Globule State Research Articles

Changes in the Hydration States of Poly(N-alkylacrylamide)s during Their Phase Transitions in Water Observed by FTIR Spectroscopy

Phase transitions of poly(N-n-propylacrylamide) (PnPA), poly(N-isopropylacrylamide) (PiPA), and poly(N-cyclopropylacrylamide) (PcPA) in H2O and D2O were investigated by Fourier transform infrared (FTIR) spectroscopy. IR spectra of these solutions were measured as a function of temperature (T) both in the heating and cooling processes. Subtraction of a spectrum at starting temperature (T0) from a spectrum at T gives an IR difference spectrum (ΔAT-T0). The magnitudes of ΔAT-T0 for IR absorption bands attributable to the alkyl and amide groups of these polymers critically increased at their lower critical solution temperatures (LCSTs). Redshifts of the amide II, C−H stretching, and C−H deformation bands were observed during the coil-to-globule transitions of these polymers. The amide I‘ bands of N-deuterated amide groups (−COND−) in PnPA and PiPA measured below their LCSTs could be fitted with single Gaussian components centered at 1630 and 1625 cm-1, respectively. In contrast, two components were observed in the solutions of PnPA (1630 and 1650 cm-1) and PiPA (1625 and 1650 cm-1) above their LCSTs. The low-frequency and high-frequency components of the amide I‘ bands of PnPA and PiPA can be attributed to the carbonyl groups that form hydrogen bonds with water (CO···D−O−D, polymer−water hydrogen bond) and with the amide N−D groups on the polymers (CO···D−N, polymer−polymer hydrogen bond), respectively. The molar fractions of the CO···D−N species in the globule states were estimated to be 0.30 and 0.13 for PnPA and PiPA, respectively. Although the phase transition temperatures of PnPA solutions depend on the polymer concentration, the fractions of the CO···D−N species are independent of the concentration (5−20 wt %), suggesting that the states of globules of PnPA at different polymer concentrations are identical with respect to hydration and water contents. The change in the profiles of the amide I‘ band of PcPA during the phase transition was quite different from those of PnPA and PiPA. Although the amide I‘ band of PcPA observed below the LCST was composed of a single component that could be assigned to the CO···D−O−D species at 1634 cm-1, the IR absorptions at the higher (ca. 1667 cm-1) and lower (ca. 1600 cm-1) wavenumbers increased above the LCST. The hydrogen bonding states about the CO group of PcPA in the globule state are suggested to be different from those of PnPA and PiPA. As for effects of salts on the phase transition, although the LCSTs of the polymers linearly decreased and the heats of transition (ΔH) slightly decreased with an increase in NaCl concentration, NaCl had no influence on the profiles of the IR spectra of these polymers. Moreover, linear relationship between the parameters that represent the strength of anion−water interactions and the LCSTs of the polymer solutions containing one of the potassium halides (KF, KCl, KBr, and KI) was observed. The effects of ions on the LCSTs of the polymers can be explained by the strength of the interactions between the ions and water.

Reentrant coil-to-globule-to-coil transition of a single linear homopolymer chain in a water/methanol mixture.

Both water and methanol are good solvents for poly( N-isopropylacrylamide) (PNIPAM) at room temperature, but strangely not a mixture of them. Using narrowly distributed linear PNIPAM chains (M(w) = 2.6x10(7) g/mol and M(w)/M(n)<1.1), we have, for the first time, observed a coil-to-globule-to-coil transition of a single PNIPAM chain when methanol was gradually added into water. This novel reentrant transition leads to insight into the complexation between methanol and water. We also found that the chain was crumpled in its globule state and the globule still contained approximately 80% of solvent in its hydrodynamic volume.

Change in Hydration State during the Coil−Globule Transition of Aqueous Solutions of Poly(N-isopropylacrylamide) as Evidenced by FTIR Spectroscopy

Coil−globule transition of poly(N-isopropylacrylamide) (PIPA), followed by intermolecular association in H2O and D2O, was investigated by Fourier transform infrared (FTIR) spectroscopy. IR spectra of the solutions were measured as a function of temperature, and spectral changes induced by the transition were observed. The intensities of the difference IR bands due to the vibrational modes of isopropyl and amide groups critically increased at the lower critical solution temperature (LCST). Heating of the PIPA solutions above the LCST led shifts of the amide II, C−H-stretching, and C−H-bending bands to lower wavenumbers during a shift of the amide I band to a higher wavenumber. The amide I band of the PIPA observed below the LCST could be fitted with a single component centered at 1625 cm-1, whereas two components (1625 and 1650 cm-1) were necessary to fit the band above the LCST. These components may be assigned to the CO group which is bound to water molecules as the solvent (1625 cm-1) and to the N−H in the side chain (1650 cm-1) via hydrogen bonding. About 13% of the CO group is estimated to form the intra- or interchain hydrogen bonding, and the remaining CO group forms a hydrogen bond with water in the globule state. Red shifts of the antisymmetric and symmetric C−H-stretching bands for the isopropyl group also indicate dehydration of the hydrophobic moiety during the transition. A mechanistic hypothesis of the coil−globule transition, insisting that above the LCST the polymer chain is dehydrated and hydrophobic interaction between isopropyl groups induces the collapse of the chain, is strongly supported. Though the presence of metal halides (NaCl, KCl, KBr, KI) lowered the LCST, the profiles of IR difference spectra of the PIPA in those solutions were similar to those measured in pure water. Specific interactions between the amide groups on the polymer chain and the ions were unlikely. Importance of the structure and properties of water in the solution to determine the LCST of the polymer solutions is suggested.

Copolymers of Poly(N-isopropylacrylamide) Densely Grafted with Poly(ethylene oxide) as High-Performance Separation Matrix of DNA

Two high molecular weight copolymers of poly(N-isopropylacrylamide) (PNIPAM) densely grafted with short poly(ethylene oxide) (PEO) chains (PNIPAM-g-PEO) were studied by NMR and laser light scattering. The long PNIPAM chains with densely grafted PEO branches had a random coil conformation at very dilute concentrations and low temperatures (i.e., T ≤ 30 °C), When the temperature was increased above 31 °C, the copolymers could undergo a broad coil-to-globule transition. The collapsed copolymer chains had a / value of about 1.0 with PNIPAM chains inside the core and the hydrophilic PEO chains on the surface. This kind of PNIPAM-g-PEO copolymers was studied as a DNA separation medium in capillary electrophoresis. Several advantages of the copolymers as a separation medium for DNA fragments were achieved, such as an automatic coating ability for the capillary inner wall, an easier injection into the capillary channel due to the slightly adjustable viscosity with temperature (up to 31 °C), a high resolution (i.e., one base pair resolution), and fast separation time. In contrast, the homo-PNIPAM or PEO showed worse DNA separation efficiency under similar conditions. The high DNA separation efficiency of the PNIPAM-g-PEO copolymers is related to the polymer chain conformation. The long copolymer chains in a random coil conformation with densely grafted PEO branches could form a physical network with a relatively stable and uniform pore size at high concentrations (i.e., ≥10 wt %). The separation medium has a high sieving ability for DNA separation in terms of DNA migration mechanisms. The collapsed copolymer chains in the globule state could destroy the chain network and thus lose the DNA separation ability.

Hydrophobic photolabeling as a new method for structural characterization of molten globule and related protein folding intermediates.

Recent advances in attempts to unravel the protein folding mechanism have indicated the need to identify the folding intermediates. Despite their transient nature, in a number of cases it has been possible to detect and characterize some of the equilibrium intermediates, for example, the molten globule (MG) state. The key features of the MG state are retention of substantial secondary structure of the native state, considerable loss of tertiary structure leading to increased hydrophobic exposure, and a compact structure. NMR, circular dichroism, and fluorescence spectroscopies have been most useful in characterizing such intermediates. We report here a new method for structural characterization of the MG state that involves probing the exposed hydrophobic sites with a hydrophobic photoactivable reagent--2[3H]diazofluorene. This carbene-based reagent binds to hydrophobic sites, and on photolysis covalently attaches itself to the neighboring amino acid side chains. The reagent photolabels alpha-lactalbumin as a function of pH (3-7.4), the labeling at neutral pH being negligible and maximal at pH 3. Chemical and proteolytic fragmentation of the photolabeled protein followed by peptide sequencing permitted identification of the labeled residues. The results obtained indicate that the sequence corresponding to B (23-34) and C (86-98) helix of the native structure are extensively labeled. The small beta-domain (40-50) is poorly labeled, Val42 being the only residue that is significantly labeled. Our data, like NMR data, indicate that in the MG state of alpha-lactalbumin, the alpha-domain has a greater degree of persistent structure than the beta-domain. However, unlike the NMR method, the photolabeling method is not limited by the size of the protein and can provide information on several new residues, for example, Leu115. The current method using DAF thus allows identification of stable and hydrophobic exposed regions in folding intermediates as the reagent binds and on photolysis covalently links to these regions.

Globule-to-Coil Transition of a Single Homopolymer Chain in Solution

Using a nearly monodisperse high molar mass poly( $N$-isopropylacrylamide) (PNIPAM) sample, we successfully made the conformation change of individual PNIPAM chains from a coil to a fully collapsed stable single chain globule in an extremely dilute aqueous solution, which enabled us to study for the first time the globule-to-coil transition of a single homopolymer chain in solution. A comparison to the coil-to-globule and the globule-to-coil transitions revealed a hysteresis in the globule-to-coil transition. Our results also confirmed the existence of two additional thermodynamically stable states between the coil and the globule states, namely, the crumpled coil and the molten globule.

Comparison of the Coil-to-Globule and the Globule-to-Coil Transitions of a Single Poly(N-isopropylacrylamide) Homopolymer Chain in Water

Using a newly prepared nearly monodisperse (Mw/Mn < 1.05) high molar mass (Mw = 1.3 107 g/mol) poly(N-isopropylacrylamide) (PNIPAM) sample, we successfully, for the first time, made the conformation of individual PNIPAM chains change from a coil to a fully collapsed thermodynamically stable single chain globule and then back to a coil in an extremely dilute aqueous solution (6.7 10-7 g/mL). The average chain density in the globule state is 0.34 g/mL, close to 0.40 g/cm3 predicted on the basis of a space-filling model, indicating that the globule still contains 66% water even in its fully collapsed state. At a given temperature around the lower critical solution temperature, the chains are smaller in the globule-to-coil transition than in the coil-to-globule transition, revealing that the coil-to-globule transition is an irreversible process. The hysteresis can be attributed to the formation of intrachain structures, presumably the intrachain hydrogen bonding, in the globule state. We confirmed the existen...

Dynamics of polyglutamic acids in α-helical and coil states. Comparison with dynamics of some globular proteins. Rayleigh scattering of Mössbauer radiation (RSMR) data

The classical model system poly-L-glutamic acid (poly-Glu), was investigated in a disordered coil state (at pH=7.0) and in helix state (at pH=2.0) by the RSMR technique. By considering that the coil state of poly-Glu models unfolded (random coil) state and α-helix state models the fluctuating secondary structure (during consequent folding of protein), a comparative analysis of the dynamical properties of poly-Glu in different states with the dynamical properties of different proteins in the native state (α-helical myoglobin and HSA, partially β-sheet lysozyme) and in intermediate (molten globule) state (α-lactalbumin) was performed. This comparison brings some unpredicted results: native α-helical proteins behave close to random coil, native partially β-sheet proteins behave close to fluctuating secondary structure (α-helix) and the dynamic behaviour of molten-globule state (partially β-sheet α-lactalbumin) is not different from the behaviour of lysozyme and much more rigid than that of native α-helical proteins.

Transportation of DNA Molecule by Dielectrophoretic Force in Micro Flow Channel. Utilization of Conformational Transition in the Higher Order Structure of DNA.

In this paper, we propose a new methodology on noncontact transportation of DNA molecules by dielectrophoretic force together with the Micro DNA Flow System. We utilize the conformational transition in the higher oder structure of DNA for transportation. Such systems can be constructed by using Microfabrication technology. At first, we designed a simple micro electrode-flow system and carried out experiments. Experimental demonstration of DNA transportation in the globule state using dielectrophoretic force and direct observation of the DNA molecule in a non-uniform electric field were carried out with fluorescence microscopy. Motion of the DNA molecule is modelled in terms of the spatial gradient in the electric field depending on the electrode design. We discuss the experimental result on the motion of the DNA molecule compared with theoretical prediction. We show that transportation of DNA with the state of compacted globule is profitable in the future practical application for the separation of giant DNAs such as human gene.

Discrete phase transition of giant DNA dynamics of globule formation from a single molecular chain

We found that a single chain of double strand DNA molecule undergoes a first-order phase transition by the direct observation of the conformation in individual DNA with fluorescence microscopy. It has become clear that a single DNA chain changes its higher order structure between expanded coil and shrunken globule in a discrete manner. The effective volume occupied by single DNA chain in the coil state is ca. 10 4 times greater than that in the globule state. The dynamic process of the globule formation, or nucleation and growth, is analyzed by measuring individual DNA molecules with fluorescence microscopy. We show that the apparent contour length of the DNA coil decreases with a constant speed, accompanied by the growth of the globular structure.

Kinematics and thermodynamics of a folding heteropolymer.

In order to elucidate the folding dynamics of protein, we have carried out numerical simulations of a heteropolymer model of self-interacting random chains. We find that folding propensity depends strongly on sequence and that both folding and nonfolding sequences exist. Furthermore we show that folding is a two-step process: the transition from coil state to unique folded state takes place through a globule phase. In addition to the continuous coil-globule transition, there exists an abrupt transition that separates the unique folded state from the globule state and ensures the stability of the native state.

Energy level structure of two-dimensional self-interacting random chains

The authors study the energy level structure of the lowest-lying states for a two-dimensional version of the random chain model proposed by Iori, Marinari and Parisi (1991). The authors find that the multifold degeneracy of the ground state is lifted as the random noise interaction is switched on and a solitary global minimum emerges in a glassy phase as the strength of the noise term is increased. This indicates that the model mimics the characteristics expected of a native protein. They also show that the transition from the globule state (with no noise term) to the glassy phase takes place smoothly.