminal L -Ala-Δ^zPhe moiety participate in intramolecular hydrogen bonding in chloroform. This suggests that the peptides n = 2–4 have a 4 → 1 hydrogen-bonding pattern characteristic of 3₁₀-helical structures. The uv spectra of all these peptides recorded in chloroform and in trimethyl phosphate showed an intense maximum around 276 nm assigned to the Δ^zPhe chromophores. The corresponding CD spectra of the peptides n = 2–4 showed exciton couplets with a negative peak at longer wavelengths, whereas that of the peptide n = 1 showed only weak signals. Theoretical CD spectra were calculated for the peptides n = 2–4 of several helical conformations, on the basis of exciton chirality method. This calculation indicated that the three peptides form a helical conformation deviating from the perfect 3₁₀-helix that contains three residues per turn, and that their side chains of Δ^z Phe residues are arranged regularly along the helix. The center-to-center distance between the nearest phenyl pair(s) was estimated to be ～ 5.5 Å. The chemical shifts of the Δ^zPhe side-chain protons (H^β and aromatic H) for the peptides n = 2–4 indicated anisotropic shielding effect of neighboring phenyl group(s); the effect also supports a regular arrangement of the Δ^z Phe side chains along the helical axis. © 1993 John Wiley & Sons, Inc.     

Peptide backbone cleavage by α‐amidation is enhanced at methionine residues

Cleavage reactions at backbone loci are one of the consequences of oxidation of proteins and peptides. During α‐amidation, the C_α–N bond in the backbone is cleaved under formation of an N‐terminal peptide amide and a C‐terminal keto acyl peptide. On the basis of earlier works, a facilitation of α‐amidation by the thioether group of adjacent methionine side chains was proposed. This reaction was characterized by using benzoyl methionine and benzoyl alanyl methionine as peptide models. The decomposition of benzoylated amino acids (benzoyl‐methionine, benzoyl‐alanine, and benzoyl‐methionine sulfoxide) to benzamide in the presence of different carbohydrate compounds (reducing sugars, Amadori products, and reductones) was studied during incubation for up to 48 h at 80 °C in acetate‐buffered solution (pH 6.0). Small amounts of benzamide (0.3–1.5 mol%) were formed in the presence of all sugars and from all benzoylated species. However, benzamide formation was strongly enhanced, when benzoyl methionine was incubated in the presence of reductones and Amadori compounds (3.5–4.2 mol%). The reaction was found to be intramolecular, because α‐amidation of a similar 4‐methylbenzoylated amino acid was not enhanced in the presence of benzoyl‐methionine and carbohydrate compounds. In the peptide benzoyl‐alanyl‐methionine, α‐amidation at the methionine residue is preferred over α‐amidation at the benzoyl peptide bond. We propose here a mechanism for the enhancement of α‐amidation at methionine residues. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Succinimide formation from aspartyl and asparaginyl peptides as a model for the spontaneous degradation of proteins

Nonenzymatic intramolecular reactions can result in the deamidation, isomerization, and racemization of protein and peptide asparaginyl and aspartyl residues via succinimide intermediates. To understand the sequence dependence of these reactions, we measured the rate of succinimide formation in a series of synthetic peptides at pH 7.4. These peptides (Val-Tyr-Pro-X-Y-Ala) contained an internal aspartyl, asparaginyl, aspartyl beta-methyl ester, or aspartyl alpha-methyl ester residue (X) followed by a glycyl, seryl, or alanyl residue (Y). The rates of succinimide formation of the asparaginyl peptides were found to be 13.1-35.6 times faster than those of the aspartyl peptides. The rates of succinimide formation for the glycyl peptides were 6.5-17.6 times faster than those of the alanyl peptides, while the rates for the seryl peptides were 1.6-4.5 times faster than those of the alanyl peptides. The overall 232-fold range in these reaction rates for aspartyl and asparaginyl residues suggests that sequence can be an important determinant in their stability in flexible peptides. In proteins, there may be a much larger range in the rates of succinimide formation because specific conformations may greatly enhance or inhibit this reaction.     

The effect of amphiphilic peptide surfactants on the light-harvesting complex II

The peptide surfactants are amphiphilic peptides which have a hydrophobic tail and a hydrophilic head, and have been reported to stabilize and protect some membrane proteins more effectively than conventional surfactants. The effects of a class of peptide surfactants on the structure and thermal stability of the photosynthetic membrane protein lightharvesting complex II (LHCII) in aqueous media have been investigated. After treatment with the cationic peptide surfactants A₆K, V₆K₂, I₅K₂ and I₅R₂, the absorption at 436 nm and 470 nm decreased and the absorption at 500–510 nm and 684–690 nm increased. Moreover, the circular dichroism (CD) signal intensity in the Soret region also decreased significantly, indicating the conformation of some chlorophyll (Chl) a, Chl b, and the xanthophyll molecules distorted upon cationic peptide surfactants treatment. The anionic peptide surfactants A₆D and V₆D₂ had no obvious effect on the absorption and CD spectra. Except for A₆D, these peptides all decreased the thermal stability of LHCII, indicating that these peptides may reconstitute protein into a less stable conformation. In addition, the cationic peptide surfactants resulted in LHCII aggregation, as shown by sucrose gradient ultracentrifugation and fluorescence spectra.     

Secondary Structure of Nuclease P1

The molecular conformation of nuclease P₁ in aqueous solution was investigated by measuring the optical rotatory dispersion (ORD) and circular dichroism (CD). The optical rotatory dispersion constant, λ was 281 nm. The Moffit-Yang parameters, a₀ and b₀, were ?2 and ?195, respectively. The ORD spectrum showed a minimum at 234 nm and the reduced mean residue rotation at 233 nm, [m]233, was ?5880. The CD spectrum showed a double minimum at 213 and 226 nm and the molecular ellipticity at 222 nm, [θ]22, was -11,900. From these data, the α-helix content was calculated to be 29 to 31 %. The computer fit of CD suggests that the α-structure is about 6% and the random coil is about 63%. The helical structure was found to be quite stable to denaturing reagents such as urea and guanidine hydrochloride. However, removal of zinc atoms from the enzyme resulted in disruption of the helical structure with inactivation.