Structural changes in poly(L-alanine) induced by heat treatment

Structural changes in poly(L -alanine)(PLA), a model compound related to tussah silk fibroin, induced by heat treatment have been studied by differential thermal analysis, x-ray diffraction, and infrared spectroscopy. PLA heated below 300°C shows x-ray patterns very similar to those of the α-helix crystalline phase, in addition to the diffraction patterns due to the β structure. Samples heated at 368°C exhibit predominantly the diffraction patterns due to the β crystalline phase. From infrared spectra, PLA samples heated below 280°C are found to be composed of all three molecular conformations: β sheet, random coil, and α helix. The intensity of the infrared absorption at 650 cm^?1 (amide V) assigned to the random coil conformation is decreased by heat treatment above 325°C. The content of the β-sheet structure remains almost constant when the specimen is heated below 325°C, and increases abruptly on heating to around 335°C, while the random coil content is decreased abruptly by heat treatment above 335°C. The α-helix content does not change, regardless of heat treatment. It is suggested therefore that the random coil conformation of PLA transforms directly into the β sheet on heat treatment above 335°C.     

Frequency dependence of intrinsic viscosity of macromolecules with finite internal viscosity

In order to explain the observed nonvanishing limiting value of dynamic intrinsic viscosity of polymer solutions at ω = ∞ one has considered the necklace model with finite resistance to the rate of coil deformation introduced long ago by Cerf for the study of gradient dependence of intrinsic viscosity and streaming birefringence. The calculation need not take into account change of hydrodynamic interaction as a consequence of coil deformation because the experimental data are always either obtained at very low gradient or extrapolated to zero gradient so that in the experiment the macromolecule has the same conformation as in the solution at rest. The model indeed yields a finite [η]′_{ω = ∞} in good agreement with experiments on polystyrene in Aroclor. According to the theory [η]′_{ω = ∞}/[η]₀ decreases with increasing molecular weight as M^?1 and M^?1/2 for the free-draining and impermeable coil, respectively. The absolute limiting value [η]_∞′, therefore turns out to be nearly independent of M, at least for small values of internal viscosity. From the observed value [η]_∞′/[η₀] one can obtain the coefficient of internal viscosity of the macromolecule. The value for polystyrene in Aroclor calculated from dynamic experiments on rather concentrated solutions is close to that derived by Cerf from streaming birefringence observations of polystyrene in a series of solvents of widely differing viscosity.     

Temperature selectivity effects in reversed-phase liquid chromatography due to conformation differences between helical and non-helical peptides

In order to characterize the effect of temperature on the retention behaviour and selectivity of separation of polypeptides and proteins in reversed-phase high-performance liquid chromatography (RP-HPLC), the chromatographic properties of four series of peptides, with different peptide conformations, have been studied as a function of temperature (5-80 degrees C). The secondary structure of model peptides was based on either the amphipathic alpha-helical peptide sequence Ac-EAEKAAKEX(D/L)EKAAKEAEK-amide, (position X being in the centre of the hydrophobic face of the alpha-helix), or the random coil peptide sequence Ac-X(D/L)LGAKGAGVG-amide, where position X is substituted by the 19 L- or D-amino acids and glycine. We have shown that the helical peptide analogues exhibited a greater effect of varying temperature on elution behaviour compared to the random coil peptide analogues, due to the unfolding of alpha-helical structure with the increase of temperature during RP-HPLC. In addition, temperature generally produced different effects on the separations of peptides with different L- or D-amino acid substitutions within the groups of helical or non-helical peptides. The results demonstrate that variations in temperature can be used to effect significant changes in selectivity among the peptide analogues despite their very high degree of sequence homology. Our results also suggest that a temperature-based approach to RP-HPLC can be used to distinguish varying amino acid substitutions at the same site of the peptide sequence. We believe that the peptide mixtures presented here provide a good model for studying temperature effects on selectivity due to conformational differences of peptides, both for the rational development of peptide separation optimization protocols and a probe to distinguish between peptide conformations.     

The pressure tolerance of different poly-l-lysine conformers in aqueous solution: Infrared spectroscopy and two-dimensional correlation analysis

Poly-l-lysine can form either of three different conformers as α-helix, anti-parallel β-sheet and random coil stably under appropriate conditions. In buffer solution poly-l-lysine exists in a random coil at about pH 4, an α-helix above pH 12, and transforms from α-helix to β-sheet when the sample is heated to 46 °C for 30 min. The effects of elevated hydrostatic pressure on three different initial conformers of poly-l-lysine are investigated with Fourier transform infrared spectroscopy and two-dimensional correlation analysis. Changes observed in the amide I′ band indicate that the α-helix conformer undergo hydration enhancement at low pressure (<400 MPa), then gradually transition into an α′-helix. Two initial conformers, the β-sheet and random coiled polypeptide, undergo conformational changes to an α-helix at low pressure and to an α′-helix at high pressure. Moreover, the conversion occurred at a lower pressure for the β-sheet (∼250 MPa) than for the α-helix (∼300 MPa) and the random coil (∼850 MPa).     

Photoinduced conformational changes of azoaromatic polyaspartates containing octadecyl side chains

Copolymers of p-(phenylazo)benzyl-L-aspartate and n-octadecyl-L-aspartate exist as right- and left-handed α-helices in solution at 25°C depending on the copolymer composition: the reversal of helix sense from a right- to left-handed one occurs with increasing the azobenzene content. The α-helices of the copolymers are very sensitive to trifluoroacetic acid (TFA), and are converted into random coil below 2.0% of TFA. Among the copolymers, the copolymer containing 47% azobenzene groups is unique since it exhibits a TFA-induced conformational change from right-handed α-helix to random coil via left-handed α-helix. Upon UV light irradiation at 25°C, the copolymers containing 68 and 89% azobenzene groups caused the reversal in helix sense from a left- to right-handed one. The conformations of the copolymers were dependent on temperature, mostly right-handed and left-handed α-helices at lower and higher temperatures, respectively. On this basis, the copolymer containing 47% azobenzene groups could be made to undergo a photoinduced helix reversal at high temperatures.     

Effect of ureas on the viscometric behavior of water-soluble isotactic poly(2-hydroxyethyl methacrylate)

Isotactic poly(2-hydroxyethyl methacrylate) (PHEMA) is soluble and has the compact random coil structure in water solution. The effect of six ureas (thiourea, urea, methylurea, ethylurea, 1,3-dimethylurea, and tetramethylurea) on the viscometric behavior of aqueous solutions of isotactic PHEMA as a function of urea concentrations at 25°C has been investigated. The compact random coil structure is found to disappear as ureas are added. The result is discussed in terms of water structure breaking and making effect of solutes, and hydrophobic interactions. In addition, experiments were performed with aqueous isotactic PHEMA solutions at four different temperatures. Isotactic PHEMA in pure water has the most compact conformation at 20°C.     

Equilibrium Shift in Solution: Molecular Shape Recognition and Precipitation of a Synthetic Double Helix Using Helicene‐Grafted Silica Nanoparticles

Chiral silica nanoparticles (70 nm) grafted with (P)‐helicene recognized the molecular shape of double helix and random coil (P)‐ethynylhelicene oligomers in solution. A mixture of the (P)‐nanoparticles and double helix precipitated much faster than a mixture of the (P)‐nanoparticles and random coil, and the precipitate contained only the double helix. The mixture of the (P)‐nanoparticles and (P)‐ethynylhelicene pentamer reversibly dispersed in trifluoromethylbenzene upon heating at 70 °C and precipitated upon cooling at 25 °C. When a 10:90 equilibrium mixture of the double helix and random coil in solution was treated with the (P)‐nanoparticles, the double helix was precipitated in 53 % yield and was accompanied by equilibrium shift.     

Solution Behavior of Poly(n-Isopropylacrylamide) in Water: Effect of Additives

The effect of different kinds of additives (electrolytes, nonelectrolytes, hydrotropes, and surfactants) on the cloud point (CP) of low molecular weight and narrow dispersed poly(n-isopropylacrylamide) (PNIPAM) synthesized via reversible addition-fragmentation chain transfer (RAFT) controlled radical polymerization was examined. The CP showed a concentration dependent variation and it is greatly modified in the presence of additives. The size of the random polymer coil at 30°C obtained from dynamic light scattering (DLS) measurements is often influenced by the presence of additives. We have explained the effects of different additives on PNIPAM in terms of their interaction with polymer and resultant changes in the coil structure.     

Concentration Threshold and Amplification Exhibited by a Helicene Oligomer during Helix‐Dimer Formation: A Proposal on How a Cell Senses Concentration Changes of a Chemical

Sulfonamidohelicene tetramer (M)‐ 1 exhibits a concentration threshold and amplification phenomena in solution during helix‐dimer formation from a random‐coil. The (M)‐tetramer is a random‐coil below a threshold concentration, and the concentration of the helix‐dimer is irreversibly amplified once the threshold concentration is exceeded. For example, a 15 % increase in (M)‐tetramer total concentration from 0.6 to 0.7 mM induces an 8‐fold increase in the concentration of the helix‐dimer, being 8:0.15=53‐fold amplification, under temperature oscillation conditions between 47 and 49 °C. Experiments without oscillation also exhibit concentration amplification. The threshold and amplification phenomenon involves concentration hysteresis, being away from equilibrium, and self‐catalysis. On the basis of this study, a proposal on how a biological cell senses concentration changes of a chemical substance is provided.     

The study of Turnip Mosaic virus coat protein by surface enhanced Raman spectroscopy

Using Turnip Mosaic virus (TuMV) coat protein as material, the secondary structure has been studied by both normal Raman spectroscopy (NRS) and surface enhanced Raman spectroscopy (SERS). The NRS of TuMV coat protein under certain conditions showed the α-helix, β-sheet and random coil structure. The CSSC comformations are trans—gauche—gauche and gauche—gauche—gauche. The SERS spectrum of TuMV coat protein under certain conditions reveals the α-helix structure. By studying SERS at different adsorbing times, we have observed the amide III vibration of α-helix, β-sheet and random coil structure. The CSSC conformations drawn from the SERS spectra are trans—gauche—gauche and trans—gauche—trans. Besides the amide I, amide III and CSSC bands, the CαCN band, aromatic amino acid bands and some other bands can also be seen in the SERS spectra.     

Monitoring folding transitions of synthetic,branched-chain polypeptides by capillary zone electrophoresis

The coil/helix transition of a synthetic, branched-chain polymeric polypeptide (poly (Lys(Glu(1)-DL-Ala(3))EAK), 50-Lys residues long in the backbone, as a function of increasing molarities of methanol in solution, is here studied by both, circular dichroism (CD) and capillary zone electrophoresis. CD spectra showed that, at 75% v/v methanol, the transition from random coil to fully helical structure was obtained, in a pH 1.1 HCI solution in the presence of 20 mM NaCI. CZE studies, run in parallel, exhibited the classical unfolding to folding sigmoidal transition, with mid-point at 60% v/v methanol concentration, plateauing at ca. 80% v/v organic solvent. Surprisingly, though, such unfolding to folding transition was accompanied by an expansion, rather than a contraction, of the resulting ordered polypeptide. As the charge of the polypeptide (a pure polycation at a pH of 2.1 in CZE) was kept rigorously constant, a plot of the radius of the polymer along the sigmoidal transition clearly showed that the radius of gyration of the helical, structured polypeptide was in fact larger than that of the random coil. Such results were confirmed by molecular dynamics simulations, which indicated that the dimensions of such polypeptide, in alpha-helix configuration, were 8.5 nm (in length) and 3.2 nm (in diameter), whereas those of the corresponding random coil were 7.2 nm (in length) and 5.1 nm (length of shorter axis). It would thus appear that the randomized structure assumes the shape of a more compact object, roughly resembling a "rugby ball".     

Predicting 15N amide chemical shifts in proteins. I. An additive model for the backbone contribution

Because proteins adopt unique structures, chemically identical nuclei in proteins exhibit different chemical shifts. Amide ¹⁵N chemical shifts have been shown to vary over 20 ppm. The cause of these chemical shift inequivalencies is the different intra‐ and intermolecular interactions that individual nuclei experience at different locations in the protein structure. These chemical shift inequivalencies can be described as structural shifts, the difference between the actual chemical shift and the random coil chemical shift. As a first step toward the prediction of these amide ¹⁵N structural shifts, calculations have been carried out on acetyl‐glycine‐methyl amide to examine how a neighboring peptide group influences the amide ¹⁵N structural shifts. The ϕ,ψ dihedral angle space is completely surveyed, while all other geometrical variables are held fixed, to isolate the effect of the backbone conformation. Similar calculations for a limited number of conformations of acetyl‐glycine‐glycine‐methyl amide were carried out, where the effects of the two terminal peptide groups on the central amide ¹⁵N structural shift are examined. It is shown that the effect of the two adjacent groups can be accurately modeled by combining their individual effects additively. This provides a quite simple method to predict the backbone influence on amide ¹⁵N structural shifts in proteins. © 2001 John Wiley & Sons, Inc. J Comput Chem 22: 366–372, 2001     

Sequence-dependent correction of random coil NMR chemical shifts.

Random coil chemical shifts are commonly used to detect secondary structure elements in proteins in chemical shift index calculations. While this technique is very reliable for folded proteins, application to unfolded proteins reveals significant deviations from measured random coil shifts for certain nuclei. While some of these deviations can be ascribed to residual structure in the unfolded protein, others are clearly caused by local sequence effects. In particular, the amide nitrogen, amide proton, and carbonyl carbon chemical shifts are highly sensitive to the local amino acid sequence. We present a detailed, quantitative analysis of the effect of the 20 naturally occurring amino acids on the random coil shifts of (15)N(H), (1)H(N), and (13)CO resonances of neighboring residues, utilizing complete resonance assignments for a set of five-residue peptides Ac-G-G-X-G-G-NH(2). The work includes a validation of the concepts used to derive sequence-dependent correction factors for random coil chemical shifts, and a comprehensive tabulation of sequence-dependent correction factors that can be applied for amino acids up to two residues from a given position. This new set of correction factors will have important applications to folded proteins as well as to short, unstructured peptides and unfolded proteins.     

Nanofibers of silk fibroin controlled by the crystallization of polyethylene glycol in frozen solution

The conformation of silk fibroin(SF) frozen with polyethylene glycol(PEG) at a molecular weight from 2kDa to 20kDa and a mass ratio of PEG:SF from 1:5 to 10:1 was studied by spectral and microscopic methods. It is found that the conformation transition of SF from random coil to b-sheet could be induced by the stress resulting from PEG crystallization at-20 °C, and greatly depended on the cooling rate, PEG:SF mass ratio and PEG molecular weight. These findings provide a new method for the preparation of desired SF nanofibers.     

Virtual bond probe to study ordered and random coil conformations of nucleic acids

Recognition of the stereochemical features inherent to a nucleotide, viz., the preferred trans character of the two C? O bonds and the approximate chemical and conformational symmetry, has facilitated the representation of a nucleotide in terms of two nearly equal blocks or two virtual bonds spanning the atoms P to C4' on the 5' side and C4' to P on the 3' side. The scheme, by virtue of its unique ability to account for the main sources of flexibility and to incorporate their interdependence, could be effectively applied to probe ordered, as well as random coil conformations of polynucleotide chains. Using this scheme, conformations of nucleotide, dinucleotides, helices, and yeast tRNA^Phe have been characterized by virtual bond parameters. The two blocks of nucleotide have also been described in terms of two approximate planes similar to peptides. Unperturbed end-to-end dimensions and persistence lengths of random coil polynucleotides have been computed by considering short-range as well as near-neighbor bond long-range correlations and are in excellent agreement with the experimentally determined values. A striking finding of these studies is the relevation that random coils comprise of a large proportion of stacked A-type helical segments in sharp contrast to earlier interpretations which invoked a high fraction of unstacked extended conformations.     

Conformational preference of polyglycine in solution to elongated structure

Do polypeptide chains ever behave like a random coil? In this report we demonstrate that glycine, the residue with the fewest backbone restrictions, exhibits a strong preference for an extended conformation in solution when polymerized in short segments of polyglycine. A model peptide system comprised of two unique tripeptide units, between which 1 to 18 glycine residues are inserted, is characterized by NMR and by small-angle X-ray scattering (SAXS). The residual dipolar coupling (RDC) values of the two tripeptide units are insensitive to changes in number of intervening glycines, suggesting that extension of the linker does not alter the average angular relationship between the tripeptides. Polyglycine segments longer than nine residues form insoluble aggregates. SAXS measurements using synchrotron radiation provide direct evidence that polyglycine peptides adopt elongated conformations. In particular, the construct with a linker with six glycines showed a scattering profile indicative of a monomeric state with a radius of gyration and the maximum dimension of 9.1 A and approximately 34 A, respectively. The ensemble averaged global structure of this 12-mer peptide can best be approximated by a cylinder with a radius of 4 A and a length of approximately 33 A, making it intermediate in extension between a beta strand and an alpha helix.     

The studies of FT-IR and CD spectroscopy on catechol oxidase I from tobacco

A novel copper-containing enzyme named COI (catechol oxidase I) has been isolated and purified from tobacco by extracting acetone-emerged powder with phosphate buffer, centrifugation at low temperature, ammonium sulfate fractional precipitation, and column chromatography on DEAE-sephadex (A-50), sephadex (G-75), and DEAE-celluse (DE-52). PAGE, SDS-PAGE were used to detect the enzyme purity, and to determine its molecular weight. Then the secondary structures of COI at different pH, different temperatures and different concentrations of guanidine hydrochloride (GdnHCl) were studied by the FT-IR, Fourier self-deconvolution spectra, and circular dichroism (CD). At pH 2.0, the contents of both alpha-helix and anti-parallel beta-sheet decrease, and that of random coil increases, while beta-turn is unchanged compared with the neutral condition (pH 7.0). At pH 11.0, the results indicate that the contents of alpha-helix, anti-parallel beta-sheet and beta-turn decrease, while random coil structure increases. According to the CD measurements, the relative average fractions of alpha-helix, anti-parallel beta-sheet, beta-turn/parallel beta-sheet, aromatic residues and disulfide bond, and random coil/gamma-turn are 41.7%, 16.7%, 23.5%, 11.3%, and 6.8% at pH 7.0, respectively, while 7.2%, 7.7%, 15.2%, 10.7%, 59.2% at pH 2.0, and 20.6%, 9.5%, 15.2%, 10.5%, 44.2% at pH 11.0. Both alpha-helix and random coil decrease with temperature increasing, and anti-parallel beta-sheet increases at the same time. After incubated in 6 mol/L guanidine hydrochloride for 30 min, the fraction of alpha-helix almost disappears (only 1.1% left), while random coil/gamma-turn increases to 81.8%, which coincides well with the results obtained through enzymatic activity experiment.     

Static and dynamic light scattering of a random coil polymer in rodlike polymer solution

We have studied ternary solutions of rodlike and random coil polymers in a common solvent. In these solutions, the rods scatter weakly, and almost all scattering comes from the coil component. From light scattering measurements, we gained information about the thermodynamic and hydrodynamic properties of the coil in rodlike polymer solutions.     

Microstructure elucidation of historic silk (Bombyx mori) by nuclear magnetic resonance

¹H NMR cryoporometry and solid-state ¹³C cross-polarization (CP) magic-angle spinning (MAS) NMR spectroscopy were used to characterize the microstructure of historic and fresh silk samples. Silk is a polymeric bicomponent material composed of fibroin and water located in micropores. According to the ¹H NMR cryoporometry method, the intensity of the water resonance as a function of the temperature was used to obtain the pore size distribution, which was strongly asymmetric with a well-defined maximum at 1.1 nm. Compared with the fresh silk samples, the volume of pores around 1.1 nm decreased distinctly in the historic silk, and more pores larger than 2 nm emerged accordingly. In addition, these results correlated well with solid-state ¹³C CP/MAS NMR spectroscopy as the percentage of random coil in the historic silk sample was much less than that in the fresh silk samples. Therefore, it is suggested that the water-filled microvoids grow larger as the random coil conformation fades away in the degradation process.