Side-chain effect on conformation of ionizable polypeptides in aqueous solution

In order to study the effect of side-chain length on the conformation of polypeptides, conformational changes of various ionic polypeptides with various lengths of side chain, poly-N^ε-glutaryl-L -lysine (PGL), poly-N^δ-glutaryl-L -ornithine (PGO), poly-N^ε-succinyl-L -lysine (PSL), and poly-N^δ-succinyl-L -ornithine (PGO), were investigated by ORD, potentiometric titration, and dilatometric measurements in aqueous solution. The results of optical rotation and potentiometric titration measurements indicate strongly that the α-helix stability increases in the sequence PSO < PSL ～ PGO < PGL, which corresponds to increased side-chain length. The volume change associated with the helix–coil transition also increased in the above sequence. This series of polymers seems to be more hydrophobic compared with poly-L -glutamic acid or poly-L -lysine, as suggested from the values of enthalpy and entropy changes for coil–helix transitions.     

Optical properties of polypeptides in the β-conformation

The rotational strengths and oscillator strengths of the nπ* band and ππ* exciton bands have been calculated for antiparallel and parallel β-structures of varying length and width. The results are compared with experiment and with previous theoretical treatments of β-structures. The generally good agreement of calculations on the antiparallel β-structure with experimental results on poly-L -lysine and poly-L -serine indicates that these systems are indeed in the antiparallel conformation. It is found that the exciton component strongest in absorption shifts to longer wavelengths as the width of an antiparallel structure increases, and it is suggested that the position of the ππ* absorption band may be a useful criterion of sheet width. The results also reconcile the linear dichroism measurements of Rosenheck and Sommer on poly-L -lysine films with an anti-parallel structure. Calculations on parallel β-structures indicate that the CD spectra of this form will be rather similar to that of the antiparallel form. However, the major absorption band in the antiparallel form is associated with a small positive CD band, while in the parallel form it coincides with a large negative CD band. Finally, it is pointed out that the large positive CD bands predicted for single-stranded parallel and antiparallel β-structures at about 200 mμ render unlikely the suggestion that random-coil polypeptides contain a substantial fraction of extended chain.     

Theoretical π-π* absorption and circular dichroic spectra of polypeptide β-structures

Absorption and circular dichroic (CD) spectra of the π-π* transition near 200 nm are calculated for poly(Gly), poly(Ala), poly(Abu), and poly(Val) in the β_P (paralle) and β_A (antiparallel) pleated-sheet structures using the dipole interaction model and including interactions among all atoms; optical parameters were obtained from previous studies of related molecules. Most of the calculations are for structures with one or three chains of six residues each. The oscillator strength and splitting of the π-π* modes are found to be affected only to a small extent by variations in side-chain structure and conformation, whereas the CD spectrum is very sensitive to these variations. Poly(Gly) and poly(Ala) β-structures in uniform, planar lattices do not show sufficient rotational strength to account for observed CD spectra. Poly(Abu) and poly(Val) β-structures in uniform, planar lattices show rotational strengths comparable to experiment when χ¹ is near ?60° for β_A-structures or in a broad range near 140° for β_P-structures. Poly(Ala) in uniformly twisted β_A- and β_P-structures or in irregular β_A-structures corresponding to regions of the crystal structure of concanavalin A also show enhanced rotational strengths in the principal π-π* CD band. Absorption and CD spectra calculated for poly(Abu) in uniform β_A- and β_P-structures are compared with experimental data on poly(Lys) in the β-form, assuming side-chain conformations in Abu that maximize the intensity of the principal CD band. The calculations for the β_A-form show the better agreement with experiment for both types of spectra.     

A simple model for the optical properties of chiral amides

The intramolecular charge-transfer model for the electronic structure of amides has been extended to include the effects of torsion about the peptide bond (Δω = ω – 180) and pyramidalization at the peptide nitrogen (θ_N). The model is used to calculate the absorption and CD spectra of nonplanar amides as a function of the angles Δω and θ_N. For cis-amides, the rotational strengths of the nπ* and ππ* transitions are much more sensitive to θ_N for a given value of Δω than is the case for trans-amides. The results from the model are compared with available experimental and previous theoretical treatments of such systems. The contributions of nonplanarity in amides to the CD of various polypeptide conformations is discussed. The effects should be negligible for α-helixes but may be quite significant in β-sheets and β-turns. The CD spectrum of unordered peptides would be readily explained if there were a significant bias toward negative Δω values in polypeptides of L-amino acids. However, the evidence currently available does not indicate such a bias.     

Conformations and interactions of excited states. II. Polystyrene, polypeptides, and proteins

Previous fluorescence and phosphorescence studies of aromatic model compounds have been extended to polymers: “atactic” and isotactic polystyrene, seven aromatic poly-(amino acids), and two proteins. We have confirmed previous observations that both forms of polystyrene exhibit strong excimer fluorescence emission at room temperature but not at 77°K. Of the poly(amino acids) (all observed in helix-supporting solvents), poly-L -phenylalanine, poly(α-benzyl-L -aspartic acid), and poly-1-benzyl-L -histidine likewise show excimer emission at room temperature but not at 77°K., while poly-L -tyrosine, poly-L -tryptophan, poly(γ-benzyl-L -glutamic acid), and poly-S-benzyl-L -cysteine exhibit no excimer emission at either temperature. The aromatic residues of bovine serum albumin exhibit only “normal” fluorescence, but, lysozyme appears to be unique among proteins in showing excimer-like tryptophan emission in the native state; its luminescence becomes “normal” upon denaturation. It appears very probable that none of these polymers has a ground-state conformation in which the aromatic groups have face-to-face orientations appropriate for excimer interaction. It is concluded that at room temperature absorption of light can cause local “melting” of regular (usually helical) structures and thus, in some polymers, permit the attainment of a face-to-face arrangement of aromatic rings within the radiative lifetime of their excited singlet states. In certain other polymers (for reasons not clear at present), and in all polymers at 77°K., this does not occur. This concept is extended to provide a bettor basis for understanding the mechanism of formation of the photodimer of thy mine in irradiated DNA.     

Theoretical π-π* absorption and circular dichroic spectra of helical poly(L-proline) forms I and II

Absorption and CD spectra of the π-π* transition near 200 nm are calculated for helical (Pro)_n I and II (n = 6, 10) using the dipole interaction model, including interactions among all atoms, with optical parameters obtained from previous studies of related molecules. Calculated spectra for (Ala)_n and (Pro)_n in the same conformation show marked differences. The spectra for (Pro)_n are sensitive to side-chain structure but are found to agree reasonably well with exeriment for forms I and II when the side-chain C? C bond length is set at 1.54 Å, with structural data otherwise obtained from x-ray diffraction studies.     

Polymerization of amino acid derivatives by polymer catalysts. V. Polymerization of DL-β-phenylalanine N-carboxyanhydride by several poly(N-alkylamino acid) diethylamides

The polymerization of DL -β-phenylalanine N-carboxyanhydride (NCA) initiated by poly(N-benzylglycine)diethylamide (DEA) and poly(N-methyl-DL -alanine)DEA has been investigated. As previously reported, polysarcosine DEA, poly-N-ethylglycine DEA, and poly-N-n-propylglycine DEA showed marked accelerations in the polymerization of DL -β-phenylalanine NCA as compared with the polymerization initiated by low molecular weight, amines having similar base strength. However, this phenomenon (the chain effect) was not observed with the two polymer catalysts studied in the present investigation With poly-N-methyl-DL -alanine DEA, adsorption of DL -β-phenylalanine NCA onto the polymer chain takes place, though not so effectively as with other polypeptides, so the absence of chain effect was ascribed to a reduced flexibility of the polymer chain. With poly(N-benzylglycine)DEA, the reactivity of terminal base group was found to be much lower than that of other polymer catalysts. However, the absence of the chain effect would be attributed to the rigidity of polymer chain of poly-N-benzylglycine DEA due to the bulkiness of the N-benzyl group.     

Optical Rotatory Dispersion of Polypeptides in the Near-Infrared Region

For the first time ORD measurements in the near-infrared region from 0.7 to 2.0 μ for well-known polypeptides, namely, poly(γ-benzyl L -glutamate), poly(L -glutamic acid), poly-L -lysine·HCl, poly-S-carbobenzoxymethyl-L -cysteine, and Bombyx mori silk fibroin, were carried out. It was found that the value of the optical activity infrared term, which is proportional to the sum of rotational strengths of vibrational transitions, depends on polypeptide conformation. The optical activity infrared term value is equal to zero for the random-coil conformation, it is small but exceeds the measurement error for the α-helical state, and finally, for the β conformation it is an order of magnitude higher than for the α-helical state. The obtained results permit one to hope that on the basis of ORD measurement in the near-infrared region it will be possible to suggest a method of determining the β-form content in polypeptides and proteins     

Linear dichroism studies of nucleic acids. III. Reduced dichoism curves of DNA in ethanol–water and in poly(vinyl alcohol) films

The uv linear dichroism of calf thylus DNA has been studied at different degrees of orientation both in flow-oriented ethanol–water solutions and in a stretched aqueous host of poly(vinyl alcohol) (PVA). The reduced dichroism (LD^R) curves in the region 250–290 nm for DNA in PVA films at 75 and 100% relative humidity (r.h.) are in fair agreement with the curves calculated for the A- and B-forms of DNA, based on the fiber structures and the π-π* transitions of the free bases. This suggests that DNA adopts its A and B conformations in PVA at 75 and 100% r.h. In ethanol, on the other hand, a deviation from the A-form spectrum shows that the conformation of DNA in the solution can differ from the fiber structure. At shorter wavelenghts, a positive contribution to LD^R is explained in terms of an out-of-plane polarized n-π* transition.     

The effect of various amino acids and drugs on thepara-andmeta-hydroxyphenylacetic acid concentrations in the mouse caudate nucleus

Injection ofl-p-tyrosine (800 mg/kg, 2 h) increased the mouse striatalpara-hydroxyphenylacetic acid (p-HPAA) concentrations. A smaller dose ofd,l-m-tyrosine (20 mg/kg, 2h) produced a larger increase in mouse striatalmeta-hydroxyphenylacetic acid (m-HPAA) concentrations. The administration ofl-phenylalanine to mice caused a slight increase in thep-HPAA concentrations in the corpus striatum after 2h while a larger dose ofl-phenylalanine (800 mg/kg) produced a greater increase. Eight hours followingl-phenylalanine injection,p-HPAA concentrations were still elevated. Withd-phenylalanine a significant increase was observed at eight hours after drug administration.Two drugs which reduce dopamine synthesis, -methyl-para-tyrosine and apomorphine, decreasedm-HPAA striatal concentrations without affectingp-HPAA concentrations. From these results, it is proposed that tyrosine hydroxylase activity determinesp-HPAA concentrations by regulatingp-tyrosine availability. This enzyme may also synthesizem-tyrosine which is subsequently decarboxylated to formm-tyramine and then oxidatively deaminated to formm-HPAA.     

Theoretical optical properties of poly-L-proline

A non-perturbational technique is used to calculate the circular dichroism and absorption spectra of polypeptide chains having conformations similar to that of poly-L -proline II. The method employs a Bogoliubov exciton formalism, from which the various optical terms associated with parallel and perpendicular components of the exciton band are obtained. A simple model for the peptide unit, consisting of three Gaussian absorption bands, leads to reasonable results for the polymer spectra, provided the lowest energy peptide π → π* transition is taken at 207 mμ and the value of the Ramachandran angle Ψ is taken to be 390°. The calculations suggest that the polymer circular dichroism spectrum is the resultant of strong interference among the two Gaussian exciton terms and the non-Gaussian helix term. Consequently, the CD spectrum is very sensitive to the value of Ψ. It is found that the small positive CD band in the vicinity of 230 mμ arises partly from the effect of the static (crystal) field interactions on the n → π* CD band.     

The calculated circular dichroism of polyproline II in the polarizability approximation

The circular dichroism (CD) spectrum of polyproline II (PPII) has heretofore been moderately well calculated from exciton theory only at the expense of assuming unreasonable chain conformations and accepting a conservative spectrum in the 180–250-nm region (which is not observed). We have incorporated far uv transitions in the polarizability approximation and, together with the π₂π* transition, have calculated the resulting correction to the exciton model. This has been accompanied by a modified assignment of the ππ* transition in PPII, and a simultaneous calculation of the absorption and CD spectra of the α-helix, β structure, PPI, and PPII. We obtain good agreement with the observed CD spectrum of PPII in the 180–250-nm region for acceptable chain conformations. In addition, we predict a negative CD into the far uv, in agreement with recent experimental observations. Our calculations also reproduce features of the far uv CD spectrum of the α-helix, and are in agreement with the CD spectra of the β chain and PPI. The calculated CD of the unordered polypeptide chain is not significantly influenced by far uv contributions, indicating that our previous calculation is valid for such a system. These results demonstrate the importance of incorporating far uv transitions in order to achieve an adequate theoretical explanation of the CD spectra of polypeptides.     

Theoretical pi-pi absorption, circular dichroic, and linear dichroic spectra of collagen triple helices

Absorption, CD, and LD spectra of the π-π* transition near 200 nm are calculated for poly(Gly-X-Y) (X,Y = Gly, Ala, Pro) in four conformations proposed for collagen like triple helices in the recent literature. A dipole interaction model is used with the same optical paramenters as in previous studies of polypeptide spectra. The CD spectra are sensitive to backbone structure and amino acid composition, although the experimentally observed negative peak near 200 nm is a general feature of most the calculated spectra. Interchain interactions significantly affect the CD spectra in most cases. Calculations for (Gly-Pro-Ala)₃ and (Gly-Ala-Pro)₃ in the triple helical structure of Fraser, MacRae, and Suzuki [(1979) J. Mol. Biol. 129 , 463–481] show absorption, CD, and LD spectra in fairly good agreement with experiment. The characteristics of the π-π* normal modes responsible for the calculated spectra are compared with those of the component bands resolved from the experimental spectra of collagen by Mandel and Holzwarth [(1973) Biopolymers 12 , 655–674].     

Aromatic side-chain contributions to the far ultraviolet circular dichroism of peptides and proteins

The rotational strength of the L_a transition in phenylalanine and tyrosine side chains has been calculated for dipeptides with various backbone and side-chain conformations. Similar calculations have also been performed for tripeptides in the β-turn conformation with aromatic residues at the corners of the turn. The interaction of the aromatic ring with neighboring peptides generates rotational strengths in the L_a transition of the order of 0.1 Debye-Bohr magneton. When the preferred backbone and side-chain conformations are considered, it is found that the most probable conformations have positive L_a bonds. This result accounts for the observation that the N-acyl amino acid amides of L -Tyr and L Phe have positive L_a bands. It also suggests that, although other interactions may affect the numerical value and even the sign, there will be a significant positive contribution to the rotational strength of aromatic residues in globular proteins from nearest-neighbor interactions. Calculations on proteins of known conformation at the nearest-neighbor level confirm the tendency toward positive L_a contributions for Phe and Tyr residues. This contribution can be of the order of 10% of the observed CD even in proteins with rather strong amide contributions. In some proteins, such as the gene 5 protein from bacteriophage fd and many snake-venom toxins, side-chain contributions from Tyr and Trp residues manifest themselves as positive CD bands in the 225–250-nm region. The magnitude of the nearest-neighbor contributions and the trend toward positive contributions are consistent with the observation of such CD bands in globular proteins. No special stacking interaction among aromatic side chains needs to be invoked.     

Conformation of poly-L-tyrosine in trimethyl phosphate solution

Absorption, circular dichroism (CD), and optical rotatory dispersion (ORD) measurements were carried out on poly-L -tyrosine in trimethyl phosphate solution over the spectral range 185–600 mμ. There is evidence in the CD spectrum for side chain-side chain interactions in poly-L -tyrosine. ORD and CD data in dimethylformamide and pyridine closely parallel those in trimethyl phosphate, indicating a similarity in conformation of the polymer in all three solvents. In the polarized infrared spectrum both position and polarization of amide A, I, and II bands are characteristic of α-helical polypeptides. Bands corresponding to side chain also exhibit dichroism, suggesting that the side chains are not randomly oriented. Viscosity and light-scattering studies are consistent with α-helical structure for the polymer that, remains rigid over a temperature range of 15–50°C and becomes somewhat flexible at higher temperatures. Optical rotatory properties were found to vary gradually and continuously with temperature over the range of ?30 to +100°C. This suggested that all three electronic transitions of tyrosyl side chain are optically active, and that the side chains have some freedom of motion that decreases with decreasing temperature, disappearing only at about ?30°C.     

Empirical calculations of dinucleotide optical activity

In this paper we present calculations of dinucleotide optical activity based on the exciton-coupled oscillator and generalized susceptibility theories of CD. Our application of these methods utilizes experimental transition moment data for the nucleic acids and does not require explicit wavefunctions for the monomers. In addition to near-uv π-π* states, the calculations include n-π* and far-uv π-π* transitions recently characterized by Clark. We also show that correlated motions of electrons in the ground state of a system is reflected in the net CD summed over all transitions. Sample calculations for dinucleotides in several conformations illustrate the methods.     

Conformational behavior of poly-N5-(3-hydroxypropyl)-L-glutamine in water-methanol mixtures studied by 13C Nmr and CD spectroscopy

The helix–coil transition of poly-N⁵-(3-hydroxypropyl)-L -glutamine (PHPLG) has been studied in methanol–water by CD and cmr spectroscopy. For polydisperse PHPLG, two separate peaks arising from residues in helical and random-coil conformations are observed during the transition for both main-chain carbons. These results are discussed and compared to those observed in the case of a polymer sample obtained by racemization of PHPLG in 0.1 M NaOH and of PHPLG samples of controlled molecular weight and dispersity. The dominant influence of the molecular-weight heterogeneity on the double-peak phenomenon has been verified. The linewidths and chemical shift of the ¹³C resonances are discussed in terms of side-chain–main-chain interactions and side-chain solvation.     

Optical activity of single-stranded polydeoxyadenylic and polyriboadenylic acids; dependence of adenine chromophore cotton effects on polymer conformation

Circular dichroism (CD) curves are reported for poly dA, (pdA)₆, (pdA)₂, poly A, ApAp, ApA, AMP, dApA, pdApA, A-2′-O-methyl pA, and A-2′-O-methyl pAp. Analysis of these curves indicated the presence of single CD bands at 228–230 mμ and at 278–280 mμ in oligomers longer than dinucleotides. In the case of dinucleotides and mononucleotides (from the literature, in addition to those studied here), the 230 mμ CD of band appears but the 280 mμ CD band does not. We assign the 230 mμ band to a very weak π–π* transition at this wavelength. From theoretical considerations, we show that the 280 mμ band is not an exciton component of the strong π–π* transition at 260 mμ in adenine. We conclude that the 280 mμ CD band must be assigned to a distinct absorption, not previously reported, which we suggest arises from an n–π* transition. The fact that the n–π* CD band at 280 mμ is not seen in mononucleotides or dinucleotides is ascribed to solvation of the adenine ring by water, which shifts the band to shorter wavelengths. Therefore, only interior residues of oligomers have the 280 mμ band, and the optical activity of a polymer cannot be computed from that of a dinucleotide, by using a nearest-neighbor approximation. The existence of this end effect hag been tested, by taking it into account in computing the rotational strengths of the 278 mμ n–π* transition for several oligomers; it is pointed out that a more sensitive test of this end effect would require CD data for the oligo dA series of 3 to 5 residues. We speculate about the structural and optical differences between poly dA and poly A, and point out the need for a theoretical treatment of n–π* Cotton effects in polynucleotides.     

Optimization of the solvent-tolerant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> S12 as host for the production of <Emphasis Type="Italic">p</Emphasis>-coumarate from glucose

A Pseudomonas putida S12 strain was constructed that is able to convert glucose to p-coumarate via the central metabolite l-tyrosine. Efficient production was hampered by product degradation, limited cellular l-tyrosine availability, and formation of the by-product cinnamate via l-phenylalanine. The production host was optimized by inactivation of fcs, the gene encoding the first enzyme in the p-coumarate degradation pathway in P. putida, followed by construction of a phenylalanine-auxotrophic mutant. These steps resulted in a P. putida S12 strain that showed dramatically enhanced production characteristics with controlled l-phenylalanine feeding. During fed-batch cultivation, 10 mM (1.7 g l⁻¹) of p-coumarate was produced from glucose with a yield of 3.8 Cmol% and a molar ratio of p-coumarate to cinnamate of 85:1.     

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.