Polypeptides. LVI. Effect of lithium bromide and of temperature on the conformation of a copolymer of glutamic acid and lysine

By using optical rotatory dispersion measurements, the helix content of poly Glu⁵⁰Lys⁵⁰ has been investigated and compared with that of poly Glu²⁰Lys²⁰Ala⁶⁰ in aqueous solutions. Measurements were made at pH 3 and at pH 8 in various concentrations of lithium bromide. Various factors affecting helix stabilization are considered and their perturbation by lithium bromide is related to the shape of the observed transition curves. A residual helix content of 12% in 8M LiBr, based upon a b₀ of +100 for a fully random conformation, was observed for poly Glu⁵⁰Lys⁵⁰ at pH 3 and 8. The loss of helix content of poly Glu⁵⁰Lys⁵⁰ as a function of temperature is also reported. ΔH is approximately ?6.9 kcal./mole for the overall transition, compared to ?6.5 kcal./mole for poly Glu²⁰Lys²⁰Ala⁶⁰. The midpoint of the broad transition is near 40°C. at pH 3, but much lower, at ?10 to 0°C., at pH 8. These results are discussed in terms of the stabilizing factors for the partial helix content of the polypeptides.     

Optical activity of simple peptides. I. Glycine oligopeptides with internal L-alanyl or L-glutamyl residues

Oligopeptides containing glycine and one or two L -alanyl or L -glutamyl residues have been studied by circular dichroism (CD) and optical rotatory dispersion (ORD) in aqueous solution at pH 1.0, pH 6.0, and pH 10.0 and in aqueous ethanol. Two glycyl residues are required to remove effects of α-carboxyl or amino titration on the optical activity of the internal alanyl or glutamyl residues. The CD spectra of the alanyl and protonated glutamyl residues are similar, having two regions of negative ellipticity around 215 nm resulting in a spectrum reassembling that of poly-α-L -glutamic acid (PGA) at high pH. Another large positive band below 190 nm was observed for gly₂-glu₂-gly₂ in water at pH 6 and 10 and for several peptides in aqueous ethanol. Residue ellipticities were approximately additive in every case except for peptides containing intrenal glutamyl residu at pH 6.0.     

Trypsin and chymotrypsin inhibitor from chick peas. Selective chemical modifications of the inhibitor and isolation of two isoinhibitors.

The trypsin and chymotrypsin inhibitor from chick peas (CI) is stable in HCl 0.001 M -- 0.01 M and in KOH 0.01 M -- 0.05 M even after 24 h. Increased KOH concentrations decrease considerably the inhibitory activity already after 1 h. Maleyation and succinylation of the inhibitor resulted in almost full loss of its trypsin-inhibitory activity but had no effect on the chymotrypsin-inhibitory activity. A series of modifications directed towards tyrosyl residues showed that iodination influenced only the chymotrypsin-inhibitory activity; however, nitration and arsanilation affected not only the chymotrypsin-inhibitory activity but also the trypsin-inhibitory activity. Treatment of the inhibitor with CNBr and chloramine T resulted only in a decrease in the chymotrypsin-inhibitory activity indicating that the only methionine is involved in the chymotrypsin-inhibitory activity. When CI-fragment A, previously treated with trypsin at pH 3.75, was further treated with carboxypeptidase B, a release of three lysyl residues per mole protein was found. CI was separated by equilibrium chromatography on SP-Sephadex column into two isoinhibitors, CII and CIII, respectively. Both inhibited trypsin and chymotrypsin with the same specific activity as CI. They differed from each other only in a glutamyl, aspartyl, glycyl and alanyl residue.     

The role of an active-site lysyl residue of spinach phosphoribulokinase as explored by site-directed mutagenesis

Based on selective labeling by ATP analogues, Lys68 of the Calvin Cycle enzyme phosphoribulokinase (PRK) from spinach has been assigned to the active-site region [Miziorkoet al. (1990),J. Biol. Chem. 265, 3642–3647]. The equivalent position is occupied by lysyl or arginyl residues in the PRK from both prokaryotic and eukaryotic sources, suggesting a requirement for a basic residue at this location. To examine this possibility, we have replaced Lys68 of the spinach enzyme with arginyl, glutaminyl, alanyl, or glutamyl residues by site-directed mutagenesis. All of the mutant enzymes retain substantial kinase activity; and even in the case of the radical substitution by glutamate, theK _m values for ATP and ribulose 5-phosphate are not perturbed significantly. Glutamate at position-68 may destabilize tertiary structure, because the yield of this mutant protein from transformedE. coli is quite low compared to that of the other proteins in this series. Despite the active-site proximity of Lys68, our results show that this residue does not play a key role in catalysis or substrate binding.     

Conformational studies of poly-L-alanine in water

The conformational properties of poly-L -alanine have been examined in aqueous solutions in order to investigate the influence of hydrophobic interactions on the helix–random coil transition. Since water is a poor solvent for poly-L -alanine, water-soluble copolymers of the type (D , L -lysine)_m–(L alanine)_n-(D , L -lysine)_m, having 10, 160, 450, and 1000 alanyl residues, respectively, in the central block, were synthezised. The optical rotatory dispersion of the samples was investigated in the range 190–500 mμ, and the rotation at 231 mμ was related to the α-helix content, θ_H, of the alanine section. In salt-free solutions, at neutral pH, the three large polymers show high θ_H values, which are greatly reduced when the temperature is increased from 5 to 80°C. No helicity was observed for the small (n = 10) polymer. By applying the Lifson-Roig theory, the following parameters were obtained for the transition of a residue from a coil to a helical state: ν = 0.012; ΔH = ?190 ± 40 cal./mole; ΔS = ?0.55 ± 0.12 e.u. Since ΔH and ΔS differ from the values expected for a process involving only the formation of a hydrogen bond, and in a manner predicted by theories for the influence of hydrophobic bonding on helix stability, it is concluded that a hydrophobic interaction is also involved. In the presence of salt (0.2M NaCl), or when the ε-amino groups of the lysyl residues are not protonated (pH = 12), the helical form of the two large polymers (n = 450 and n = 1000) is more stable than in water. Since the electrostatic repulsion between the lysine end blocks is greatly reduced under these conditions, the alanine helical sections fold back on themselves, and this conformation is stabilized by interchain hydrophobia bonds. This structure was predicted by the theory for the equilibrium between such interacting helices, non-interacting helices, and the random coil.     

Conformational aspects of polypeptides. XXV. Solvent and temperature effects on the conformations of copolymers of benzyl and methyl L-aspartate with nitrobenzyl L-aspartate

A series of copolymers of β-p-nitrobenzyl L -aspartate with β-benzyl L -aspartate and with β-mcthyl L -aspartatc in helix-supporting and helix-breaking conditions have been reexamined by using ultraviolet isotropic, absorption, optical rotatory dispersion, and circular dichroism techniques. Many different conformations are apparent, depending on solvent and temperature. Chloroform, trifluoroethanol, and methylene dichloride support the left-handed helical conformation of the copolymers containing less than about 20 mole-% nitroaromatic residues and the right-handed helical conformation of the copolymers containing more than approximately 30 mole-% nitroaromatic residues. In trifluoroacetic acid all the copolymers are in a random-coil conformation. In hexa-fluoroacetone trihydrate and in trimethyl phosphate, the copolypeptides with low nitroaromatic residues content are predominantly in a disordered conformation, while those with high nitroaromatic residues content show a right-handed helical array. Reversible helix-ramlom-coil transitions are observed with increasing temperature in trimethyl phosphate. An example of right-handed-left-handed helix reversible transition with temperature is reported in a chloroform-trimethyl phosphate (2:1) mixture. Nitrobenzyl-nilrobenzyl side-chain interactions in chloroform, but not in trifluoroacetic acid or in trimethyl phosphate, have been confirmed. For the first time we report the circular dichroism spectra in which the n-π* peptide band of a left-handed helical conformation is almost completely evident.     

Characterization of molecular motions in 13C-labeled aortic elastin by 13C-1H magnetic double resonance

Purified insoluble elastin samples labeled with [1-¹³C]valine, [1-¹³C]alanine, and [1-¹³C]-lysine were prepared from chick aorta in culture. The molecular mobility at the labeled sites was investigated using ¹³C-¹H magnetic double-resonance spectroscopy. Linewidths, T₁, and nuclear Overhauser effect (NOE) values of the labeled carbons alone were obtained from dipolar decoupled difference spectra. Analysis of these parameters together with signal intensity measurements showed that essentially all the valyl residues, ca. 75% of the alanyl residues, and ca. 60% of the lysyl residues were characterized by rapid backbone motions having τ = 65 nsec. Resonances due to the remaining alanyl and lysyl residues were detected in cross-polarization experiments, which enhance the signals of motionally restricted carbons. Since lysyl and alanyl residues are found in the crosslink regions of elastin, whereas valyl residues are not, we conclude that crosslinks rather than secondary structures in the extensible region of the protein are the main source of motional restrictions in the protein. Elastin chain mobility was monitored by linewidth measurements over the range ?90 to +70°C. When the swelling solvent (0.15M NaCl) was fixed at 0.6 g/g of elastin, a rapid monotonic reduction in chain mobility was observed as the temperature was lowered from 50 to 5°C. Liquidlike mobility was completely lost at 5°C. In contrast, the same sample in contact with excess solvent retained its liquidlike molecular mobility until ?13°C, where it abruptly became rigid. The molecular mobility of this sample was temperature insensitive in the physiologically interesting range, 20–40°C, as a consequence of the opposing influences of temperature and swelling. Taken together these nmr data indicate that under physiological conditions, elastin is a network of mobile chains whose motions are strongly influenced by protein–solvent interactions.     

A peptide containing a reactive lysyl group from ox liver glutamate dehydrogenase

1. Inhibition of ox liver glutamate dehydrogenase with N-(N'-acetyl-4[(35)S]-sulphamoylphenyl)maleimide (ASPM) is more specific at pH7.3 than at pH6.9. At pH7.3 inhibition accompanies the incorporation at 1 mole of ASPM residues into about 53000g. of protein. 2. Digestion of the modified protein with chymotrypsin and trypsin yields a unique radioactive peptide. 3. Acid hydrolysis of 1 mole of this peptide yields 1 mole of N(in)-succin-2-yl-lysine. The in-amino group of a lysyl residue is thus the site of modification of the protein. 4. The sequence containing the modified lysyl residue is: [Formula: see text] where Asx respresents either aspartic acid or asparagine.     

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.     

Reaction of myosin with salicylaldehyde I. The effect of salicylaldehyde on the physicochemical properties of myosin

The specificity and nature of the reaction between salicylaldehyde and myosin and the effect of salicylalation on the molecular parameters of myosin were studied. The following observations were made.

1. 1. The reaction of salicylaldehyde with the lysyl residues of myosin is specific, since no salicylaldehyde is bound if the lysyl residues of myosin are trinitrophenylated.

2. 2. Salicylaldehyde is bound by myosin through the formation of an azomethine linkage (Schiff's base). This was established from the measured difference absorption spectrum of the myosin-salicylaldehyde complex.

3. 3. Three groups of lysyl residues can be distinguished with respect to the reaction with salicylaldehyde, namely, (a) residues with high association constant (K_ass = 1.8 ± 0.9·10⁵ M^-1), (b) residues with moderate association constant (K_ass = 2.2·10³ M^-1) and (c) residues that react with salicylaldehyde only after the denaturation of the protein. Their numbers could be estimated as 10 ± 5, 130 ± 5 and 260 ± 5 per mole myosin, respectively. The first group of residues was found to be absent from heavy and light meromyosin, the proteolytic fragments of myosin.

4. 4. The reaction is reversible. The complex formation rate constant, evaluated from the formula for second order reaction, is 2.2 sec^-1·M^-1, and the decomposition rate constant for first order reaction is 1.1·10^-3 sec^-1 at 22°.

5. 5. The reaction is pH dependent, the reaction yield increasing at higher pH.

6. 6. The solubility of myosin at low ionic strength decreases with increasing degree of salicylalation at slightly alkaline pH.

7. 7. The intrinsic viscosity of myosin does not change on salicylalation.

8. 8. A second peak due to polymerization appears on the sedimentation profile of the protein if more than 70 lysyl residues are salicylalated per mole of myosin.

Studies on conformational changes in the DNA structure induced by protonation: reversible and irreversible acid titrations and sedimentation measurments

Reversible and irreversible conformational changes in the acid-induced denaturation of DNA were studied by spectrophotometric titration, sedimentation, and melting measurements. A GC-rich DNA (72 mole-%) shows complete or partial reversibility of the titration profiles within the pH region of transition from helix to coil, while AT-rich DNA (29 mole-%) is irreversible in its titration behavior at each acid pH below the onset of the transition. The results for GC-rich DNA further indicate distinct differences in the titration behavior, which can be attributed to differences in the frequency of GC clusters along the DNA molecule. Plots of the sedimentation coefficient and the parameter a_s^app against pH lead to the conclusion that conformational changes occur before the onset of the acid-induced helix–coil transition. These alterations are more pronounced upon protonation of larger GC-rich domains than of smaller ones, as concluded from very marked differences observed in the sedimentation–pH behavior of two GC-rich DNA's. An acid denaturation scheme for a GC-rich DNA segment is suggested. Reversibility of the acid denaturation is explained by the existence of stable, protonated, single GC base pairs in nonprotonated stacked single-stranded domains formed in the acid-induced transition region.     

Polynucleotides. 13. Stoichiometric and thermodynamic studies of polynucleotide helices with non-complementary residues

Stoichiometry and thermodynamic properties of polyadenylate–polyuridylate helices containing varying proportions of near-randomly distributed non-complementary uridine residues were charactrized from an analysis of their mixing curves and melting profiles measured at 259 nm and at appropriate longer wavelength isochromic points. The noncomplementary residues in this homopolymer–copolymer system (in which the homopolymer has the capacity to readjust with respect to the residues with which it is in opposition) show absolute preference for an extrahelical conformation even when situated in … AAUAA … sequences and must occur therefore as single loops. As the frequency of extrahelical residues in creases, the electrostatic energy of these complexes becomes greater, and is particularly severe for the three-stranded helices. Thus, an adenyl-ate-uridylate copolymer containing 35.2 mole percent uridine residues does not form a three-stranded complex with polyuridylate even in 0.7M Na⁺at O°C. The imperfections introduced into the helix lattice by extrahelical residues decrease the cooperativity of thermal denaturation as well as T_m. However, for the helices with extrahelical residues in low frequency (～1 per helix turn) only small increases in concentration of charge-neutralizing ions are required to bring T_m to the level of their perfect analogs. Two-stranded helices with a higher density of extra helical residues (～5 per helix turn) show [Na⁺] dependence of T_m characteristic of perfect three-stranded helices. These findings together with the absence of an effect of these imperfections on the hypochromicity per base-pair suggest only minimal disruption of helix continuity or distortion of stacking interactions that normally in volve the base pairs or triplets.     

Absorption and fluorescence spectra ofS-quinolylethylated Kunitz soybean trypsin inhibitor

Disulfide bonds in soybean trypsin inhibitor (Kunitz) were simultaneously reduced and alkylated using tri-n-butylphosphine and 2-vinylquinoline at pH 7.6 in 0.11 M Tris-4.4 M urea, 41% ethanol. The resulting S--2-quinolylethylated protein (2-QE-STI) has a new absorption peak at 315–318 nm. Its quinoline fluorescence can be excited above 310 nm independently of intrinsic protein fluorescence. Free 2-quinolylethylcysteine (2-QEC) shows unexpectedly weak fluorescence. Quinoline absorption in 2-QEC and 2-QE-STI changes with pH. The apparentpK values determined spectrophotometrically are near 5 for 2-QEC and 3 for 2-QE-STI. Fluorescence decreased with increasing pH and in the presence of chloride ions. Both structural and charge effects thus appear to influence the absorption and fluorescence of the quinoline group. Corrected fluorescence emission (excited at 316 nm) of neutral 2-QE-STI diluted in 0.1 N H₂SO₄ was directly proportional to concentration in the range 0.4–8 m 2-QEC. The 2-QEC content of the protein derivative determined by UV absorption at pH 1.5 was in agreement with the expected value of four residues per mole. Fluorescence measurements ofS-2-quinolylethylated proteins may be especially useful as a sensitive, specific assay for cyst(e)ine residues.Reference to a company or product name does not imply approval or recommendation of the product by the U.S. Department of Agriculture to the exclusion of others that may be suitable.Abbreviations used are Mops: 3-(N-morpholino)propanesulfonic acid; STI: soybean trypsin inhibitor (Kunitz); 2-PE-STI:S--2-pyridylethylated STI; 2-QEC:S--(2-quinolylethyl)-l-cysteine; 2-QE-STI:S--2-quinolylethylated STI; TosPheCH₂-trypsin: bovine trypsin treated withp-toluenesulfonyl phenylalanine chloromethyl ketone.     

Succinylchymotrypsin

Succinylchymotrypsin was prepared from succinylchymotrypsinogen with trypsin and purified by DEAE-Sephadex chromatography.

The total 15 lysyl and N-terminal amino residues of chymotrypsinogen were succinylated. About 23.5 out of 49 seryl and threonyl residues and none in the total 4 tyrosyl residues in succinylchymotrypsin were modified

The helix content of succinylchymotrypsin was about 8.4%.

The pK_app of N-terminal amino group in succinylchymotrypsin did not change and that of tyrosyl residues shifted to alkali side, in comparison with those of unmodified α-chymotrypsin.

The k_cat of succinylchymotrypsin increased and its Km did not change within experimental error, in comparison with those of α-chymotrypsin.     

Thermal and charge-induced coil to -helix transition of poly-L-glutamic acid and random L-glutamic acid-L-alanine copolymers

Thermal and charge induced random coil to α-helix transitions of poly-L -glutamic acid (PGA) were measured by optical rotatory dispersion in various solvents. The data of PGA in 0.1M Nacl were analyzed by the Zimm-Rice theory. Enthalpy and entropy changes for the coil-to-helix transition in the unionized state were obtained: ΔH° = ?1020 ± 100 cal/residue mole; ΔH° = ?3.0 ± 0.4 e.u./residue mole. The initiation parameter, σ, of the Zimm-Rice theory was given by a value of 5 ± 1 × 10^-3. Random copolymers of L -glutamic acid and L -alanine containing 10, 30, and 40 molar percents of alanyl residue were synthesized. Stabilities of α-helix of the copolymers were compared to that of PGA. In water and water-ethanol solutions, stabilities of the polymers were almost equal after the simple correction about the ionized charge density of the polymers. In 0.1 M NaCl solution these copolymers showed some deviations from the transition curve of PGA, which would suggest the hydrophobic contribution of the alanyl residues.     

Iodination of Ricin D

Approximately five tyrosine residues of ricin D were iodinated preferentially under appropriate conditions probably forming diiodotyrosine. Iodination of this toxin carried out in 0.1 m phosphate buffer at pH 7.0 and 0°C for 60 min with a 20 fold molar excess of iodine per mole of protein, yielded a main component which appeared as a single band on polyacrylamide gel disc electrophoresis. Analysis of protein-bound radioactivity and the content of diiodotyrosine of ¹⁸¹I-labeled ricin D revealed that two tyrosine residues in the isoleucyl chain and three in the alanyl chain were substituted. The toxicity of iodinated ricin D decreased to one hundredth of that of native protein, However, the hemagglutinating activity of this protein was not affected by the iodination reaction.     

Investigations of primary and secondary structure of porcine ubiquitin. Its N epsilon-acetylated lysine derivative

N epsilon-acetylation in vitro of internal lysyl residues of Ub by p-nitro-phenyl acetate at pH 8.0 was performed. The position of acetylation sites are determined. (e.g. Fully acetylated: Lys-6, Lys-11 and Lys-33; partially free internal lysines: Lys-27, Lys-29; Lys-48 and probably Lys-63.) 55 cycles Edman degradation were performed and the first 53 N-terminal residues were identified. Secondary structural studies of ubiquitin have been carried out using the circular dichroism (CD) technique. No changes are noted upon heating to 100 degrees C at neutral pH even in the presence of 8 M urea but in 6 M guanidine-HCl extensive modification results. Ubiquitin with an average of 4.4 of its 7 lysines in the N epsilon-acetyl form shows little deviation from native protein. After reduction with dithiothreitol and subsequent removal of the mercaptan, significant changes in the secondary structure are noted. Circular dichroic measurements of ubiquitin indicated an alpha-helical content of about 10% whereas the secondary structural predictions of Chou and Fasman suggest a level of about 45%.     

Isolation and chemical characterization of lipopolysaccharides from four Mycoplana species (M. bullata,M. segnis,M. ramosa and M. dimorpha)

Lipopolysaccharides (LPS), isolated from four Mycoplana species, i.e. the type strains of M. bullata, M. segnis, M. ramosa and M. dimorpha, were characterized onto their chemical composition and their respective lipid A-types. Those of M. bullata and M. segnis showed on DOC-PAGE an R-type character and had lipid A's of the Lipid A_DAG-type which exclusively contained 2,3-diamino-2,3-dideoxy-d-glucose as lipid A sugar. LPS's of M. ramosa and M. dimorpha showed, although only weakly expressed, ladder-like patterns on DOC-PAGE indicating some S-type LPS's and lipid A of the d-glucosamine type (Lipid A_GlcN). M. bullata LPS contained mannose and glucose in major amounts and additionally l-glycero-d-mannoheptose, whereas M. segnis LPS was composed of rhamnose, mannose and glucose together with both, d-glycero-d-manno- and l-glycero-d-manno-heptoses in a molar ratio of 1:2. All LPS's contained 2-keto-3-deoxy-octonic acid (Kdo), phosphate and an unidentified acidic component X. In addition to X, M. segnis LPS contained glucuronic and galacturonic acids, whereas M. ramosa LPS contained only galacturonic acid. Acetic acid hydrolysis of the LPS resulted in splitting off lipid A moieties, very rich in 3-hydroxy fatty acids, in particular in 3-OH-12:0 (in Lipid A_DAG), or in 3-OH-14:0 (in Lipid A_GlcN). Analysis of the 3-acyloxyacyl residues revealed major amounts of amide-linked 3-OH(3-OH-13:0)12:0 in lipid A of M. bullata and 3-OH(12:0)12:0 in lipid A of M. segnis. The rare 4-oxo-myristic acid (4-oxo-14:0) was observed only in M. bullata LPS, where it is ester-linked. Amide linked diesters could not be traced in M. ramosa and M. dimorpha. All four lipid A's lacked erster-bound acyloxyacyl residues.Non-standard abbreviations DAG 2,3-diamino-2,3-dideoxy-d-glucose - Kdo 2-keto-3-deoxy-octonate - LPS lipopolysaccharide - PITC phenyl isothiocyanate - NANA N-acetyl neuraminic acid     

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.     

Double-headed protease inhibitors from black-eyed peas. III. Subunit interactions of the native and half-site chemically modified proteins.

The chemical modification of two new double-headed-protease inhibitors from black-eyed peas, a trypsin-chymotrypsin inhibitor (BEPCI) and a trypsin inhibitor (BEPTI) with dansyl chloride was investigated under various conditions. The NH2-terminal serine of both BEPCI and BEPTI, the 4 lysyl residues of BEPCI, and 4 of the 5 lysyl residues of BEPTI, could not be dansylated in the absence of urea. The single tyrosine per subunit of BEPCI and BEPTI was unreactive even in the presence of urea but could be labeled with half-site reactivity by the Celite method. Lysine, NH2-terminal serine, and tyrosine were reactive in fully reduced, carbamidomethylated BEPCI and BEPTI. Gel filtration was used to study the subunit interactions of BEPCI and BEPTI. At pH 8 or pH 3.0 there is a complex set of multiple equilibria with widely differing rates of attainment. We have found evidence for a rapid dimer-tetramer equilibrium, a distinct moderate rate dimer-tetramer equilibrium, a very slow monomer-dimer equilibrium, and postulate slow isomerization of the two forms of dimer and the two forms of tetramer. The monomer-dimer equilibrium is quite unusual in that the dimer is stabilized by chaotropic ions and even slightly by guanidine HC1. In contrast to the complex pattern seen in native BEPCI, the half-site, dansylated BEPCI exists at similar concentration exclusively as a tetramer at neutral pH.