Effect of counterion concentration on the dielectric behavior of a polypeptidic chain in the helix–coil transition

On the basis of the two-state model of a polyelectrolyte solution, the ion concentration in the polymer domain has been calculated by using the spherical Poisson–Boltzmann equation. The ion accumulation in the neighboring of the polyion influences, on different time scales, various electrical properties of the solution, in particular the low-frequency electrical conductivity and the high-frequency dielectric dispersion. These predictions have been compared with recent dielectric measurements on poly (L -glutamic acid) aqueous solutions during the conformational transition from the α-helix to random coil, and a satisfactory agreement has been found. This finding suggests that counterion distribution plays a different role in determining the electrical properties of charged polymer solutions, causing a electrophoretic contribution of the polymer domain to the electrical conductivity and influencing the high-frequency dielectric dispersion. © 1993 John Wiley & Sons, Inc.     

Conformation and stability of alpha-helical membrane proteins. 1. Influence of salts on conformational equilibria between active and Inactive states of rhodopsin

We studied the influence of salts on the pH-dependent conformational equilibria between the active and the inactive photoproduct states of rhodopsin, Meta II and Meta I, respectively, and between the active and inactive conformations of the apoprotein opsin. In both equilibria, the active species is favored in the presence of medium to high concentration of salt. The ion selectivity for the Meta I/Meta II equilibrium is particularly pronounced for the anions and follows the series trichloroacetate > thiocyanate > iodide > bromide > sulfate > chloride > acetate. The Hill coefficient of this salt-induced transition is close to 2.0. Both ion selectivity and Hill coefficient suggest that the transition is mainly regulated by ion binding to two specific charged binding sites in the protein with smaller contributions being due to the Hofmeister effect. We propose that these putative ion binding sites are identical to those sites that are titrated in the corresponding pH-dependent conformational transition. They presumably function as ionic locks, which keep the receptor in an inactive conformation, and which may be disrupted either by pH-dependent protonation or by salt-dependent ion binding.     

Raman spectroscopic studies on the interaction between counterion and polyion

The interaction between alkali metal ions and the polyacrylate ion was investigated by means of Raman spectroscopy. in comparison with the Raman spectra of propionate salts. The Raman bands due to the metal-oxygen bond were not apparent and no significant difference was observed among the Raman spectra of several univalent salts of polyacrylate. except in the case of the lithium salt. The apparent degree of dissociation of lithium polyacrylate, as determined from the relative intensity of a specific band characteristic of the carboxylate ion, was lower than those of the other alkali metal salts. It is concluded from the Raman data that the electrostatic interaction between counterions and a polyion is not specifically modified by forces of a nonionic nature. Moreover, it is pointed out that the local conformation of polyacrylate changes gradually with the degree of neutralization, but that the change is not like a conformational transition between globular and random coil forms.     

Conformational studies of alternating copoly(Leu-Lys) and copoly(Leu-Orn) in alcohol-water solvent mixtures

Conformational transitions of alternating copoly(l-leucyl-l-lysine) and copoly(l-leucyl-l-ornithine) in organic solvents and in alcohol-water mixtures were determined by c.d. measurements and the results compared with those from random copoly(Leu^48.3, Lys^51.7). As reported previously^16,17, in salt-free water these alternating copolymers undergo a conformational transition from a disordered to β-structure when the pH is raised or when various salts are added, whereas random copolymers adopt an α-helix conformation under similar conditions. However, both alternating copolymers reveal a tendency to form α-helix in 2,2,2-trifluoroethanol and in alcohol-water mixtures at neutral pH, as does the random copolymer. The alcohol concentration at which the α-helix can be induced is dependent on the kind of alcohol, the α-helix promoting power follows the the series: 2,2,2-trifluoroethanol > isopropanol > ethanol > methanol. In addition, these alternating copolymers in methanol-water mixtures below 50% (by volume) methanol form the β-structure when the pH is raised. On the other hand, above 60% methanol the fraction of α-helix already formed at neutral pH is enhanced at higher pH-values.     

The effect of neutral salts on the conformational transition of poly-alpha-amino acids. I. Potentiometric titration and optical rotatory dispersion studies

The effect of salts on the coil-to-helix transition of poly-α-amino acids was investigated by optical rotatory dispersion and potentiometric titration techniques. Both charge-dependent and charge-independent contributions to the free energy were considered. The free energy of formation ΔF° of the uncharged α-helix from the uncharged random coil for poly-L -glutamic acid (PGA) decreases very rapidly in the limit of zero added salt concentration. This effect probably depends on the uncertainty affecting the choice of the extrapolation of the apparent pK for the random coil at low ionic strength. Above 0.1 M salt, where the free energy determination becomes meaningful, the anions and cations investigated do not affect the value of ΔF°, with the exception of Li⁺. Our data support the point of view that this cation binds to the peptide group. A class of salts produces an increase of the helical content of poly-L -ornithine (PO) both at low and high degree of ionization. This effect appears to be anion dependent. It is shown that (1) no change of ΔF° is involved; (2) recent theories of polyelectrolyte solutions cannot account for our results. We suggest that a true site binding of the anions to the charged amino groups occurs. The role of electrostatic binding in determining the conformational stability of proteins in the presence of some anions is stressed, and a general treatment for the electrostatic binding equilibria is outlined.     

Carbon-13 and proton NMR studies of helix-coil transition of poly(gamma-benzyl-L-glutamate).

The helix–coil conformational transition undergone by poly(γ-benzyl-L -glutamate) in solutions of trifluoroacetic acid and deuterated chloroform was studied by proton and carbon-13 nmr. The results indicate that in the case of the solvent-induced helix–coil transition, the side chain assumes a helical conformation before the backbone. In the thermally induced helix–coil transition, the results indicate the existence of an intermediate state, which is between the α-helix and random coil and is free from intramolecular hydrogen bonding.     

Ultrasonic study of the kinetics of counterion site binding in aqueous solutions of polyelectrolytes

The excess ultrasonic absorption due to counterion binding has been studied as a function of frequency for a series of polysalts in the range 1–150 MHz. All the relaxation spectra can be represented by a relaxation equation with two relaxation terms. The relaxation frequencies appear concentration independent and the relaxation amplitudes seem proportional to concentration. The low frequency relaxation process appears to depend mainly on the nature of the counterion while the high frequency relaxation process seems to be mostly dependent on the nature of the polyion. These results are quite similar to those obtained in ultrasonic studies of ion-pairing in solutions of divalent sulfates. The kinetic model used for the quantitative analysis of these results has been modified for polysalts through introducing the concept of“counterion condensation”. In this modified model the excess absorption is assigned to the perturbation by the ultrasonic waves of the equilibria between the three states of hydration of ths complex formed by a counterion and that part of the polyion where it is bound. Analytical expressions of the relaxation amplitudes have been derived using classical procedures for this modified kinetic model. In the case of cobalt-polyphosphate (Co-PP), the ultrasonic data together with the results of NMR measurements on either Co²⁺ or Co-PP have been used for the evaluation of the volume changes, the rate constants and the fractions of counterions in the three states of hydration involved in the binding equilibria. The volume changes obtained in this manner depend only slightly on the method of calculation and appear to be consistent with volume changes for outer-sphere and inner-sphere complex formation. These results are discussed.     

Intramolecular conformational transitions "random coil-helix-folded structure" in polypeptides.

A general theory has been developed for conformational intramolecular transitions in a single macromolecule with a high degree of polymerization (an infinite length model) capable of forming two types of ordered structures: the α-helix and the folded β-structure, as well as acquiring the random coil conformation. The phase diagram analysis of this system has shown that the regular β-structure state is separated from all other states of the chain by the phase boundary line. Any intersection of the phase boundary is a phase transition which can be either of the first order or second order, depending on values of the energy parameters of the system. Mechanisms of intramolecular rearrangements: β-structure–random coil and α-helix–β-structure have been discussed. It has been shown that there exist two different mechanisms for each of these rearrangements, and the regions of parameter variation corresponding to each mechanism have been specified.     

Flow-induced conformational changes and phase behavior of aqueous poly-L-lysine solutions

Poly-L -lysine exists as an α-helix at high pH and a random coil at neutral pH. When the α-helix is heated above 27°C, the macromolecule undergoes a conformational transition to a β-sheet. In this study, the stability of the secondary structure of poly-L -lysine in solutions subjected to shear flow, at temperatures below the α-helix to β-sheet transition temperature, were examined using Raman spectroscopy and CD. Solutions initially in the α-helical state showed time-dependent increases in viscosity with shearing, rising as much as an order of magnitude. Visual observation and turbidity measurements showed the formation of a gel-like phase under flow. Laser Raman measurements demonstrated the presence of small amounts of β-sheet structure evidenced by the amide I band at 1666 cm⁻¹. CD measurements indicated that solutions of predominantly α-helical conformation at 20°C transformed into 85% α-helix and 15% β-sheet after being sheared for 20 min. However, on continued shearing the content of β-sheet conformation decreased. The observed phenomena were explained in terms of a “zipping-up” molecular model based on flow enhanced hydrophobic interactions similar to that observed in gel-forming flexible polymers. © 1998 John Wiley & Sons, Inc. Biopoly 45: 239–246, 1998     

Photoresponsive polypeptides: Photochromism and conformation of poly (L-glutamic acid) containing spiropyran units

Spirobenzopyran units were bound to the side chains of poly (L -glutamic acid) and partially methylated poly(L -glutamate)s. The modified polymers were found to exhibit “reverse photochromism” in hexafluoro-2-propanol (HFP), so the samples kept in the dark were characterized by an intense absorption band in the visible range of the spectrum, which was completely erased upon exposure to sunlight or irradiation at 500–550 nm. The CD spectra showed that the macromolecules adopted a random coil conformation in the dark, whereas the bleached solutions after exposure to light displayed the typical CD pattern of the α-helix. The back reaction in the dark was accompanied by the progressive decrease of the helix content and recovery of the original disordered conformation. The photoinduced conformational changes resulted in large and reversible viscosity variations. When spiropyran side chains were converted to “spiropyran salts” of trifluoroacetic acid, the system was still photochromic, but the macromolecules were disordered both in the dark and light conditions. However, when appropriate amounts of methanol were added as a cosolvent to the HFP solutions, the system responded to light, giving reversible variations of the α-helix content. Irradiation at appropriate solvent compositions allowed modulation of the extent of the photoresponse. © 1993 John Wiley & Sons, Inc.     

Conformation of poly-L-tyrosine in aqueous solution

The conformational transition of poly-L -tyrosine in 0.1M KCl was investigated by ORD and infrared spectroscopy, potentiometric titration, and sedimentation velocity experiments. It is shown that the fully ordered conformer is obtained by slow titration of the random coil with 0.1N HCl at 25°C. The charge-induced transition, at variance with other poly-α-amino acids, is completed in a narrow range of α. An aggregation process was detected both by potentiometric titration and sedimentation velocity. The polyamino acid aggregates around α = 0.7 at 25°C when the conformational transition is almost complete. Infrared spectra, in the region of the amide I band (1650 cm^?1) showed that the transition is a random coil → antiparallel β one. Evidence exists that the form is of the intramolecular type. The foregoing interpretations of ORD and CD spectra in terms of the α-helix conformation are discussed.     

Circular dichroism studies of the fatty acid synthetase complex from the insect Ceratitis capitata

Circular dichroism spectra of the native fatty acid synthetase complex from the insect Ceratitis capitata and of the lipidated and cholate- and SDS-treated enzyme have been obtained. Native enzyme has a calculated structure of 43% α-helix, 23% β structure and 31% random coil. Lipidation and cholate-treatment did not modify the structure of the enzyme complex whereas the SDS-treatment changed the native conformation into a structure based on 42.8% α-helix, 8.4% β structure and 48.8% random coil. These data are interpreted in terms of both the enzyme activity and the quaternary structure of the complex.     

Vibrational circular dichroism spectra of three conformationally distinct states and an unordered state of poly(L-lysine) in deuterated aqueous solution

Fourier transform ir vibrational circular dichroism (VCD) spectra in the amide I′ region of poly(L-lysine) in D₂O solutions have confirmed the existence of three distinct conformational states and an unordered conformational state in this homopolypeptide. Characteristic VCD spectra are presented for the right-handed α-helix, the antiparallel β-sheet, an extended helix conformation previously referred to as the so-called “random coil,” and a completely unordered conformation characterized by the absence of any amide I′ VCD. VCD for the antiparallel β-sheet in solution and the unordered chain conformation are presented for the first time. Each of the four different VCD spectra is unique in appearance and lends weight to the view that VCD has the potential to become a sensitive new probe of the secondary structure of proteins in solution.     

Correlation between computed conformational properties of cytochrome c peptides and their antigenicity in a T-lymphocyte proliferation assay

By means of conformational energy calculations, we previously showed that the antigenic strength of a series of oligopeptides (derived from the carboxyl terminal sequence of cytochrome c) in a T-lymphocyte proliferation assay depends on their ability to adopt the α-helix conformation. Using experimentally determined statistical weights (within the framework of the Zimm–Bragg theory for the helix–coil transition), here we present a simple free energy analysis of the ability of these peptides to adopt the α-helix conformation in water. The experimental statistical weights have been modified to include the effect of long-range charge–dipole interactions on helix stability. We find that there is a close correlation between the tendency of a peptide to adopt the α-helix conformation and its ability to stimulate antigen-primed T cells. The shortest peptide with a tendency to adopt the α-helix conformation is also the shortest one that exhibits antigenic activity. The rapid and simple method presented here can thus be used to predict relative antigenicities for different peptides derived from cytochrome c.     

Conformational studies of poly(L-tyrosine). A helix–coil transition in dimethyl sulfoxide–dichloroacetic acid mixtures

Poly(L -tyrosine) is a random coil in dimethyl sulfoxide. Upon addition of dichloroacetic acid, poly(L -tyrosine) undergoes a conformational transition centered at about 10% dichloroacetic acid. The transition is nearly complete at 20% dichloroacetic acid. Further addition of dichloroacetic acid leads to precipitation of poly(L -tyrosine). We have characterized this transition by optical rotation, viscosity, circular dichroism, and infrared. The optical rotation at 350 nm and the intrinsic viscosity increase sharply to values that are consistent with a transition to the α-helix conformation. The circular dichroism of poly(L -tyrosine) in dimethyl sulfoxide and in dimethyl sulfoxide/dichloroacetic acid (80:20 v/v) agrees with previous reports for random-coil and α-helix conformations, respectively. The infrared spectrum of poly(L -tyrosine) in dimethyl sulfoxide/dichloroacetic acid (80:20 v/v) shows no evidence of β-structure. We conclude that the transition on going from dimethyl sulfoxide to 20% dichloroacetic acid in dimethyl sulfoxide is a coil → α-helix transition. The amide-I band of poly(L -tyrosine) in dimethyl sulfoxide/dichloroacetic acid (80:20) is found to be at 1662 cm^?1. It has been suggested that this high frequency may be indicative of a left-handed α-helix. However, this high amide-I frequency is consistent with conformational energy calculations of Scheraga and co-workers. The mechanism of the dichloroacetic acid-induced transition to an α-helix is discussed. Dichloroacetic acid and dimethyl sulfoxide interact strongly and the transition presumably involves a marked decrease in the ability of dimethyl sulfoxide to solvate the peptide backbone and aromatic side chains upon complex formation with dichloroacetic acid.     

Anions stabilize a metarhodopsin II-like photoproduct with a protonated Schiff base.

In rhodopsin, the retinal chromophore is covalently bound to the apoprotein by a protonated Schiff base, which is stabilized by the negatively charged counterion Glu113, conferring upon it a pK(a) of presumably >16. Upon photoexcitation and conformational relaxation of the initial photoproducts, the Schiff base proton neutralizes the counterion, a step that is considered a prerequisite for formation of the active state of the receptor, metarhodopsin II (MII). We show that the pK(a) of the Schiff base drops below 2.5 in MII. In the presence of solute anions, however, it may be increased considerably, thereby leading to the formation of a MII photoproduct with a protonated Schiff base (PSB) absorbing at 480 nm. This PSB is not stabilized by Glu113, which is shown to be neutral, but by stoichiometric binding of an anion near the Schiff base. Protonation of the Schiff base in MII changes neither coupling to G protein, as assessed by binding to a transducin-derived peptide, nor the conformation of the protein, as judged by FTIR and UV spectroscopy. A PSB and an active state conformation are therefore compatible, as suggested previously by mutants of rhodopsin. The anion specificity of the stabilization of the PSB follows the series thiocyanate > iodide > nitrate > bromide > chloride > sulfate in order of increasing efficiency. This specificity correlates inversely with the strength of hydration of the respective anion species in solution and seems therefore to be determined mainly by its partitioning into the considerably less polar protein interior.     

Helix conformation of a small peptide melittin in a methanol-water mixed solvent studied by NMR

Temperature dependence of the α-helix conformation of bee venom melittin in methanol-water mixed solvents has been examined by NMR, in order to elucidate conformation stability and a phase diagram. At high methanol concentration of 100 - ca. 80 wt.%, melittin forms a full α-helix conformation in the temperature range from 25 °C to 60 °C. At intermediate methanol concentration of ca. 80 - ca. 25 wt.%, it undergoes a thermal transformation from a full α-helix to a partial α-helix. In solutions of low methanol concentrations of ca. 25 - 0 wt.%, partial α-helix monomers and their self-aggregated conformers coexist at low temperatures, and the relative number of the monomers increases with increase in temperature. The monomers turn to a random coil state at high temperatures only below ca. 10 wt. % methanol concentrations. The thermal transitions are discussed from the viewpoint of stability of intra-molecular hydrogen bonds and inter-molecular hydrophobic interactions.     

Acetonitrile-induced conformational transitions in poly-l-lysine

The effect of acetonitrile on the random coil, α-helix and β-sheet conformations induced in poly-

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-lysine is studied. It is found that acetonitrile at higher concentrations transforms the backbone of polylysine from a random coil to a helical conformation. Addition of acetonitrile to polylysine (pH 11.5) in the α-helix conformation, induces conformational changes in two stages. At concentrations below 60% v/v, acetonitrile stabilizes the helical conformation and at higher concentrations (>70% v/v), it destabilizes the helix. β-sheet→α-helix→random coil conformational transitions are found to occur when polylysine in the heat-induced conformation is titrated with acetonitrile. The possible mechanism(s) of action of acetonitrile in inducing these structural transitions is discussed.     

Chiral switch of enzymatic ketone reduction by addition of γ-cyclodextrin

We report a chiral switch in the configuration of 1-(p-methoxyphenyl)-propan-2-ol, synthesized in aqueous media by ketoreductase in the presence of high concentration of γ-CD. NMR, ECD and fluorescence spectrometry were used in the effort to explain this unexpected effect. A comparison has been made between the catalytic activity of the enzyme and α-helix content in the conformation and it has been observed that enzyme is most active at the maximum α-helix content. The β-sheet content and random coil conformation in the enzyme were found to be dependent on cyclodextrin concentration.     

Salt‐promoted protein folding,preferential binding,or electrostatic screening?

The extended coil/molten globule conformational equilibrium exhibited by ferricytochrome c in 10 to 20 mM HCl was examined using free boundary capillary electrophoresis. Addition of the osmolyte glucitol, also called sorbitol, to shift the conformational equilibrium toward the molten globule markedly diminished the mobility of the protein. This diminution can be entirely assigned to the relative viscosity of the added glucitol. The insensitivity of the viscosity corrected protein mobility to added glucitol suggests that both the extended coil and molten globule conformations of cytochrome c are free draining in an electrophoresis measurement. Addition of a neutral salt to shift the conformational equilibrium toward the molten globule conformation also markedly diminished the mobility of the protein. This diminution can be entirely assigned to the electrostatic screening afforded by the added salt. The onset of the conformational transition observed by optical measurements and the onset of electrostatic screening observed by mobility measurements appear to be in common for some but not all neutral salts. The exception suggests that preferential binding of the anion of a neutral salt to the molten globule conformation and not electrostatic screening is principally responsible for the shift in the conformational equilibrium of cytochrome c in acidic solutions.