Gold nanoparticles (AuNPs) have been of interest due to their biocompatibility and surface plasmon resonance. Biomolecules can spontaneously adsorb to their surface, a trait that could be exploited for drug targeting. It is unclear, however, whether protein-AuNP interactions at the nanoparticle surface are dependent on nanoparticle size. In this project, we investigate whether surface curvature can induce protein unfolding and multilayer binding in citrate-coated AuNPs of various sizes. An NMR-based approach was utilized to determine the adsorption capacity, and protein NMR spectra were compared to determine whether nanoparticle size influences protein interactions. Transmission electron microscopy (TEM) was used to support the results. Over a range of AuNP sizes (15-100 nm) proteins appear globular on the nanoparticle surface. Additionally, a single layer of proteins is adsorbed regardless of AuNP size. Our results are consistent for two differently sized proteins, GB3 (6 kDa) and bovine carbonic anhydrase (BCA, 29 kDa).

The rhomboid family of intramembrane serine proteases can catalyze proteolysis of substrates that are normally embedded in the cell membrane, making them key players in a diverse range of biological processes. While X-ray crystal structures provide detailed insights into the mechanism of intramembrane hydrolysis, questions remain concerning how transmembrane (TM) substrates are able to gain access to the rhomboid active site, and whether interactions with the membrane environment can influence its structure and function. In this thesis, these questions were investigated using the E. coli rhomboid ecGlpG. In Chapter 3, the effect of hydrophobic mismatch between lipid and protein was investigated using families of amphiphiles with saturated alkyl chains. While ecGlpG displayed maximal activity against a water-soluble model substrate when solubilized in detergents containing 10-12 carbon atoms, shorter and longer chain detergents led to loss of activity. An even larger effect was observed when ecGlpG was reconstituted into phospholipid bicelles, with no proteolytic activity being detected in 14-carbon lipids. These results suggest that mismatch between the hydrophobic regions of the catalytic TM domain (TMD) and the local membrane environment is detrimental to proteolysis. To obtain further insight into the structure and dynamics of ecGlpG, sample conditions were identified in Chapter 4 that enabled, for the first time, the acquisition of NMR spectra showing signals from the ecGlpG TMD. While significant peak broadening prevented chemical shift assignment, the sensitivity and resolution of peaks corresponding to the tryptophan indole NH group allowed their use as structural probes. These were employed in Chapter 5 to characterize the open conformation of ecGlpG that is postulated to facilitate substrate entry. These spectra showed evidence of an open conformation in which the intact α5 is laterally displaced. Interactions with a substrate-derived peptide also appeared to stimulate gate opening; however, activity assays suggested that formation of the open state could compromise catalytic activity against water-soluble substrates, and that interactions with TM substrates could counter this effect. Taken together, these results provide new insight into the role of both the local membrane environment and α5-conformational dynamics on intramembrane proteolysis, and suggest a mechanism to prevent cleavage of off-target rhomboid substrates in vivo.

The ability of mutations to change the fold of a protein provides evolutionary pathways to new structures. To study hypothetical pathways for protein fold evolution, we designed intermediate sequences between Xfaso1 and Pfl6, two homologous Cro proteins that have 40% sequence identity but adopt all–α and α+β folds, respectively. The designed hybrid sequences XPH1 and XPH2 have 70% sequence identity to each other. XPH1 is more similar in sequence to Xfaso1 (86% sequence identity) while XPH2 is more similar to Pfl6 (80% sequence identity). NMR solution ensembles show that XPH1 and XPH2 have structures intermediate between Xfaso1 and Pfl6. Specifically, XPH1 loses α-helices 5 and 6 of Xfaso1 and incorporates a small amount of β-sheet structure; XPH2 preserves most of the β-sheet of Pfl6 but gains a structure comparable to helix 6 of Xfaso1. These findings illustrate that the sequence space between two natural protein folds may encode a range of topologies, which may allow a protein to change its fold extensively through gradual, multistep mechanisms. Evolving a new fold may have consequences, such as a strained conformation. Here we show that Pfl6 represents an early, strained form of the α+β Cro fold resulting from an ancestral remnant of the all-α Cro proteins retained after the fold switch. This nascent fold can be stabilized through deletion mutations in evolution, which can relieve the strain but may also negatively affect DNA-binding function. Compensatory mutations that increase dimerization appear to offset these effects to maintain function. These findings suggest that new folds can undergo mutational editing through evolution, which may occur in parallel pathways with slightly different outcomes.

Inhibition of the intrinsically disordered proteins (IDP) is a recognized issue in drug research. Standard approaches, based on key-lock model, cannot be used in the absence of rigid structure and defined active site. Here a basic helix-loop-helix leucine zipper (bHLHZip) domain of c-Myc was studied, which is intrinsically disordered and prone to aggregation. Chemical denaturation of proteins is a widely accepted technique to study protein folding, but here this methodology was applied to IDP, observing its effect on the structural ensemble of c-Myc by NMR spectroscopy. Nonlinear chemical shift changes indicated cooperative unfolding of the helical structure of the part of the leucine zipper domain in parallel with the melting of the N-terminal helix. Paramagnetic relaxation enhancement (PRE) was used to probe long-range structure and revealed presence of long-range contacts. The following search for inhibitors can be directed to the search for ligands, locking c-Myc in its more compact conformation. Protein self-association is a problem typical for IDPs and intrinsic process for all proteins at high concentrations. It leads to increased viscosity, gelation and possible precipitation, which cause problems in protein manufacturing, stability and delivery. If protein drugs require high dosing, special approaches are needed. At high concentrations proteins experience conditions close to the crystal state, therefore interactions in solution could potentially coincide with crystal lattice contacts. A range of diverse methods is used to study this process, but the complexity of the mechanism makes it hard to build a reliable model. Here, the self-association of streptococcal Protein G (PrtG) was studied using Nuclear Magnetic Resonance (NMR) spectroscopy in solution. The properties of protein-protein interactions at high concentration, up to ~ 160 mg/ml, were studied at residue-level resolution. Residue specific information on protein dynamics was obtained using 15N relaxation measurements. The experiments were carried out at multiple concentrations. Variation in the rotational correlation time over these concentrations showed changes in the protein dynamics, which indicated weak protein-protein interactions occurring in solution. Pulsed-field gradient NMR spectroscopy was used to monitor translational diffusion coefficients in order to estimate the degree of protein self-association. Oligomer formation was also monitored by looking at variations in 1H and 15N amide chemical shifts. Better understanding of protein self-association mechanisms under different conditions could assist in developing methods to reduce the level of reversible protein self-association in solution at high protein concentrations.

<p>The overall goal of this project is to enhance the physical accuracy of individual models in macromolecular NMR (Nuclear Magnetic Resonance) structures and the realism of variation within NMR ensembles of models, while improving agreement with the experimental data. A secondary overall goal is to combine synergistically the best aspects of NMR and crystallographic methodologies to better illuminate the underlying joint molecular reality. This is accomplished by using the powerful method of all-atom contact analysis (describing detailed sterics between atoms, including hydrogens); new graphical representations and interactive tools in 3D and virtual reality; and structural bioinformatics approaches to the expanded and enhanced data now available.</p> <p> The resulting better descriptions of macromolecular structure and its dynamic variation enhances the effectiveness of the many biomedical applications that depend on detailed molecular structure, such as mutational analysis, homology modeling, molecular simulations, protein design, and drug design.</p> / Dissertation

Prostate apoptosis response factor-4 (Par-4) is a ubiquitously expressed pro-apoptotic and tumour suppressive protein. Par-4 contains a highly conserved coiled coil (CC) region at the Cterminus, particularly the distal 40 residues fulfil the criteria for a leucine zipper (LZ). This Cterminal domain serves as the primary recognition domain for a large number of binding partners. Par-4 is tightly regulated by the aforementioned binding partners and also by posttranslational modifications. Biophysical data presented here describe Par-4 as primarily an intrinsically disordered protein (IDP). Bioinformatic analysis of the highly conserved Par-4 reveals low sequence complexity and enrichment in polar and charged amino acids. High proteolytic susceptibility and increased hydrodynamic radii are consistent with largely extended structures in solution. Spectroscopic measurements using circular dichroism (CD) and nuclear magnetic resonance (NMR) also reveal characteristic features of intrinsic disorder. Under physiological conditions, data show that Par-4 self-associates via the C-terminal domain possibly through coiled coil formation. Analysis of various constructs comprising the Par-4 LZ domain by NMR, CD, light scattering and other techniques reveals an environment-dependent conformational equilibrium between primarily disordered monomers and predominantly coiled coil dimers. Whereas the disordered monomers are easily observed by NMR, the coiled coil fraction is not amenable to NMR studies possibly due to intermediate exchange processes. Mutational approaches that stabilise the coiled coil fraction result in NMR spectra of lower quality compared to the wild-type form. The high degree of sequence conservation suggest that coiled coil formation and intrinsic disorder are essential for Par-4 to function as an effective regulator of apoptosis.

Rhomboids are a family of intramembrane serine proteases that cleave transmembrane protein substrates within the lipid membrane. They are involved in a wide range of biological processes, including signal transduction, parasite invasion, bacterial quorum sensing and apoptosis. While previous X-ray crystal structures and functional studies have provided some detailed insights into the mechanism of intramembrane hydrolysis, it is still not clear how the transmembrane substrate can gain access into the active site from the lipid environment. While several modes of action have been suggested, one hypothesis proposes a lateral movement of the fifth transmembrane helix, causing a displacement that would allow transmembrane substrates to enter the rhomboid active site. A powerful method that has the potential to yield insights into rhomboid dynamics is solution NMR; however, the large size of rhomboid protease samples has complicated conventional methods typically used to assess protein structure and dynamics. ¹⁹F NMR could allow the study of rhomboid conformational dynamics by providing a simplified spectrum with high sensitivity to changes in local chemical environments. In this thesis various methods of ¹⁹F incorporation were evaluated for utility in studying rhomboid conformational dynamics, focusing on the GlpG rhomboid from E. coli. First, GlpG samples were prepared with ¹⁹F incorporated into tryptophan sidechains, and 1D ¹⁹F NMR spectra were acquired. While spectra with decent spectral dispersion were obtained, the assignment process was complicated by low signal-to-noise, and multiple changes in the spectrum introduced by the mutation. Chemoselective labelling of cysteine residues with probes containing a trifluoromethyl group was also investigated and found to give rise to well resolved ¹⁹F NMR spectra with promising characteristics. In addition, protocols for incorporation of trifluoromethyl-phenylalanine using unnatural amino acid incorporation at introduced amber codon sites were also explored, since one of the long-term goals of this work is to study ¹⁹F-labelled GlpG in its native lipid environment. For this purpose, some protocol development was also performed to introduce GlpG into lipid nanoparticles using styrene maleic acid co-block polymers. However, low expression yields of trifluoromethyl-phenylalanine-labelled GlpG and the large size of the lipid nanoparticles are not yet compatible with solution NMR. Nonetheless, this thesis lays the groundwork for further development of these samples to allow the future study of conformational exchange of GlpG in native lipid membranes.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19315号 / 工博第4112号 / 新制||工||1634(附属図書館) / 32317 / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 白川 昌宏, 教授 跡見 晴幸, 教授 梶 弘典 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

The overall goal of my work was to develop and apply new chemical methods for the study of epigenetic DNA and protein modifications. In Chapter 3 the development of Suzuki-Miyaura cross coupling (SMcc) for the post-synthetic modification of DNA is described. DNA modification by SMcc is efficient (4-6h) and proceeds under mild conditions (37°C, pH 8.5). The incorporation of various groups useful for biological investigations is demonstrated using this methodology. Using a photocrosslinker, introduced into the DNA by SMcc capture experiments are performed to identify potential binding partners of modified DNA. In Chapter 4 a dehydroalanine (Dha) based chemical protein modification method is described that enables the introduction of posttranslational modification (PTM) mimics into histones. The PTM mimics introduced by this method are tested using western- and dot-blot and binding and enzymatic assays, confirming they function as mimics of the natural modifications. Chapter 5 describes the use of a generated PTM mimics to elucidate the function of O-linked β-Nacetylglucosamine (GlcNAc) of histones in transcriptional regulation. It is shown that GlcNAcylation of Thr-101 on histone H2A can destabilize nucleosome by modulating the H2A/B dimer – H3/H4 tetramer interface. N- and C-terminal histone tails play an important role in transcriptional regulation. In Chapter 6, nuclear magnetic resonance is used to investigate the structure of the histone H3 N-terminal tail in a nucleosome. The H3 tail, while intrinsically disordered, gains some α-helical character and adopts a compact conformation in a nucleosome context. This H3 tail structure is shown to be modulated by Ser-10 phosphorylation. The effect of a new covalent DNA modification, 5- hydroxymethylcytosine (5hmC), on transcription factor binding is investigated in Chapter 7. 5hmC influences HIF1α/β, USF and MAX binding to their native recognition sequence, implying involvement of this modification in epigenetic regulation.

Les métaux lourds, et en particulier les actinides, sont toxiques pour l'homme. La compréhension des mécanismes responsables de leur toxicité constitue un champ d'investigation important dans le domaine de la toxicologie. La compréhension des interactions de l’uranyle à l’échelle moléculaire est nécessaire pour prédire sa toxicité et pour concevoir des agents décorporants efficaces. Ce travail a pour objectif de contribuer à la caractérisation des sites d’interaction protéine-uranyle et à l’identification des facteurs clés gouvernant ces interactions. Pour obtenir des données thermodynamiques et structurales sur ces sites, deux stratégies ont été adaptées à l’étude des deux protéines humaines prédites comme cibles majeures de l’uranyle et dont les propriétés et structures sont très différentes. Les deux domaines structurés de la fétuine-A, ont été produits puis étudiés indépendamment par des méthodes physico-chimiques complémentaires incluant la spectroscopie RMN multidimensionelle afin d’obtenir des informations structurales sur les sites de liaison du métal dans la protéine. Afin d’élucider les interactions entre l’uranyle et l’ostéopontine, une protéine phosphorylée intrinsèquement désordonnée, nous avons conçu des peptides préorganisés en feuillet β comme modèles de sites de liaison de l’uranyle. Des acides aminés phosphorylés ont été introduits dans ces structures, permettant ainsi de reproduire l’environnement de coordination du métal dans la protéine. Les différences de structures et de propriétés entre biomolécules peuvent représenter un frein aux études d’affinité. Une sonde fluorescente non naturelle a donc été développée pour mettre au point une méthode de hiérarchisation des cibles de l’uranyle s’affranchissant de ces différences. / Heavy metals, especially actinides, are toxic for humans. Understanding the mechanisms responsible for their toxicity is an important field of research in toxicology. Uranyl toxicity is still not well understood. The understanding of uranyl interactions at the molecular level is necessary to predict its chemical toxicity and to develop efficient chelating agents. This work aims at identifying uranyl binding sites in proteins and key factors that govern these interactions. To obtain thermodynamic and structural data, strategies were developed to study two proteins predicted as major uranyl targets which present different structures and properties. We took advantage of fetuin-A structure and studied the two structured domain of the protein by complementary physico-chemical methods including multidimensional NMR spectroscopy to acquire structural information on uranyl binding sites in this protein. In order to elucidate interactions between the metal and disordered phosphorylated proteins such as osteopontin, we designed peptides preorganized in β-sheet optimized to coordinate uranyl cation. We introduced amino acids containing phosphate groups and demonstrated that these peptides are relevant models to mimic uranyl binding sites found in phosphorylated proteins. Biomolecules display different structures and properties which may constitute an obstacle to affinity studies. A tool based on a non-natural fluorescent probe was developed to investigate and compare uranyl targets affinities.