Control and Characterization of the Compactness of Single-Chain Nanoparticles

Polymers when self-cross-linked into single-chain nanoparticles bear some resemblance to folded proteins; yet proteins have clear energy landscapes that determine precisely folded structures, while single-chain polymer nanoparticles (SCNPs) have more undefined structures. There have been initial reports showing that some structural parameters in SCNPs can be controlled, for example, compactness. Here, we construct SCNPs from poly(allylamine) (M-w similar to 22 000 Da) with dicarboxylic acids (HOOC-R-COOH) in solvent conditions, where initial chains adopt either extended or collapsed conformation. The spacer groups R that we used were -CH2CH2-, -(CH2S)(2)-, or -(CH2CH2)(3)-, whose length can be estimated to vary from similar to 4 to similar to 12 A. We present a systematic study that uses several characterization techniques (H-1 NMR diffusion-ordered spectroscopy (DOSY), viscometry, analytical ultracentrifugation, and H-1 NMR T-2 relaxation) to show that both initial reaction conditions as well as length of cross-linking molecules determine the final compactness of SCNPs. Specifically, when short cross-linking molecules are applied, short-range loops dominate and the cross-linking process fails to achieve global chain compaction, leading to less compact SCNPs. When the chain is precollapsed (0.1 M water solution of NaCl or 10 vol % ethanol as opposed to DI water), the particles resulting after cross-linking are more compact. Of utmost practical relevance, we show that particles that are essentially chemically identical but differ only in compactness have different toxicity when interacting with HeLa cells, the more compact ones being less toxic.

Study of fast exchanging processes by NMR Spectroscopy

Estel Canet Martinez

Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful and informative methods to probe molecular dynamics. Specifically, chemical exchange is an important phenomenon and one of the earliest and most vigorously investigated in NMR spectroscopy. Its effects can be observed in many NMR spectra; lines in a spectrum are broadened or averaged because the nuclei are exchanging magnetic environments. A wide variety of experimental methods exists to determine exchange rates of protons that hop between amino acids and water in the slow or intermediate exchange regime. However, so far there is a lack of methods to investigate hydrogen exchange in the fast exchange regime. In this thesis, we focus on the determination of very fast chemical exchange rates from the effects of scalar relaxation on a coupled nucleus. We quantify the effects of scalar relaxation caused by exchanging hydrogens, by detecting the decay of a 15N coherence under a multiple-refocusing Carr-Purcell-Meiboom-Gill (CPMG) pulse train in the presence or absence of hydrogen decoupling. In particular, we have adapted to the case of deuterium a method that was originally designed to study the exchange rate of the indole proton of tryptophan in water as a function of pH and temperature. We will present the exchange rates of the indole deuterium in tryptophan with solvent heavy water. After that, the proton exchanges rates have been compared with the deuterium exchange rates in order to describe the kinetic isotope effect. In a similar manner, we have quantified the proton exchange rates in pure water by using multiple refocusing of transverse 17O magnetization as a function of pH and temperature. Such measurements are widely used to obtain valuable information about molecular dynamics and structure of proteins, protein complexes and nucleic acids. The knowledge of the kinetic isotope effect may contribute to the characterization of reaction mechanisms and other dynamic parameters that can give insight into the stability of hydrogen bonded secondary structures in biomolecules. This information could form a foundation for a more thorough theoretical or modeling development of a number of long and outstanding fundamental questions. In the second part of the thesis, we combine fast exchange with dissolution dynamic nuclear polarization (D-DNP) to study intrinsically disordered proteins (IDPs). IDPs constitute a very large and functionally important class of proteins that lack stable secondary and tertiary structures. As a consequence of the broad dynamics and flexibility of IDPs, and specially under physiological conditions, the NMR spectra of IDPs typically show a strong signal overlap and broadening due to proton exchange. We present a method to overcome these limitations by means of hyperpolarized HDO produced by D-DNP. We could study the effects of conformational adaptations in Osteopontin (OPN), a metastasis- associated intrinsically disordered protein (IDP) by chemical exchange. The magnetization from hyperpolarized water is transferred to the rapidly exchanging protons of the solvent-exposed residues of the protein. The exchange with hyperpolarized water boosts the signals of fast exchanging residues above the detection limits. This allows one to follow individual resonances and draw conclusions regarding conformational adaptations due to ligand binding under physiological conditions. Thus, with our approach, exchange with the solvent is turned into an advantage

EPFL2017

Explicit solvent effects on protein physics - modeling lattice proteins in aqueous environments

Protein folding stands as one of the major interdisciplinary challenges of the last ten years, involving biology, chemistry, medicine and physics. In this PhD thesis, we investigate the solvent effects and the resulting hydrophobic effect on proteins. In particular, we focus on a simple lattice model of proteins (the HPW model), where the solvent is (semi)explicitly taken into account (by means of the model of Muller, Lee and Graziano (MLG)), investigating thermodynamical quantities of the modeled proteins and comparing the results with those of real proteins. We approach the protein design problem using the HPW model and we investigate some statistical and thermodynamic properties of the designed sequences. In particular we show that the HPW model is able to capture, within a single framework, both warm and cold denaturation of proteins. We further adapt the MLG model model to include chaotrope cosolvents in the solution. We show that the alteration of the network of hydrogen bonds between water molecules due to the presence of chaotropic agents induces destabilization of proteins. Moreover, we find that the presence of chaotropes originates an effective interaction between the chaotropes and the protein. In addition, we use a common method to extract effective amino-acid interactions on real proteins: a perceptron algorithm. The method applied to proteins designed with the HPW model reveals that the solvent effects can not be reproduced by effective residue interactions. Indeed, we show that it is not possible to determine a set of global interaction values able to stabilize all proteins at once.

EPFL2004

Extending the concept of template-assembled synthetic proteins

A review with 56 refs. The creation of native-like macromols. in copying nature's way represents a fascinating challenge in protein chem. today. In the absence of detailed knowledge of the complex folding pathway, the ultimate goal in protein de novo design, the construction of artificial proteins with predetd. three-dimensional structure and tailor-made functions based on a defined, generally valid set of rules, appears to still be out of reach. With progress in synthesis strategies and biostructural characterization methods, topol. templates have become a versatile tool for inducing and stabilizing secondary and tertiary structures, such as protein loops, b-turns, a-helixes, b-sheets and a variety of folding motifs. In this article, we extend the concept of template-assembled synthetic proteins for the construction of protein-like topologies with multiply bridged, oligocyclic chain architectures termed locked-in tertiary folds that exhibit unique physicochem. and folding properties because of the highly confined conformational space. Furthermore, we show that some fundamental questions in protein assembly can be approached applying the template concept. Using covalent template trapping of self-assocd. peptide assemblies in aq. soln. the structural and phys. forces guiding protein folding, supramol. assembly and mol. recognition processes can be studied on a mol. level. [on SciFinder (R)]

1999

Study of fast exchanging processes by NMR Spectroscopy

Estel Canet Martinez

EPFL2017