NMR studies of multi-component macromolecular solutions

Barhoum, Suliman Abdallah (2012) NMR studies of multi-component macromolecular solutions. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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    Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission.
    (Original Version)

Abstract

My doctoral studies deals with model systems in soft matter. In these model systems, we address questions that are on the interface between physics and biology. The primary focus of the studies is to explore the dynamics and structural changes of aggregations of biologically relevant multi-component polymer, protein, peptide, and micellar systems using nuclear magnetic resonance (NMR) based techniques. Diffusometry, relaxometry, and deuterium NMR techniques were chosen since they complement one another in understanding the dynamics and structure in the process of macromolecular self-assembly. Moreover, identifying the size of nanoparticle clusters in these model systems is a challenging problem and there is no technique that gives complete answers . Therefore, my doctoral work is a systematic attempt to use NMR as a tool to make quantitative statements about the nature of macromolecular clustering. -- During my PhD, we used a model polymer-surfactant solution in order to identify surfactant concentration regimes relevant to understanding the behavior of biomolecules in a "crowded environment" consisting of multiple species of macromolecules. Such crowded environments are a characteristic of intracellular plasma . We studied a system composed of nonionic polymer poly(ethylene glycol) (PEO) and an anionic sodium dodecyl sulfate (SDS) at different SDS concentrations. Using NMR diffusometry, we measured the self-diffusion coefficients of the polymer (PEO) and surfactant (SDS) components simultaneously as a function of SDS concentration. Also, we obtained NMR relaxation rates for PEO and the chemical shifts as a function of SDS concentration. Using a simple model to interpret our experimental results, we developed a precise method to identify the onset of aggregation of SDS on PEO chains (critical aggregation concentration CAC=3.5 mM) and a crossover concentration (C2 = 60 mM) which is associated with a sharp change in the relaxation behavior, as well as an increase in free surfactant concentration. Moreover, in the context of a simple model, we identified the SDS concentration (Cm = 145 mM) beyond which the diffusion of the aggregates was strongly hindered by the presence of other structures such as free micelles. -- Another source of hindered diffusion, apart from macromolecular crowding, is diffusion in the presence of geometrical constraints. In the case of cylindrical or wormlike micelles, the constraint is to diffuse within the cylinder. Aggregates in a multi-component wormlike micelle are difficult to study via scattering techniques because they would require contrast matching. Therefore, We studied a system composed of a zwitterionic surfactant N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (TOPS) and an anionic surfactant SDS in brine. NMR diffusometry indicates that the self-diffusion coefficients of surfactant is consistent with restricted diffusion within a reptating micelle. Using a simple model to interpret our diffusometry results, we estimated the average end-to-end micellar distance to be ͌ 1 μm. Using NMR relaxometry, we obtained the wormlike micelle overlap concentration (a characteristic concentration Cthreshold = 4.5 mM). Deuterium NMR spectra indicate that the internal structure of the wormlike micellar system includes domains with different orientation orders with slow exchange between domains. Our experimental results (NMR diffusometry and rheometry) revealed that the wormlike micelles exhibit remarkable polymer-like scalings with a crossover from Zimm-like (diffusion) to Rouse-like (rheology) regimes. -- During the latter part of my PhD, we explored protein solutions . We were motivated by the fact that scattering techniques in concentrated lysozyme protein solutions show conflicting reports over the existence of an equilibrium cluster phase. It is recognized that protein aggregation is essential in understanding the biophysics of proteins. Also, it must be noted that protein aggregations and misfoldings, which are implicated as a root cause of some diseases that are thus sometimes called “conformational diseases” are technically challenging to quantify. To address this problem, we worked on diffusion of lysozyme proteins in concentrated lysozyme solutions. Lysozyme is a common mammalian protein, which is found in human amylases. Using NMR diffusometry, we obtained the self-diffusion coefficients of lysozyme in concentrated lysozyme solutions. Diffusometry and relaxometry showed that both the observed diffusion coefficient and relaxation rates are a weighted average of monomer and aggregate fraction s. Therefore, our studies gave a strong evidence for both lysozyme monomers and thermodynamically stable lysozyme clusters in the protein solution with a rapid exchange between monomer and aggregate on the NMR time scale. -- Finally, we addressed a practical problem of relevance to biochemists and biophysicists. Studying the dynamics of macromolecules and aggregates in multicomponent systems composed of peptide or protein and surfactants is challenging. Many peptides and proteins are synthesized in tiny quantities. We used NMR diffusometry and relaxometry to explore systems composed of peptides that are very long compared to the surfactant, dissolved with an anionic surfactant in an aqueous solution. We compared the results to those for shorter dipeptides whose size is comparable to the surfactant. Diffusometry shows that the longer peptide behaves as if there is no free fraction of peptide molecules in the solution. Based on that, we extracted reliable physical quantities such as the true hydrodynamic radius of long peptide-surfactant aggregate as a function of surfactant concentration. On the other hand, the smaller dipeptides are partitioned between the bound and free state such that the fraction of the bound peptide varies with surfactant concentration.

Item Type: Thesis (Doctoral (PhD))
URI: http://research.library.mun.ca/id/eprint/2317
Item ID: 2317
Additional Information: Includes bibliographical references.
Department(s): Science, Faculty of > Physics and Physical Oceanography
Date: 2012
Date Type: Submission
Library of Congress Subject Heading: Macromolecules--Properties; Nuclear magnetic resonance; Surface active agents--Properties; Microclusters--Properties

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