A CD study of the role of metal ions in the conformation of poly(L-lysine)

The effect of LiCl, NaCl, and CsCl as univalent salts, and of CaCl₂, ZnCl₂, and MgCl₂ as divalent salts, on the α and antiparallel β-sheet, and random conformations of poly(L-lysine) (PLL), in water at room temperature were examined by means of CD and compared quantitatively on the basis of elliptical strength at the maximal peak. Changes in the α-helical and antiparallel β-sheet helical conformations of PLL were markedly dependent on the salt concentrations of LiCl, NaCl, and CsCl, which induced decreases in negative intensity in that order. The CD spectrum of the random conformation, the most disordered form, displayed positive cotton effect in concentrations of these salts up to 3.0M and a negative peak in concentrations of 6.0M. The effect of these salts on the random conformation of PLL was stronger than that on the α- and β-conformations in higher concentrations. The CD spectrum of the random conformation in the presence of CaCl₂, ZnCl₂, and MgCl₂, on the other hand, showed negative cotton effect in salt concentrations as low as 3.0M. It was impossible, however, to measure the effect on α- and β-conformations of ZnCl₂ and MgCl₂ above concentrations of 10 mM because of a solubility problem with salts in alkaline solution.     

A type II β-turn in a flexible peptide: Proton assignment and conformational analysis of the α-factor from saccharomyces cerevisiae in solution

Two-dimensional ¹H-nmr spectra of the α-mating factor [in dimethyl sulfoxide-d₆ (DMSO) and in water] and several dodecapeptide analogues (in DMSO) were obtained. Homonuclear correlated spectroscopy resulted in the complete and unequivocal assignment of all backbone and side-chain resonances of the peptides. The solution conformation of the pheromones was probed using two-dimensional (2D) nuclear Overhauser effect spectroscopy (NOESY) and rotating frame nuclear Overhauer effect spectroscopy (ROESY). The 2D NOE results, and results of complementary one-dimensional experiments, suggest that a type II β-turn is assumed by the central portion of active pheromones in both DMSO and water. Inactive analogues of the α-factor do not exhibit this structural feature. Except for this one β-turn, the nmr parameters for α-factor are indicative of a conformationally flexible molecule in both solvents. This conclusion is in contrast to that of other researchers who have proposed a highly structured conformation of α-factor in aqueous solution.     

The effect of methylmercury (II) binding on the conformation of poly(L-glutamic acid) and poly(L-lysine: a Raman spectroscopic study

The binding of the methylmercury cation CH₃Hg⁺ by poly(L -glutamic acid) (PGA) and by poly(L -lysine) (PLL) has been investigated by Raman spectroscopy. Coordination on the side-chain COO^? and NH groups of these polypeptides gave characteristic ligand–Hg stretching modes at ca. 505 and 450 cm^?1, respectively. Precipitation generally occurred upon formation of the complexes and changes of conformation were common. The solid complex obtained from PGA at pH 4.6 was found to have a mostly disordered conformation, which differed from the respective α-helical and β-sheet structures of the dissolved and precipitated uncomplexed polypeptide in the same conditions. An α-helical structure was generally adopted by the complex formed with PLL, even in pH and temperature conditions where the free polypeptide normally exists in another conformation. The addition of a stronger complexing agent, glutathione, to the PLL/CH₃Hg⁺ complex caused a migration of the bound cations and a restoration of the polypeptide to its original state.     

Conformation of poly-S-carboxyethyl-L-cysteine in aqueous solutions

Poly-S-carboxyethyl-L -cysteine, a higher side-chain homolog of poly-S-carboxymethyl-L -cysteine, has been prepared from poly-S-carbobenzoxyethyl-L -cysteine with hydrogen bromide in chloroform or acetic acid. The polymer is found to be in the β-conformation of an antiparallel arrangement of polypeptide chains in solid films, both in acid and salt forms, when examined by infrared spectra. Aqueous solutions of t he polymer have been investigated by measurements of rotatory dispersion and circular dichroism as well as by infrared spectra in D₂O. These properties show sharp changes around pH 5.5, as the pH of solution is varied. At higher ionization the polymer is randomly coiled, but at lower ionization it is in the β-conformation. Dependence of the rotatory properties upon polymer concentration as well as on ionic strength has been observed even at the lowest degree of ionization attained, and this has been attributed to the formation of intermolecular β-conformation in solutions. The β-structure is characterized by a negative circular dichroic band at 223 mμ and a positive dichroic band at a wavelength lower than 200 mμ, and furt her by a negative b_o value, ?140°. The pH-induced coil-β transition of the polymer is compared with that of poly-S-carboxymethl-L -cysteine.     

Interaction of water with poly-alpha-amino acids. I. Relationships between conformation and relaxation processes.

The relaxation behaviour of the sodium salt of poly (L -glutamic acid) in the solid state has been examined by means of dynamic mechanical spectroscopy. Bound water was found to exert a profound influence on the relaxation behaviour and on a bulk property, the rigidity. Certain features of the loss spectrum have been identified with the hydration-dependent β-to-α conformational transition. Thus two side-chain relaxations are observed below ambient temperature, one associated with the β from (β₁^β) and a second at a lower temperature associated with the α form (β₁^α). The greater rigidity of the α form below the relaxation temperature and the larger rigidity drop accompanying the β₁^α can be explained in terms of the structural differences of the two conformations.     

Chain motions in lipid-water and protein-lipid-water phases: a spin-label and x-ray diffraction study

An attempt has been made to establish a relation between the rapid reorientational motions of the lipid chains and the structure of the lipid-water and the protein-lipid-water phases. The movements were studied by electron paramagnetic resonance spectra of the fatty acids labelled at different positions along the chain; the structure of the phases, established by X-ray diffraction, differs by the long-range organization (lamellar, hexagonal, etc.) and by the short-range conformation of the chains (liquid-like, α; stiff and hexagonally packed, β or β′). The rapid motions of the hydrocarbon chains are found to be largely independent of the long-range order and highly dependent on the short-range conformation. The mobility of the nitroxide group incorporated within regions in the β (or β′) conformation is low and independent of its position along the chain. On the contrary, the mobility of the nitroxide group incorporated in regions in the α-conformation is high and increases as the position is moved along the chain away from the polar end. Temperature and water content affect the motions of the chains in the α but not in the β-conformation. Moreover, the labels appear to be perfectly soluble in the α regions, whereas the solubility in the βregions varies with the position of the nitroxide group and with the nature of the lipid. As a consequence, among the numerous phase transitions observed in these systems only those which involve a change in the conformation of the chains are accompanied by profound perturbations of the spin-label spectra; when the label is soluble in the β-regions, a discontinuity in chain motions is observed at the α-β transition; when the label is less soluble in the β than in the α-regions, its concentration in the α-regions increases as the α-β transition proceeds and magnetic interactions can be observed. In lipoprotein phases with electrostatic interactions the motions of the chains appear to be unperturbed by the presence of the protein. If the interactions are of the hydrophobic type, the electron paramagnetic resonance spectra indicate that some regions of the chains come in contact with the proteins.     

Calculation and use of protein-derived conformation-related spectra for the estimate of the secondary structure of proteins from their infrared spectra

This paper probes the calculation of conformation-related basis spectra from infrared spectra (amide I′band) of reference proteins of known conformational composition and, with their aid, the computation of conformations from the amide I′ band of globular proteins using in both approaches a least-squares, curve-fitting computer program for the analysis of the spectra. The following results were obtained. The infrared basis spectra for the α-helix conformation, the β-(antiparallel-chain pleated sheet) conformation and the ρ-conformation were calculated and their physical reality was substantiated. The basis spectra were shown to be similar when the absorption contributions of the side chains of amino acids were either neglected or taken into account (uncorrected or corrected basis spectra). The mutual correlation of the basis spectra, quantified by the roots of the diagonal elements of the inverse matrix, was found to be low enough only for the β-conformation to allow a statistically reliable estimate of the β-conformation content of proteins. The comparison of the percentages of the β-conformation derived from x-ray structural analysis or calculated from infrared spectra showed the suitability of the basis spectra for the rough estimate of the β-conformation percentages of proteins. The results were not significantly different when using the uncorrected or corrected basis spectra.     

Molecular conformation of poly-S-carboxymethyl-L-cysteine in aqueous solutions

Poly-S-carboxymethyl-L -cysteine has been prepared by debenzylation of poly-S-carbobenzoxymethyl-L -cysteine with hydrogen bromide in acetic acid. By the infrared spectroscopic method the polymer is found to be in the extended β-conformation with an antiparallel arrangement of polypeptide chains in solid film, if it has been regenerated from dimethyl sulfoxide solution. Aqueous solutions of the polymer have been investigated by measurements of optical rotatory dispersion and viscosity. Various properties sharply change around pH 5 at different ionic strengths. By combining these with infrared studies in D₂O solutions, it has been shown that the polymer exists in the random coil conformation at higher ionization but associates into the intermolecular β-conformation at lower ionization. At the lowest pH attainable in solution, the β-form is partly converted into the random coil as the temperature is raised. The rotatory dispersion of the polymer is described by the Moffitt equation. While the random coil form has a large negative a₀ value and a zero b₀ value, the β-form is characterized by a positive a₀ value and a negative b₀ value, ?130°.     

Studies on the conformation of amino acids. IX. Conformations of butyl, seryl, threonyl, cystennyl, and valyl residues in a dipeptide unit

Potential energies of conformation of a dipeptide unit with butyl, seryl, threonyl, eysteinyl, and valyl side groups have been computed by using classical energy expressions. The presence of a γ-atom introduces characteristic restrictions on the backbone rotational angles ? and ψ the γ-atom itself is restricted to three staggered positions about the C^α—C^β bond. The important results are that a γ-carbon in position I (χ¹ ? 60°) cannot be accommodated in the standard right-and left-handed α-helices, whereas a γ-oxygen or sulfur could easily be accommodated in the right-handed α-helix. Further, a γ-carbon or a heteroatom in position II (χ¹ ? 180°) does not favor a conformation ψ ? 180°, compared to two other positions. The valyl side group significantly reduces the allowed ? and ψ values and energetically prefers a β-conformation compared to right-or left-handed α-helical conformations. The less favorable α-helical conformation is possible only for γ (III, II) combination of the valyl residue. The observed ?, ψ, and χ¹ values of all the amino acid residues in the three protein molecules, lysozyme, myoglobin, and chymotrypsin are compared with the theoretical predictions and the agreement is excellent. The results bring out the important fact that even in large molecules, the conformation of local segments are predominantly governed by the short-range intramolecular interactions.     

Conformational studies on pertrimethylsilyl derivatives of some 6-deoxyaldohexopyranoses and of four less-common aldohexopyranoses by 220-MHz P.M.R. spectroscopy. Substituent and configurational effects on the chemical shifts of the ring protons

The complete interpretation of 220-MHz p.m.r. spectra and the accurate chemical shifts and coupling constants, obtained after computer simulation of the spectra, of the per-O-trimethylsilyl (Me₃Si) derivatives of a number of 6-deoxy-aldohexopyranoses and of β-D-altro-, β-D-allo-, and α- and β-D-talo-pyranose are given. By means of an adapted Karplus equation, the structure of the derivatives has been studied in detail. All of the pyranoid rings occur in the ⁴C₁(D) or ¹C₄(L) chair conformation. The preferred conformation of the C-5—CH₂OSiMe₃ group in the four aldohexopyranoses was found to be dependent on the configuration at C-4. By comparison of Me₃Si-aldohexopyranoses with the corresponding 6-deoxy analogues, it was found that the 6-OSiMe₃ group has no marked effect on the conformation of thering. The influence of this group on the chemical shifts of the ring protons is discussed in terms of electric field and inductive effects. Rules are presented for the estimation of the chemical shifts of the ring protons of Me₃Si-aldohexopyranoses and Me₃Si-6-deoxyaldohexopyranoses.     

Trifluoroethanol-induced conformational change of tetrameric and monomeric soybean agglutinin: Role of structural organization and implication for protein folding and stability

2,2,2-Trifuoroethanol (TFE)-induced conformational structure change of a β-sheet legume lectin, soybean agglutinin (SBA) has been investigated employing its exclusive structural forms in quaternary (tetramer) and tertiary (monomer) states, by far- and near-UV CD, FTIR, fluorescence, low temperature phosphorescence and chemical modification. Far-UV CD results show that, for SBA tetramer, native atypical β-conformation transforms to a highly α-helical structure, with the helical content reaching 57% in 95% TFE. For SBA monomer, atypical β-sheet first converts to typical β-sheet at low TFE concentration (10%), which then leads to a nonnative α-helix at higher TFE concentration. From temperature-dependent studies (5–60 °C) of TFE perturbation, typical β-sheet structure appears to be less stable than atypical β-sheet and the induced helix entails reduced thermal stability. The heat induced transitions are reversible except for atypical to typical β-sheet conversion. FTIR results reveal a partial α-helix conversion at high protein concentration but with quantitative yield. However, aggregation is detected with FTIR at lower TFE concentration, which disappears in more TFE. Near-UV CD, fluorescence and phosphorescence studies imply the existence of an intermediate with native-like secondary and tertiary structure, which could be related to the dissociation of tetramer to monomer. This has been further supported by concentration dependent far-UV CD studies. Chemical modification with N-bromosuccinimide (NBS) shows that all six tryptophans per monomer are solvent-exposed in the induced α-helical conformation. These results may provide novel and important insights into the perturbed folding problem of SBA in particular, and β-sheet oligomeric proteins in general.     

Influence of methanol on the rotational diffusion of poly(L-lysine) in the helix–coil transition

¹³C spin-lattice relaxation times of poly(L -lysine) have been obtained at 67.9 MHz in aqueous solution and in a mixed solvent (40% methanol/60% water). A concomitant determination of the conformation by CD permits the correlation of conformation and rotational diffusion of the polymer. The dependence on pH of the spin-lattice relaxation times of the ¹³C^α and the side-chain carbon resonances reflects the diffusional motion in the random-coil conformation, in the helix–coil transition, and in the conformation of the α-helix. In the mixed solvent the reorientational correlation time of the C^α-H^α vector increases from τ = 0.37 nsec (random coil) to τ = 12.0 nsec (α-helix). In aqueous solution the correlation time of this vector increases from τ = 0.33 nsec (random coil) to τ ? 11 nsec. The reorientation rates of the side-chain methylene groups in the two solvents are markedly different. The reorientation of all methylene groups is reduced in the mixed solvent.     

The two beta forms of poly(L-glutamic acid).

β-Helical poly(L -glutamic acid) in a gel state was found to be easily converted to the antiparallel β form by heating. Two β forms were obtained, depending on the temperature of heating. Temperatures between 40° and 85°C produced a β form with a spacing between pleated sheets (d₀₀₁) of 9.03 Å, termed β₁. If the heating was carried out at temperatures higher than 85°C, the β₁ form underwent another conformational transition reducing the d₀₀₁ value from 9.03 to 7.83 Å (termed β₂) without any prominent change in the fiber repeat distance (i.e., the polypeptide backbone conformation). The time course of these two transitions was followed by measuring the infrared spectra of the samples, and it was concluded that the α → β₁ transition in its initial stage obeys a pseudo-first order rate process with activation enthalpy and entropy of 54 kcal/mol and 92 eu, respectively. On the other hand, the typical sigmoidal conversion curves observed for the transition between the two types of β forms (β₁ → β₂) indicate that this transition proceeds via a socalled “nucleation and growth” process. The kinetic theory of phase transitions developed by Avrami can be applied with success to explain this transition. The infrared spectra, in the region from 1800 to 200 cm^?1, were measured for these two β forms and the results showed that the conformation of the side chains and the mode of the hydrogen bonding between the side-chain carboxyl groups undergo appreciable change during the transition. The heat-induced conformational transition of poly(L -Glu⁷⁸ L -Val²²) was also studied. The copolymer was transformed from the α-helical conformation directly to the β₂ form. The reason for this was thought to be due to the fact that the L -valine residues and the L -glutamyl residues near the L -valine residues have a strong tendency to take the more compact β₂ form.     

Conformations of poly-L-valine in solution

In order to test theoretical predictions that poly-L -valine can exist in an α-helical conformation, water-soluble block copolymers of L -valine and D , L -lysine were prepared. By carrying out the synthesis on a resin support (with the use of N-carboxyanhydrides) contamination of the individual blocks by any unreacted monomer from the previous block was avoided. A single glycine residue was incorporated at the C-terminus of the chain for use in amino acid analyses. Using optical rotatory dispersion and circular dichroism criteria, about 50% of the short valine block of (D , L -lysine HCl)₁₈-(L -valine)₁₅-(D , L -lysine-HCl)1₆-glycine was found to be in the right-handed α-helical conformation in 98% aqueous methanol, in water, the polymer appears to be a dimer, with the valine block being involved in the formation of an intermolecular β-structure.     

Conformational studies on polypeptides. The effect of sodium perchlorate on the conformation of poly-L-lysine and of random copolymers of L-lysine and L-phenylalanine in aqueous solution

Circular Dichroism measurements have been carried out on poly-L -lysine (PLL) and on random copolymers of lysine and phenylalanine at various p^H values and in the presence of different amounts of NaClO₄. The results indicate that either the homopolymer or the copolymers at p^H conditions at which the side-chain amino groups are fully protonated, assume the right-handed α-helical conformation in the presence of NaClO₄. The results are interpreted in terms of specific binding of ClO₄^? ions on charged side-chain amino groups.     

Conformational properties of L-leucine,L-isoleucine,and L-norleucine side chains in L-lysine copolymers

The structure inducing properties of L -leucine, L -isoleucine, and L -norleucine residues incorporated into poly(L -lysine) were investigated by the observation of the circular dichroism of the respective random copolypeptides. The comparison involves the coil-helix transition in water/methanol mixtures, the formation of ordered structures at higher pH, and the kinetics of the α-helix to β-conformation transition of the leucine and norleucine copolymers induced by temperature changes at pH 10.5. The results confirm the known properties of the leucine residue, strongly supporting the α-helix conformation. They also support the idea that the isoleucine residue is one of the most powerful candidates for β-structure formation, and they show that the unbranched norleucine residue has intermediate properties. The results are discussed on the basis of steric and hydrophobic properties of the three side chains.     

Crystal Structure and Molecular Conformation of the Cyclic Hexapeptide cyclo-(Gly-Aib-Gly)2

We have synthesized and crystallized the cyclic peptide (Gly-Aib-Gly) ₂. Its structure has been determined by conventional X-ray diffracti on methods. In the crystal it adopts a conformation with one β-turn (type I) and its mirror image at the other side of the ring. All conformation al angles are similar to those reported for these amino acid residues. In particular the Aib residue has a conformation intermediate between α- and 3₁₀-helical conformations. The ring is an adequate model for the β-turn conformation. A molecule of formic acid is found in the crystal which shows a very short hydrogen bond with one of the glycine carbonyl groups.     

Restriction in the conformational flexibility of apoproteins in the presence of organic cosolvents: A consequence of the formation of “native-like conformation”

The influence of n-propanol on the overall α-helical conformation of β-globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of α-helical conformation is induced into these proteins atpH 6.0 and 4°C in the presence of relatively low concentrations of n-propanol. The induction of α-helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of α-globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The α-helical conformation induced into α- and β-globin by n-propanol decreased as the temperature is raised from 4 to 24°C. In contrast, the α-helical conformation of both α- and β-chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced “α-helical conformation” of globins and the “α-helical conformation” of α- and β-chains. A cross-correlation of the n-propanol induced increase in the fluorescence of β-globin with the corresponding increase in the α-helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the α-helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a “native-like structure.” The induction of α-helical conformation into these proteins in the presence of n-propanol and the consequent generation of “native-like conformation” is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an α-helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases. Thus, the high sensitivity of apoproteins and the protein domains to assume α-helical conformation in the presence of low concentration of n-propanol with a concomitant restriction of the proteolytic susceptibility of their polypeptide chain appears to be unique to those proteins that exhibit high α-helical propensities. Apparently, this phenomenon of helix induction and the restriction of proteolysis reflects the formation of rudimentary tertiary interaction of the native protein and is unique to apoproteins or structural domains of α-helical proteins. Consistent with this concept, the induction of α-helical conformation into shorter polypeptide fragments of 30 residues, (e.g., α_1-30, which exists in an α-helical conformation in hemoglobin) is very low. Besides, this peptide exhibited neither the high sensitivity to the low concentrations of n-propanol seen with the apoproteins/protein domains nor the resistance toward proteolysis. The results suggest that the organic cosolvent induced decrease in the conformational flexibility of the apoprotein, and the consequent restriction of their proteolytic cleavage provides an opportunity to develop new strategies for protease catalyzed segment condensation reactions.     

Conformations of copoly(gamma-benzyl L-glutamate-O-benzyl-L-serine)

Four samples of copolymers of γ-benzyl is L -glutamic (BLG) and O-benxyl-L -serine (BLS) were synthesized by changing the mixing ratio of two monomer anhydrides. The conformations of these copolymers in CHCl₃, containing very small amount of dichloroacetic acid (DCA) necessary to dissolve the sample, were determined by their compositions; the copolymer which is rich in BLG is mostly helical and one which is rich in BLS is mostly in the β conformation. However, in DCA solution the conformations of these copolymers are independent of their composition. The interesting observation was that the copolymer which has least con tent of BLS is richest in β-conformation.     

Laser Raman spectra of glucagon

Laser Raman spectroscopy study indicates that in concentrated fresh acidic solution (30 mg/ml), glucagon remains predominantly α-helix and not random-coil. The splitting of the amide III band into three components in the crystal at 1262, 1275, and 1295 cm^?1 is due to the α-conformation as expected. The presence of a small fraction of β-conformation is demonstrated by the appearance of the weak band at 1230 cm^?1 in the fresh solution. This study also established the frequencies of amide III′ bands for the α- and β-conformations of glucagon: 957 and 988 cm^?1 for α and β forms, respectively. The conformations of acidic and basic glucagon solutions are apparently different.