COIL-TO-GLOBULE TRANSITION OF POLY-NIPAAM DOPED WITH AMINO-ACIDIC COMONOMERS AND OF DISORDERED POLYPEPTIDE CHAINS

 

Polymer chains in diluted solutions can experience different degree of expansion, from compact globules to swollen coils, depending on the relative strength of the intra-chain and chain-solvent interactions (see ref. 1 and 2). For some polymers in specific solvents these interactions almost balance and the chain can undergo a coil-to-globule transition varying the temperature. Typically, the transition from coil to globule occurs upon decreasing the temperature in non-aqueous solvents, as in the case of polystyrene in cyclohexane. Noticeably, polymers of N-IsoPropylAcrylAmide (p-NIPAAm) undergo a rather sharp transition upon increasing the temperature in water solvent. The unusual and pronounced thermosensitive behavior in water of p-NIPAAm has motivated several studies aimed at understanding the basic physics of coil-globule transition. The swollen-to-compact transition of p-NIPAAm is typically combined with strong inter-chain association and phase separation and can be attributed to increased chain hydrophobicity at higher temperatures.

coilglobule.png

Schematics of the pronounced decrease of the size of p-NIPAAm upon increasing the temperature.

In previous works we have investigated the temperature behavior of p-NIPAAm copolymerized with a 1/10 fraction of amino-acidic residues, valine- (MAVal) or leucine-derived (MALeu) groups randomly positioned along the chains. Copolymerization with MALeu (pNMAL) and MAVal (pNMAV) adds to pNIPAAm a double character, since these monomers are at the same time hydrophobic and become charged upon dissociation of their carboxylic group. We have estimated (ref. 7) the average degree of dissociation of carboxy groups in pNMAV and pNMAL units at pH 4 to be 50%.

monomers.png

 

By combined light scattering and circular dichroism measurements (CD), we have investigated the temperature behavior of these variants of p-NIPAAm and found that for valine-derived copolymers, the coil-globule transition is basically unmodified with respect to p-NIPAAm, whereas doping with leucine-derived groups significantly lowers the transition temperature and makes the transition discontinuous.

The scattered intensity (at 90˚) and its time-correlation function enable quantifying the coilization. As the temperature raises, the hydrodynamic radius, RH, decreases, indicating the collapse of the individual chains. The growth of the scattered light intensity, IS, normalized to IS,20, the intensity scattered at 20 °C, indicates the aggregation of multiple chains in the same globule.

lightscattering.png

Hydrodynamic radius (RH) and scattered intensity (IS) measured by laser light scattering experiments on p-NIPAAm variants. Since RH is always shorter than the wavelength, IS is sensitive to the mass of the scatterers but not to their specific structure (Rayleigh regime). Hence, the growth of IS relative to the scattered intensity at 20° C reflects mutual chain aggregation. Assuming that at the lowest temperatures the chains are insulated, the ratio IS/IS,20 provides an estimate for the mean number of chains coagulated in each cluster.

By combining the hydrodynamic radius and the aggregation number extracted from light scattering measurements, we extract the local chain density, r(T), which represents a property intrinsic to the polymer under study. We find that for both pNMAV and pNMAL, r(T) ranges from ~0.0004 g/cm3, when random coils, to 0.1 g/cm3, when globulized. The smaller density of pNMAV and pNMAL with respect to pNIPAAm (ref. 6) in both coil and globule form is due to the electric charges of the doping group, which make the polymer chain stiffer and self-repulsive.

density.png

 

Copolimerization with MALeu increases the average chain hydrophobicity (as indicated by the decrease of the transition temperature) and also introduces a significant variance of the hydrophobicity along the chain. In lattice models of hetero-polymers with random hydrophilic-hydrophobic charges interacting with the solvent (Ref. 8), the transition line between the swollen and the collapsed phase is found to become first-order for high values of the variance of the hydrophilicity along the chain. Accordingly, we surmise that the globulization of pNMAL is promoted by micelle-like intra-chain clusters of leucine-derived groups, interspersed with the (more) soluble NIPAAm sequences. The formation of such crumpled coils as an intermediate state in the globularization of leucine-doped p-NIPAAm could explain the discontinuous character of the transition.

The understanding of the basic parameters governing the onset of a sharper coil-to-globule transition of the amino-acidic variants of p-NIPAAm has clear implications in the fields of material science and drug delivery. Moreover, it can also provide important indications on the mechanism at the base of the collapse and structuring of arguably the most relevant class of hetero-polymers: proteins.

Proteins are highly heterogeneous polymers, which fold into a unique compact structure determined by their amino-acid sequence. In the last decade, novel time-resolved spectroscopy techniques have provided compelling evidences that the folded state of a simple protein is typically in thermodynamic equilibrium with a rather collapsed, globule-like, disordered state (Ref. 9). Despite the tremendous importance of proteins, not only a consolidated theory for the mechanism of folding is still missing, but also there are few hints about the effect of sequence on the globularization tendency of the chain (Ref. 10). In this vein, recent results obtained by the proposing group have shown that the statistical conformations of disordered polypeptides can be accurately described by combining different spectroscopy approaches, hence enabling the study of the polymer behavior of sequences with different amino-acid composition.

Based on the preliminary results here described, the proposed project aims to study the behavior of p-NIPAAm copolymerized with small fractions of amino-acidic groups, whose charge depends on pH. The coil-globule transition of the polymers will be characterized mostly by combined static and dynamic light scattering measurements and circular dichroism. Moreover, polypeptide chains will be designed, synthesized and tested with novel time-resolved spectroscopy approaches in order to understand the role of the sequence on the chain collapse in water and other mixed solvents.

 

Suggested reading:

1. Flory, P. J., J. Chem. Phys., 17, 303 (1949)

2. Sanchez, I., Macromolecules, 12, 980 (1979)

3. Lebon F. et al., J. Phys. Chem. B, 111, 2372 (2007)

4. Lebon F. et al., Chirality, 15, 251 (2003)

5. Penco M. et al., Macromol. Chem. Phys., 202, 1150 (2001)

6. Fujishige S. et al., J. Phys. Chem., 93, 3311 (1989)

7. Casolaro M. et al., Macromolecules, 28, 2351 (1995)

8. Trovato A. et al., Eur. Phys. J. B, 6, 63 (1998)

9. Sherman, E. and Haran G., PNAS, 103, 11539 (2006)

10. Vitalis, A. et al., Biophys J., 93, 1923 (2007)