Ion mobility-mass spectrometry (IM-MS), gas-phase hydrogen/deuterium (H/D) exchange ion molecule reactions and molecular modeling provide complimentary information and are used here for the characterization of peptide ion structure, including fine structure detail (i.e., cation-π interactions, β-turns, and charge solvation interactions). IM-MS experiments performed on tyrosine containing tripeptides show that the collision cross-sections of sodiated, potassiated and doubly sodiated species of gly-gly-tyr are smaller than that of the protonated species, while the cesiated and doubly cesiated species are larger. Conversely, all of the alkali-adducted species of try-gly-gly have collision cross-sections that are larger than that of the protonated species. The protonated and alkali metal ion adducted (Na+, K+ and Cs+) species of bradykinin and bradykinin fragments 1-5, 1-6, 1-7, 1-8, 2-7, 5-9 and 2-9 were also studied using IM-MS and the alkali metal ion adducts of these species were found to have cross-sections very close to those of the protonated species. Additionally, multiple peak features observed in the ATDs of protonated bradykinin fragments 1-5, 1-6 and 1-7 are conserved upon alkali metal ion adduction. It was observed from gas-phase H/D ion molecule reactions that alkali adducted species exchange slower and to a lesser extent than protonated species in the tyrosine- and arginine-containing peptides. Experimental and computational results are discussed in terms of peptide ion structure, specifically the intra-molecular interactions present how those interactions change upon alkali salt adduction, as well as with the sequence of the peptide. Additionally, IM-MS data suggests the presence of a compact conformation of bradykinin fragment 1-5 (RPPGF) when starting from organic solvent conditions. As water is added stepwise to methanolic solutions, a more extended conformation is populated. When the starting solution is composed of ≈90% water, two distinct mobility profiles are observed as well as a shoulder, indicating the presence of three gas-phase conformations for RPPGF. Gas-phase H/D exchange of [M+H]+ ions prepared from aqueous solvents show a bi-exponential decay, whereas samples prepared from organic solvents show a single exponential decay. The effect of solvent on gas-phase peptide ion structure, i.e., solution-phase memory effects, is discussed and gas-phase structures are compared to know solution-phase structures.

MDM2 regulates p53 predominantly by promoting p53 ubiquitination. However, ubiquitination-independent mechanisms of MDM2 have also been implicated. Here we show that MDM2 inhibits p53 DNA binding activity in vitro and in vivo. MDM2 binding promotes p53 to adopt a mutant-like conformation, losing reactivity to antibody Pab1620, while exposing the Pab240 epitope. The acidic domain of MDM2 is required to induce p53 conformational change and inhibit p53 DNA binding. ARF binding to the MDM2 acidic domain restores p53 wild type conformation and rescues DNA binding activity. Furthermore, histone methyl transferase SUV39H1 binding to the MDM2 acidic domain also restores p53 wild type conformation and allows p53-MDM2-SUV39H1 complex to bind DNA. These results provide further evidence for an ubiquitination-independent mechanism of p53 regulation by MDM2, and reveal how MDM2-interacting repressors gain access to p53 target promoters and repress transcription. Furthermore, we show that the MDM2 inhibitor Nutlin cooperates with the proteasome inhibitor Bortezomib by stimulating p53 DNA binding and transcriptional activity, providing a rationale for combination therapy using proteasome and MDM2 inhibitors.

The translocation of a biopolymer through a narrow pore exists in universal cellular processes, such as the translocations of nascent proteins through ribosome and the degradation of protein by ATP-dependent proteases. However, the molecular details of these translocation processes remain unclear. Using computer simulations we study the translocations of a ubiquitin-like protein into a pore. It shows that the mechanism of co-translocational unfolding of proteins through pores depends on the pore diameter, the magnitude of pulling force and on whether the force is applied at the N- or the C-terminus. Translocation dynamics depends on whether or not polymer reversal is likely to occur during translocation. Although it is of interest to compare the timescale of polymer translocation and reversal, there are currently no theories available to estimate the timescale of polymer reversal inside a pore. With computer simulations and approximate theories, we show how the polymer reversal depends on the pore size, r, and the chain length, N. We find that one-dimensional transition state theory (TST) using the polymer extension along the pore axis as a reaction coordinate adequately predicts the exponentially strong dependence of the reversal rate on r and N. Additionally, we find that the transmission factor (the ratio of the exact rate and the TST rate) has a much weaker power law dependence on r and N. Finite-size effects are observed even for chains with several hundred monomers. If metastable states are separated by high energy-barriers, transitions between them will be rare events. Instead of calculating the relative energy by studying those transitions, we can calculate absolute free energy separately to compare their relative stability. We proposed a method for calculating absolute free energy from Monte Carlo or molecular dynamics data. Additionally, the diffusion of a knot in a tensioned polymer is studied using simulations and it can be modeled as a one-dimensional free diffusion problem. The diffusion coefficient is determined by the number of monomers involved in a knot and its tension dependence shows a maximum due to two dominating factors: the friction from solvents and “local friction” from interactions among monomers in a compact knot. / text

Bulk imprinted polymers were synthesized using traditional small molecular weight imprinting techniques for the recognition of bovine serum albumin (BSA). Reproducibility and capacity concerns prompted the use of circular dichroism to investigate the potential effects that conditions commonly employed have on the structure of the protein prior to polymerization. These studies clearly showed a substantial change in the secondary structure of three common model protein templates when in the presence of various monomers and crosslinkers. Molecular docking was used to further examine the interactions taking place at the molecular level. Docking simulations revealed that significant amounts of non-covalent interactions are occurring between the amino acid side chains and ligands; although, the interactions taking place amongst the analyte and polypeptide backbone are responsible for the experimentally observed conformational change. The computational studies also showed that several of the ligands preferentially ‘docked’ to the same amino acids in the protein, indicating that if multiple monomers are employed, this competition for similar binding sites will potentially result in non-specific recognition. These findings are important as they offer insight into the fundamental reasons why recognition of macromolecular templates has proven difficult as well as provide guidance for future success in the field. Using this information, novel surface imprinted polymers were synthesized via a facile technique for the specific recognition of BSA. Thin films based on 2-(dimethylamino)ethyl methacrylate (DMAEMA) as the functional monomer and varying amounts of either N,N’ methylenebisacrylamide (MBA) or poly(ethylene glycol) (400) dimethacrylate (PEG400DMA) as crosslinker were synthesized via UV free-radical polymerization. A clear and reproducible increase in recognition of the template was demonstrated for these systems as 1.6-2.5 times more BSA was recognized by the MIP sample relative to the control polymers. Additionally, these polymers exhibited specific recognition of the template relative to similar competitor proteins with up to 2.9 times more BSA adsorbed than either glucose oxidase or bovine hemoglobin. These synthetic antibody mimics hold significant promise as the next generation of robust recognition elements in a wide range of bioassay and biosensor applications. / text

L'organisation de la chromatine a une importance fonctionnelle pour contrôler le programme d'expression des gènes. Par contre, les liens qui l'unissent au déroulement de la réplication de l'ADN sont beaucoup moins connus. Grâce à des approches basées sur la capture d'interactions chromosomiques et sur l'imagerie cellulaire, nous avons étudié les liens entre le repliement à grande échelle de la chromatine et le timing de réplication. Cette analyse, effectuée dans des cellules humaines lymphoblastoïdes, des cellules mononucléées du sang (PBMC) et des cellules issues d'une leucémie myéloïde à caractère érythrocytaire, a permis l'identification de domaines structuraux du noyau. Ces domaines sont relativement isolés les uns des autres et leurs frontières coïncident avec les zones d'initiation précoce. De plus, notre étude montre que ces zones d'initiation précoce interagissent préférentiellement, aussi bien entre voisins immédiats (séparation génomique de l'ordre de la mégabase) que le long du chromosome entier. Les loci répliqués tardivement interagissent eux-aussi avec leurs homologues, conduisant, dans l'espace nucléaire, à une ségrégation des loci en fonction de leur timing de réplication. Ces résultats sont soutenus par des mesures de distances sur des hybridations in-situ qui montrent que les loci répliqués en début de phase S sont plus proches qu'attendus. Nos travaux révèlent enfin que l'organisation de la chromatine est similaire dans des cellules en phase G0 (PBMC dormantes), démontrant qu'elle n'est pas spécifique des cellules en phase S. Pris ensemble, ces résultats apportent des preuves directes d'une organisation robuste de la chromatine, partagée par les cellules en cycle et dormantes, et corrélée au timing de réplication à différentes échelles.

<p>Small-angle X-ray scattering (SAXS) is a biophysical technique that allows one to study the statistical conformation of a biopolymer in solution. The two-dimensional data obtained from SAXS is a low-resolution probe of the statistical conformation- it is a population weighted orientational average of all conformers within a conformational ensemble. Traditional biological SAXS experiments seek to describe an "average" structure of a protein, or enumerate a "minimal ensemble" of a protein at the atomic resolution scale. However, for highly flexible proteins, an average structure or minimal ensemble may be insufficient for enumeration of conformational space, and may be an over-parameterized model of the statistical conformation. This work describes a SAXS analysis of highly flexible proteins and presents a protocol for describing the statistical conformation based on minimally parameterized polymer physics models and judicious use of ensemble modeling. This protocol is applied to the structural characterization of S. aureus protein A - a crucial virulence factor - and Fibronectin III domains 1-2 - an important structural protein.</p> / Dissertation

Protein PEGylation (covalent attachment of poly(ethylene glycol) or PEG to proteins) is an excellent example of a drug delivery system that improves pharmacokinetics and pharmacodynamic properties of therapeutics. However, although PEGylation is clinically proven and attracts both scientific and commercial interest, the technique is associated with many process constraints, in particular related to controlling the number of conjugated PEG chains. A novel, solid-phase PEGylation methodology was attempted to overcome the drawbacks of the commonly used solution-phase methods for preparing PEGylated products. The solid-phase PEGylation methodology involved conjugating protein onto a tethered PEG derivative attached onto a solid matrix, followed by hydrolytic cleavage of the PEG chain from the solid matrix under mild conditions to yield PEGylated protein in free solution. PEGs with molecular weights (MWs) 2000 and 4000 Da were used and a heterobifunctional PEG derivative, α-(β-alanine)-ω-carboxy PEG, with a cleavable β-alanine ester terminal was prepared for surface grafting and protein conjugation. The amine terminal of this PEG derivative was used for grafting PEG onto carboxy functionalized hydrophilic Sephadex and hydrophobic polystyrene derivatives. The free carboxyl terminal was used for protein conjugation via amine coupling. A kinetic study of PEG-surface grafting was performed to understand the influence of a number of parameters on the PEG surface concentration and its conformation, including temperature, reaction time, nature of the matrix, solvent and base, and MW of PEG. PEG grafted matrices were characterized using various surface characterization tools including Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Higher PEG grafting was observed with polystyrene matrices (up to 0.3 mmol/g) than either of the Sephadex derivatives (less than 0.15 mmol/g) using both molecular weights. Detailed surface characterization using XPS studies showed a layer thickness of 11.87 nm was achieved with polystyrene matrices using 4000 Da PEG derivatives after a grafting period of 72 hours at 40°C, indicating the presence of brush conformations for the grafted PEGs. In contrast, mushroom conformations were observed for PEG molecules grafted on both carboxymethyl and carboxypentyl Sephadex derivatives after the same reaction period, with a layer thickness of 2.62 nm and 4.14 nm respectively. Optimized PEG grafting and hydrolysis conditions were developed for solid-phase protein PEGylation using Cytochrome c as a model protein. The presence of PEGylated species were detected by size exclusion chromatography (SEC) from Sephadex derivatives but were absent when using polystyrene matrices. Both Sephadex derivatives gave mainly multi-PEGylated species with poor yields, in place of the expected mono-PEGylated products. A solution-phase PEGylation using the same PEG derivatives was performed successfully and various PEGylated species were identified and characterized using SEC and gel electrophoresis, based on their viscosity radius. An examination of the surface characteristics of the PEG-grafted was carried out by XPS, showing that protein conjugation was greatly influenced by surface force interactions, which depended on the PEG grafting densities and the nature of the solid matrices. Finally, fluorescent images obtained using confocal microscope with fluorescein isothiocyanate labelled Cytochrome c provided supporting evidence regarding the factors that constrained the solid-phase PEGylation process.

Although attractive interactions between π systems (π-π interaction) have been known for many years, understanding of its origin is still incomplete. Quantitative measuring of π-stacking is challenging due to the weak nature of the π-π interaction. This dissertation aims at elucidating a quantitative conformational analysis by NMR ring current anisotropy of an organic compound capable of intramolecular π-stacking in solution and studying charge effects on the stacking of π-systems. This dissertation offers four contributions to the area. (1) A general approach to four-state, conformational analysis based on the magnetic anisotropy of molecules undergoing fast dynamic exchange is described. (2) Study unveiled the importance of charges in the conformation of a dication in the solution. (3) Novel aromatic salt pairs of triangulene derivatives with the delocalized cation-anion interaction were synthesized and studied. (4) Study unveiled ionic π-systems preferred face-to-face stacking due to strong cation-π and anion-cation attractions. A general protocol for the application of magnetic anisotropy to quantitative multi-state conformational analysis of molecules undergoing fast conformational exchange was suggested in the current study. The reliability of this method of conformational analysis was checked by the mass balance. VT-NMR was also conducted to study the enthalpic parameters. This technique can be further used to study canonical interactions such as ion pairing, hydrogen boning, and molecular recognition. In the current study, dependence of the probe conformations on the dispersive interactions at the aromatic edges between solvent and probes was tested by conformational distributions of the fluorinated derivatives (2b and 2c) of the probe molecule (1a). Solution and solid studies of these molecules put the previous conclusion drawn by the Cammers group in question. Current studies show that the dispersive interaction at the aromatic edge could not be the predominant force on the conformational changes in the probe molecule 1a during the fluoroalkanol perturbation. This study indicated that charges might be important in the formation of the folding conformations in the solution and solid state of 1a, 2b, and 2c. A contribution of this thesis was to prepare and study a conformational model that lacked charges. The previous molecules were charged. The solid-state structures of pyridinium-derived aromatic rings from the CSD (Cambridge Structural Database) were studied to investigate the π-π interaction between cationic π-systems in solid state. Novel aromatic salt pairs of triangulene derivatives with the delocalized cation-anion interaction were synthesized to study the π-π interaction between two aromatic rings that carried opposite charges. This study showed that the interaction between ionic π-systems can be enhanced by cation-π and anion-cation attractions. The stackings of these π-systems introduce more overlap, closer packing and stronger atomic contact than that of the solid states of comparable neutral species. Cation-π and anion-cation attractions are synergistic in aromatic salts.

The Distal-less homeobox (Dlx) genes encode a group of transcription factors that are involved in various developmental processes including forebrain development. Dlx genes are arranged in convergently transcribed bigene clusters with enhancer sequences located in the intergenic region of each cluster. The expression patterns of Dlx1/Dlx2 and of Dlx5/Dlx6 are attributed in part to the activity of I12a/I12b and I56i/I56ii intergenic enhancers, respectively. In an effort to determine how Dlx intergenic enhancers interact with the promoter regions of each cluster, I employed the Chromosome Conformation Capture (3C) technique on developing forebrain at E13.5 and E15.5. My 3C analysis provided potential enhancer-promoter interaction, in cis, that are consistent with previously known regulatory mechanisms. Furthermore, trans interactions may exist between Dlx1/Dlx2 and Dlx5/Dlx6 clusters in the developing forebrain at E13.5, thus providing a possible novel cross-regulatory mechanism between these two loci. I have also investigated the phenotypic consequences of Dlx enhancer deletion(s) on forebrain development by characterizing mice with I56ii and I56ii/I12b enhancer deletions. Enhancer deletions significantly impair Dlx expression as well as that of Evf2, Gad2 and of the striatal markers Islet1 and Meis2. Enhancer deletion(s) also reduce the expression of ISLET1 and CTIP2 proteins and Semaphorin 3A, Slit1 and Ephrin A5 that are thought to provide guidance cues in the corridor cells. Overall, these changes may disrupt the guidance of the thalamocortical axons. The data presented here further our understanding of the interactions between Dlx intergenic enhancers and promoter regions. Enhancer deletion(s) furthers our understanding of Dlx regulatory networks necessary that ensure proper Dlx expression, which, in turn may be involved in a genetic pathway underlying the synthesis of GABA, which may be further essential in maintaining the GABAergic phenotype.

The biological function of a protein is determined by its amino acid sequence, structure, and internal dynamics. In turn the prediction of a protein structure from its folding pathway involves the characterization of the dynamics of the polypeptide backbone. This study addresses how the internal dynamics of arborescent polypeptides are affected by increased crowding of the interior of these branched polymer molecules. Linear, comb-branched, and arborescent poly(L-glutamic acid) (PGA) samples were analyzed by 1H NMR spectroscopy to determine their chain conformation. The PGA chains of these constructs were shown to adopt α-helical and random coil conformations in N,N-dimethylformamide (DMF) and in dimethyl sulfoxide (DMSO), respectively. The hydrodynamic diameter (Dh) of the arborescent PGAs, determined using dynamic light scattering measurements, increased with increasing generation number and when the side-chains adopted random coil instead of α-helical conformations. The PGA samples were labelled with 1-pyrenemethylamine to determine how their structure affected the internal dynamics of the arborescent polymers in solution, from the analysis of their fluorescence spectra and decays. For each pyrene-labelled polymeric construct excimer formation increased with increasing pyrene content, and the efficiency of excimer formation increased with the generation number due to the increased density of the macromolecules. Comparison of the time-resolved fluorescence results acquired in DMF and in DMSO demonstrated that the helical conformation led to slower chain dynamics in DMF and that despite the higher viscosity of DMSO, the polypeptide side-chains were more mobile as a consequence of the random coil conformation of the linear PGA segments. These results suggest that the formation of structural motives inside a polypeptide slows down its internal dynamics.