Browsing by Subject "PNIPAM"

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Open Access
Elucidation of the denaturation mechanism of urea on macromolecules in aqueous medium
(2022-09) Mutlu, Ferhat
The urea molecule is a well-known denaturant for a wide range of macromolecules. To date, there is no unified molecular-level explanation for urea-induced denaturation of all macromolecules. As a result, considerable effort has been directed toward this subject in recent years, because osmolyte protein interactions are of central interest and have implications ranging from polymer physics to cell biology. Detailed urea denaturation mechanisms, focusing on the entropically driven formation of urea clouds, urea induced cross-linking mechanism, and osmolyte clouding around macromolecules, have been proposed in the literature. However, no agreement has been reached on the molecular machinery of urea denaturation. In this thesis, the urea-induced solubility changes of macromolecules in aqueous solutions were investigated by utilizing the lower critical solution temperature (LCST) of poly (N - isopropylacrylamide) (PNIPAM) and poly (N,N-diethylacrylamide) (PDEA) as a function of urea concentration up to 6.0 M. Due to the lack of suitable probing methods for the collapsed state of interested macromolecules, a temperature-controlled ATR – FTIR spectroscopy based method was developed to explore the interaction between urea and the collapsed state of macromolecule. LCST measurements revealed that the solubility of PDEA increases with increasing urea concentrations, whereas PNIPAM salts-out from the solution monotonically, despite the fact that both polymers have similar molecular structures. Temperature gradient ATR-FTIR measurements were carried out to further investigate this discrepancy. First, no favourable urea accumulation towards the collapsed form of macromolecules was observed up to 6.0 M urea, which puts doubt on the urea clouding mechanism. Moreover, at elevated (> 3.0 M) urea concentrations, the collapsed form of the PNIPAM and PDEA accumulated towards the cooler parts of temperature gradient in solution, indicating the preferred form of the macromolecule is the soluble (uncollapsed) form. These surprising results indicate that both PNIPAM and PDEA prefers the soluble form at elevated urea concentrations above the LCST. As a molecular mechanism, the urea molecules act as a "glue" between the amide groups of PNIPAM, bringing intra- and inter-molecular parts of the macromolecule into close proximity, and act as a collapsed form of macromolecules. Apparently, such a mechanism is not valid for the PDEA. Our findings shed new light not only on aqueous phase phenomena, but also on the aggregated (collapsed) phase of macromolecules in aqueous medium.
Open Access
Ion specificity from small molecules to oligomers and beyond amide-based macromolecules
(2024-01) Farooq, Sobia
Presence of ions in aqueous solution regulate the properties of molecules in the same aqueous environment. Such alteration processes are mainly dependent on the concentration and the identity of ions. In this thesis, two parts of ion specific effects were aimed to be explored. First the synthesis and characterization of PNIPAM oligomers by using both reversible addition-fragmentation chain transfer (RAFT) and radical polymerization methods will be shown. Both of these methods give the control over molecular size of the polymer. Oligomers with charged and neutral end group were synthesized to comparatively investigate ion specific effect. These oligomers were also systematically characterized by using various analytical techniques such as phase transition temperature measurement, 1H-NMR and Gel permeation chromatography (GPC). Such oligomers were employed to investigate the specific ion effects via the salt influence on the Lower Critical solution temperature (LCST). By employing two sodium salts; NaCl (strongly hydrated) and NaSCN (weakly hydrated), it was found that strongly hydrated anions salt-out both charged and neutral oligomers, whereas weakly hydrated anions increase the phase transition temperature with a salting in mechanism. By empirical modeling with a Langmuir-type binding isotherm, a weak binding with a dissociation constant KD = 0.57 M for charged and KD = 1.13 M for neutral oligomers were demonstrated. The second part of this thesis focused on the specific ion effects beyond amide-based macromolecules i.e. hydroxypropyl cellulose (HPC) as a model for sugar-based macromolecules. Eight sodium salts were employed to demonstrate the entire Hofmeister series. Namely; NaSCN, NaI, NaNO3, NaClO4 NaCl, Na2SO4, Na2CO3, NaH2PO4 were measured on the phase transition temperature and 1H-NMR measurements. Salts of weakly hydrated anions; NaSCN, NaI, NaClO4 and NaNO3 showed a salting in mechanism and demonstrate a non-monotonic phase transition behavior. In contrast, salts of strongly hydrated anions; NaCl, Na2SO4, NaH2PO4 and Na2CO3 showed salting out mechanism with a monotonic decrease in the phase transition temperature. Additionally, the site-specific ion-macromolecule interaction was studied by 1H-NMR, and Correlation Spectroscopy (2D-COSY) NMR measurements. Although, the exact binding site cannot be specified, it was concluded that the ion binding site is at the side-chain hydroxypropyl groups and that yields the salting-in effect that was observed for the weakly-hydrated anions.
Open Access
Molecular interactions and binding mechanisms of hydrophobic hofmeister cations to macromolecules
(2023-08) Ertekin, Umay Eren
It’s been known for over a century that ions specifically affect the bulk properties of solutions, behavior of macromolecules and a myriad of interfacial phenomena occurring in solution. Yet, the molecular mechanisms underlying these so-called Hofmeister effects are only recently being realized within the last few decades. In the resurgence in specific ion effects studies, the role attributed to cations has been relatively understated in comparison to the effects of anions. Whereas various molecular mechanisms have been elucidated for a diverse spectrum of anions, cationic effects have largely remained limited to common metal ions. Within the cationic Hofmeister series for cations, strongly-hydrated cations exhibit very weak binding to polar, electronegative groups, but weakly-hydrated cations in particular are classified as non-interacting. This thesis brings a much-needed expansion to specific cation effects by investigating the interactions between the weakly-hydrated tetraalkylammonium cations and model neutral thermoresponsive polymers, principally poly(N-isopropylacrylamide) (PNIPAM). The hydrophobicity of the cations is incrementally tuned by increasing the length of their alkyl chains, thus forming the series of salts investigated herein: NR4Cl where R = H, Me, Et, n-Pr, n-Bu, and the anion is kept constant as Cl-. By using a multi-instrumental approach, it is demonstrated that the largest of these cations exhibit a significant binding to the polymers and that the resulting salting-in effects are comparable in magnitude to those observed for sodium salts of weakly-hydrated anions. Thermodynamic phase transition measurements of the polymers are complemented by ATR-FTIR and quantitative 1H-NMR spectroscopic studies to systematically investigate the nature and molecular-level mechanism of the interaction. In stark contrast to the known behavior of the strongly-hydrated cations, through the temperature-controlled ATR-FTIR investigations it is found that carbonyl moieties are not the primary sites of interaction. Instead, it is found that these weakly-hydrated, ‘greasy’ cations preferentially interact with the most hydrophobic groups on the polymer: the isopropyl group on the PNIPAM side-chain, as revealed by a quantitative externally-referenced 1H-NMR methodology developed to elucidate ion-macromolecule interactions. The binding generally follows a Langmuir-type saturation behavior and exhibits site-specific dissociation constants as low as KD ≈ 0.2 M. This unprecedented, hydrophobically-mediated interaction between weakly-hydrated tetraalkylammonium cations and neutral macromolecules is then demonstrated to be a general mechanism and is shown to extend to polymers of vastly different molecular architectures. The results presented, thus, signify a new, more expansive view of cationic Hofmeister effects, where the far weakly-hydrated region of the series interacts with a novel mechanism entirely unlike that of other cations.
Open Access
The mechanisms of ATP - biomacromolecule interactions
(2023-12) Ayvaz, Cansın
Adenosine triphosphate (ATP), one of the most important biomolecules of life, plays a vital role as the primary energy source within cells for essential biological functions. It has recently been discovered that ATP can also serve as a biological hydrotrope to destabilize protein aggregates and fibers. This thesis aims to investigate the recently discovered hydrotropic behavior of ATP and its interaction mechanisms with biomacromolecules, particularly poly(N-isopropylacrylamide) (PNIPAM), using a multi-experimental approach combined with molecular dynamics (MD) simulations. Adapting the bottom-up approach, the phase behavior of macromolecules is examined through phase transition and ATR-FTIR measurements. Additionally, site-specific interactions are identified with quantitative 1H-NMR spectroscopic studies, and the hydration shell structure and cluster morphologies of ATP molecules are explored through Multivariate Curve Resolution (MCR) Raman experiments. It is demonstrated that adenine and adenosine subgroups show negligible effect on the solubility of macromolecules, whereas ATP, AMP, and triphosphate exhibited purely salting-out behavior, and induced the aggregation of macromolecules. In stark contrast to the recently discovered hydrotropic behavior of ATP, no specific interactions between the macromolecule and ATP were observed in spectroscopic ATR-FTIR and 1H-NMR measurements, as well as MD simulations. Surprisingly, at elevated concentrations, self-association of ATP was observed leading to partial destabilization of larger PNIPAM aggregates to smaller ones. In the absence of ATP binding sites, interactions with random-coil-like structured macromolecules do not lead to effective hydrotropic action of ATP. Instead, they function more as stabilizers rather than solubilizing the macromolecules.