Random Coil State Research Articles

Influence of the solvent on the conformational-dependent properties of random coil polypeptides

Mean square optical anisotropies and molar Kerr constants were calculated for homopolypeptides of the 20 natural amino acids and of several enzymes and proteins in the random-coil state. The effect of hydration was taken into account in constructing the molecular potential that gives the conformational energies as a function of the rotational angles phi and psi of the backbone and chi(1) of the side chain. The Rotational Isomeric State model was used in calculated energies, the Valence Optical Scheme and the matrix calculus technique of Flory being employed in the evaluation of the optical properties. The results are compared with calculations for the same substances that were performed without taking into account the solvent, as well as with other similar studies. The Kerr constant is confirmed as being one of the most sensitive properties of a given polypeptide to the residue class and to the sequence of those residues.

Conformational distributions of melittin in water / methanol mixtures from frequency-domain measurements of nonradiative energy transfer

We used fluorescence energy transfer to examine the effects of solvent composition on the distribution of distances between the single tryptophan residue of melittin (residue 19) to the N-terminal alpha-amino group, which was labeled with a dansyl residue. The tryptophan intensity decays, with and without the dansyl acceptor, were measured by the frequency-domain method. The data were analyzed by a least-squares algorithm which accounts for correlation between the parameters. A wide distribution of tryptophan to dansyl distances was found for the random-coil state, with a Gaussian half-width of 25 A. Increasing concentrations of methanol, which were shown to induce and alpha-helical conformation, resulted in a progressive decrease in the width of the distribution, reaching a limiting half-width of 3 A at 80% (v/v) methanol. The distance from the indole moiety of Trp-19 to the dansyl group in 80% (v/v) methanol/water was found to be 25 A, as assessed from the center of the distance distribution. A distance of 24-25 A was recovered from the X-ray crystal structure of the tetramer, which is largely alpha-helical. At low ionic strength (less than 0.01) the CD spectra revealed a small fraction or amount of alpha-helix for melittin in water, which implies a small fraction of residual structure. This residual structure is apparently lost in guanidine hydrochloride as demonstrated by a further broadening in the distribution of distances. These results demonstrate the usefulness of frequency-domain measurements of resonance transfer for resolution of conformational distributions of proteins.

Conformational studies of a synthetic peptide corresponding to the repeat motif of C hordein.

C hordein, a storage protein from barley grains, has an Mr of about 53,000, and consists predominantly of repeated octapeptides with a consensus sequence of Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln. Previously reported hydrodynamic and c.d. studies indicate the presence of beta-turns, the repetitive nature of which may lead to the formation of a loose spiral. In order to study these turns we have compared the structures of a synthetic peptide corresponding to the consensus repeat motif and total C hordein by using c.d. and Fourier-transform i.r. spectroscopy. The synthetic peptide exhibited spectra typical of beta I/III reverse turns when dissolved in trifluoroethanol at 22 degrees C and in water at 70 degrees C, but 'random-coil'-like spectra in water at 22 degrees C. The whole protein also showed increases in beta I/III reverse turns when dissolved in increasing concentrations of trifluoroethanol (50-100%, v/v) or heated in ethanol/water (7:3, v/v). Two cryogenic solvent systems were used to determine the c.d. spectra of the peptide and protein at temperatures down to -100 degrees C. Methanol/glycerol (9:1, v/v) and ethanediol/water (2:1, v/v) were selected as analogues of trifluoroethanol/water and water respectively. The peptide exhibited beta I/III-reverse-turn and 'random-coil'-like spectra in methanol/glycerol and ethanediol/water respectively at 22 degrees C, but a spectrum similar to that of a poly-L-proline II helix in both solvents at -100 degrees C. Similarly the proportion of this spectral type also increased when the whole protein was cooled in both solvents. These results indicate that a poly-L-proline II conformation at low temperatures is in equilibrium with a beta I/III-turn-rich conformation at higher temperatures. The latter conformation is also favoured in solvents of low dielectric constant such as trifluoroethanol. The 'random-coil'-like spectra exhibited by the protein and peptide in high-dielectric-constant solvents at room temperature may result from a mixture of the two conformations rather than from the random-coil state.

Permeability Changes of a Porous Membrane by an Adsorbed Photoresponsive Polypeptide

Photoresposive polypeptides, poly(L-glutamic acid)s containing azobenzene-4-sulfonic acid residues in the side chains (azo·S-X-PGA, X is the mol% of the azobenzene sulfonate residue), were adsorbed onto a porous support membrane and photoinduced permeation changes of the membrane obtained were studied. The maximum amount of adsorption, nmax (g g−1), of azo·S-PGA in the membrane was shown to decrease by increasing the azobenzene sulfonate groups, X, resulting from decrease in the content of the L-glutamic acid residues which interact with the adsorption sites on the pore wall through hydrogen bonds. It was estimated that the value of nmax of the azo·S-14.1-PGA adsorbed membrane used for the permeation measurements was 1.40×10−2 g of the polymer per g of the support membrane. The filtration coefficient of the azo·S-14.1-PGA adsorbed membrane largely decreased at the pH where α-helix to coil transition of the polymer occurred, indicating that permeation of the membrane was mainly determined by the conformation of the adsorbed azo·S-PGA on the pore wall. Moreover, it was observed that photoirradiation of the membrane at adequate pHS could induce rapid decrease in Lp of the membrane within a few minute. The photoresposive behavior of the membrane can be explained in terms of the photoinduced helix to coil transition of the azo·S-14.1-PGA in the membrane. The permeability of the membrane containing azo·S-46.3-PGA, which was in the random coil state at any given pH value in the aqueous solution, was independent on pH and photoirradiation.

Effect of unfolding on the tryptophanyl fluorescence lifetime distribution in apomyoglobin

Proteins exhibit, even in their native state, a large number of conformations differing in small details (substates). The fluorescence lifetime of tryptophanyl residues can reflect the microenvironmental characteristics of these subconformations. We have analyzed the lifetime distribution of the unique indole residue of tuna apomyoglobin (Trp A-12) during the unfolding induced by temperature or guanidine hydrochloride. The results show that the increase of the temperature from 10 to 30 degrees C causes a sharpening of the lifetime distribution. This is mainly due to the higher rate of interconversion among the conformational substates in the native state. A further temperature increase produces partially or fully unfolded states, resulting in a broadening of the tryptophanyl lifetime distribution. The data relative to the guanidine-induced unfolding show a sigmoidal increase of the distribution width, which is due to the transition of the protein structure from the native to the random-coiled state. The broadening of the lifetime distribution indicates that, even in the fully unfolded protein, the lifetime of the tryptophanyl residues is influenced by the protein matrix, which generates very heterogeneous microenvironments.

Isolated polymer molecule in a random environment.

The shape of a polymer molecule in the presence of randomly distributed stationary impurities is investigated using the path-integral formulation. The polymer molecule is assumed to be described by the Edwards minimal model. Calculation of the mean-square end-to-end displacement 〈${R}^{2}$〉 shows that at a critical density of the scatterers the polymer molecule makes a transition from a swollen state (chain with excluded-volume interactions) to a disordered, random coiled state (a Gaussian chain). At this density there is a crossover in the value of the exponent \ensuremath{\nu} (in three dimensions), characterizing the scaling of 〈${R}^{2}$〉, with the length of the polymer from about 0.6 (corresponding to the excluded-volume regime) to 0.5 (corresponding to the coiled state). For densities below the critical density, the exponent \ensuremath{\nu} is well described by the Flory value.

Dimensional Changes of Polypeptides in the Helix-Sense Inversion Region

Dimensional changes of polypeptides in the helix-sense inversion region are analyzed by a theory based on the three-state, nearest-neighbor interaction model. Each peptide residue in a chain is assumed to take on right- and left-handed α-helical states as well as a random coil state, and the helix stabilization effect due to hydrogen-bond interactions is taken into account. Such interactions between four consecutive residues have explicitly been discussed in a 9×9 statistical-weight matrix in a previous theory [Macromolecules, 17, 1599 (1984)], while in the present model we simplify this matrix into the form of a 3×3 matrix by considering the interactions only between two consecutive residues. Calculations of the average helix fraction and the number of helix sequences are correspondingly simplified with maintaining quantitative agreement in numerical results. The new theory is also applied to the analyses of the length distribution and the higher moments of helix. Theoretical transition curves illustrating the general characteristics of the sense inversion phenomena suggest a method for evaluating the transition parameters.

Structural studies of apolipoprotein B: physical properties of the protein in guanidine hydrochloride

Apolipoprotein B was isolated from human plasma low-density-lipoprotein without precipitation by diethyl ether/ethanol extraction of the protein in 6 M guanidine hydrochloride. The physical properties of this protein, which contained a residuum of approximately 7% phospholipid, were examined in 6 M guanidine solution under reducing conditions. The circular dichroism spectrum was indistinguishable from that of a random coil protein. Sedimentation equilibrium analyses of apolipoprotein B by the meniscus depletion method of Yphantis (1984, Biochemistry 3, 297–317) were complicated by heterogeneity and nonideality despite the low concentrations employed. 63 analyses of the weight average ( M w ) and z average ( M z ) molecular weight were made on the apolipoprotein B from 12 subjects. The M w observed was a function of initial concentration, rotor speed, and a heterogeneity index (if M z/ M w). Multiple linear regression of apolipoprotein B molecular mass against these parameters suggested that an M w of 540000 ± 110000 would be observed under apparently ideal and homogeneous conditions. The sedimentation coefficient and intrinsic viscosity of the reduced protein at 25°C in 6 M guanidine were 2.13 S and 116 ml/g, respectively; these values predict molecular weights of 640 000 and 250 000, respectively, if apolipoprotein B was fully denatured into a random coil. Lack of agreement between these estimates and with the sedimentation equilibrium analysis can best be explained by compactness of structure and incomplete denaturation to a random coil state. Furthermore, an irreversible temperature dependence of apolipoprotein B reduced viscosity indicated that residual structure remained in solutions of 6 M guanidine hydrochloride/20 mM dithiothreitol. Taken together, the physical data demonstrate that apolipoprotein is a single polypeptide of approximately 540 kDa, whose structure resists denaturation under conditions where most proteins exist as random coils.

Possible role of helix-coil transitions in the microscopic mechanism of muscle contraction

Local helix-coil transitions in the coiled coil portion of myosin have long been implicated as a possible origin of tension generation in muscle. From a statistical mechanical theory of conformational transitions in coiled coils, the free energy required to form a randomly coiled bubble in the hinge region of myosin of the type conjectured by Harrington (Harrington, W. F., 1979, Proc. Natl. Acad. Sci. USA, 76:5066-5070) is estimated to be approximately 25 kcal/mol. Unfortunately this is far more than the free energy available from ATP hydrolysis if the crossbridges operate independently. Thus, in solution such bubbles are predicted to be absent, and the theory requires that the rod portion of myosin be a hingeless, continuously deforming rod. While such bubble formation in vivo cannot be entirely ruled out, it appears to be unlikely. We further conjecture that in solution the swivel located between myosin subfragments 1 and 2 (S-2 and S-1) is due to a locally random conformation of the chains caused by the presence of a proline residue at the point that physically separates the coiled coil from the globular portion of myosin. On attachment of S-1 to actin in the strong binding state, the configurational entropy of the random coil in the swivel region is greatly reduced relative to the case where the ends are free. This produces a spontaneous coil-to-helix transition in the swivel region that causes rotation of S-1 and the translation of actin. Thus, the model predicts that the actin filaments are pushed rather than pulled past the thick filaments by the crossbridges. The specific mechanism of force generation is examined in detail, and a simple statistical mechanical realization of the model is proposed. We find that the model gives a substantial number of qualitative and at times quantitative predictions in accord with experiment, and is particularly appealing in that it provides a simple means of free energy transduction--the well known fact that topological constraints shift the equilibrium between helical and random coil states.

Inactivation and proteolysis of heat-sensitive adenylate kinase of Escherichia coli CR341 T28.

Adenylate kinase from Escherichia coli K12 (strains CR341 and CR341 T28, a temperature-sensitive mutant) was purified by a two-step chromatographic procedure. Denaturation by heat above 60 degrees C of pure or crude preparations of adenylate kinase from both strains of bacteria was shown to be "reversible" if the enzyme was converted to the random coiled state by guanidinium chloride after heat treatment. Like other small monomeric proteins, adenylate kinase refolded rapidly to the native active state by dilution of guanidinium chloride. Adenylate kinase from the mutant strain was irreversibly inactivated by exposure of crude extracts at 40 degrees C. This inactivation is due to proteolysis which follows thermal denaturation (or transconformation) of mutant adenylate kinase at 40 degrees C. ATP, P1, P5-di(adenosine 5')-pentaphosphate, and anti-adenylate kinase antibodies protected the thermosensitive adenylate kinase in crude extracts against denaturation and proteolysis at 40 degrees C.

Kerr constants of random coil polypeptides

Calculations of average molar Kerr constants of polypeptides of the twenty natural α-aminoacids in the random coil state are presented. The computation was carried out according to the Rotational Isomeric States model with the values of energies, dipole moments and optical anisotropies of the repeating units reported elsewhere. In the case of homopolypeptides, the ratios /x extrapolated to x→∞ range approximately from −6000 to +5000 in units of 10−27V−2m5mol−1. Results obtained for ten actual proteins and three enzymes in the random coil state are also reported; their values of /x are very sensitive to the kind of aminoacid residues and to the sequence of those residues.

Dipole moments of random coil polypeptides

The Rotational Isomeric States model is applied to calculate dipole moments of polypeptides of the twenty natural α-amino acids in the random coil state. Dipole moments of each repeat unit (μ i), are evaluated using a quantum mechanics procedure. Dipole moment ratios ( D x = 〈μ 2〉 xμ i 2, x = number of repeat units ) of homopolypeptides are calculated and extrapolated to x →⇔. With a few exceptions, D ⇔ = 0.36 ± 0.1. Ten actual proteins and three enzymes are also studied; their dipole ratios ( D x ′ =〈 μ〉/ x) range from 7.34 to 10.57 in 10 −59 C 2 m 2 (6.6–9.5 D 2). Diffferences in the values of D x′ are due mainly to the different contributions, μ i, of the amino acid residues contained in each polymer, whereas the sequence of amino acids has a very minor effect.

Conformations of Biopolymers

Abstract Discussion of the history of biopolymers is focused on proteins and polypeptides. Rubber elasticity is discussed from the early days onward, when the first and second laws of thermodynamics were established. Insight in the elasticity of elastin, an amorphous protein occurring in ligaments and arteries, is followed against this background. Denatured proteins also fit in this category. At present, the random-coil state that underlies the elasticity is rather well understood as a result of the new methods of analyzing the dimensions in terms of the conformations of the residues. Subsequently, the discovery of the α-helix, as well as that of the other helical structures of DNA and collagen, is described. The conversion to random coils is followed with emphasis on our insight into the cooperative nature of the transition. Finally, the least understood globular proteins are considered. Major progress was made with the successful analysis of x-ray patterns. The native state is characterized by closely p...

EXPERIMENTAL EVALUATION OF THE TRANS FRACTION IN CH<sub>2</sub>-CH<sub>2</sub> BONDS OF MELT-QUENCHED POLY (ETHYLENE TEREPHTHALATE)

In a previous paper we speculated that the molecular conformation of poly (ethylene terephthalate) (PET) quenched from the melt is not in random coil state but in frozen metastable state which is easily transformed into the stable crystalline state. This speculation should be confirmed with direct structural evidences. However, experimental methods to investigate the structure of melt-quenched PET are scarce. In this paper we have evaluated the trans fraction in CH2-CH2 bonds of PET quenched from the melt by means of infrared spectroscopy.The trans fraction was determined by two methods. The first one uses the ratio of intensities of the annealed sample and the melt-quenched one for both the trans and gauche bands (At/A°t and Ag/A°g). The trans fraction of melt-quenched PET (X°t) can be obtained from the following equation; where Ft and Fg are the ratio of extinction coefficient of the annealed sample and that of the meltquenched one for trans and gauche chain respectively. These are assumed to be unity. Scbonherr used the peak heights as the absorbance values and the 1578 cm-1 band as the internal thickness band. However, our study shows that the band widths change upon annealing and the 1578 cm-1 band is a crystallization sensitive band. In order to obtain reasonable value of the trans fraction, we used the integrated intensities as the absorbance values and the 1505 cm-1 band as the internal thickness band. Namely, a computer program was written which resolves the infrared spectar in the 760-930 cm-1 region of PET into composite vibrational bands whose envelopes are assumed to be Lorenzian. The computation was based on the principle of least squares. The integrated absorption intensity for the individual band was accurately obtained. By these procedures, we evaluated 0.2 as the value of trans fraction in melt-quenched PET.The second one is newly proposed here. If one obtains the ratio of extinction coefficient of the trans and the gauche band (et /eg ), the trans fraction (X°t) can be evaluated from the following equation; where A°t and A°g are the absorbances for the trans and gauche band in melt-quenched PET. The ratio of extinction coefficient of the trans and the gauche band was determined by using intensities of the trans and the gauche band in crystals of diol type oligomers of PET in which the CH2-CH2 bonds in both molecular ends were found to be gauche. The result was consistent with that from the improved Schonherr's method. Thus, a considerably small value of 0.2 is conclusively assigned to the trans fraction in CH2-CH2 bonds of melt-quenched PET.

On the optical activity of pseudoisocyanine bound by sulfated polysaccharides in dilute aqueous solution

Visible absorption and circular dichroism spectra of the cationic dye pseudoisocyanine bound to sulfated polysaccharides in dilute aqueous solution are reported. Characteristic, strong optical activity is associated with stacks of dye molecules bound by dextran sulfate as well as by i-carrageenan in the random coil states. Conversion of i-carrageenan chains to an ordered state (by addition of NaCl) markedly changes the dye circular dichroism spectrum. The possible configurational origins of the observed effects are pointed out.

Absorption of ultrasound in protein solutions

Absorption coefficient and propagation speeds for aqueous solutions of globular proteins were measured at pH 7 and 290°K. Concentrations were approximately 100 mg/ml. Proteins measured were lysozyme, γ‐globulin, myoglobin, hemoglobin, egg albumin, β‐lactoglobulin, bovine serum albumin, and bacitracin. Specific absorption and propagation speed did not correlate with molecular weight, fraction of α‐helix content, or n‐mer structure. In addition, fibrous muscle protein paramyosin, and its subunit, PPC‐1, were measured in α‐helical and random coil state. The conformation change was produced with 6M quanidine hydrochloride. The ratio of α‐helix state absorption to random coil state absorption was approximately two for PPC‐I and six for paramyosin. [Work supported by NIGMS.]

Spectrophotometric titration of phenolic groups of pepsin

The ionization of tyrosine residues in diazotized pepsin under various solvent conditions was studied. All tyrosyl residues of the protein titrated normally with a p K of 10.02 in 6 M guanidine hydrochloride solution. On the other hand, two stages in the phenolic group titration curve were observed for the inactivated protein in the absence of guanidine hydrochloride; only about 10 tyrosine residues ionized reversibly up to pH 11, above which titration was irreversible. The irreversible titration zone corresponds to the pH range 11–13 in which unfolding, leading to the random coil state, was shown to occur by circular dichroism and viscosity measurements. The number of tyrosine residues exposed in the native and alkali-denatured (pH 7.5) states of diazotized protein were also studied by solvent perturbation techniques; 10 and 12 groups are exposed in the native and denatured states, respectively.

Segmental motions of spin‐labelled poly(ethylene oxide) in dilute solution

AbstractThe rotational correlation times (τ) of spin‐labelled poly(ethylene oxide), (Mw=20 000) were measured in benzene, pyridine and water. It was found that τ is independent of polymer concentration (c) when c<0,15 g/(ml solvent). In dilute solutions τ values of 49 ps, 43 ps and 100 ps in benzene, pyridine and water at 293 K were obtained. A comparison with results of dielectric relaxation and nuclear magnetic resonance measurements indicated that τdiel. < τ13CNMR < τspin‐labelling < τ1H NMR. Values 17.7±2,2 kJ/mol and 17,3kJ/mol for the potential barrier of local conformational changes of PEO in pyridine and water solutions were obtained.The apparent hydrodynamic radii (0,35–0,46 nm) indicated the loosely arranged random coil state of the polymer molecules in solution at 293 K.

Tyrosine fluorescence of two tryptophan-free proteins: histones H1 and H5.

The fluorescence intensity of the single tyrosine residue in histone H1 increases from RTYR = 0.3 to RTYR = 1.3 as the protein undergoes a conformational change from the random coil state to a folded form. Enhanced fluorescence in the folded state has not been observed before in ap protein. Histone H5 shows no change in fluorescence intensity on folding. This is interpreted as a result of compensation between enhanced and reduced fluorescence in the three tyrosine residues.

Thermal properties of collagen in helical and random coiled states in the temperature range from 4° to 300°K

AbstractThe low‐temperature heat capacity of collagen (in the hydrated and dehydrated states) and the large entropy of collagen in the coiled state relative to the same protein in the helical state were investigated. The heat capacity for collagen in the solid state in the temperature range 4°–50° K changes proportionally to the square of temperature (Cp ∼ T2). Above 50°K there is a linear dependence (Cp ∼ T). The differences in the character of temperature dependence of heat capacity for the hydrated and dehydrated collagen show the importance of the specific interaction of water molecules with polypeptide chains of this protein. The peculiarities of the temperature dependence of the heat capacity difference (ΔCp) of hydrated denatured (random coiled) and hydrated native (helical) collagen are observed at 15°, 120°, and 240°K. These differences are caused by the varying degree of ordering of the hydrate water molecules in native and denatured collagen macromolecules. At all temperatures (4°–300°K) the entropy of the random coiled state is higher than that of collagen in the native state and at 298°K ΔS = ∫ (ΔCp/T)dT = 0.8 cal/100 g °K.