Rozprawy doktorskie na temat „Protein-water systems”

Cell-free protein synthesis is an emerging technology that has many applications. The open nature of this system makes it a compelling technology that can be manipulated to answer many needs that are unavailable in other systems. This dissertation reports on engineering this technology for: 1) sense codon emancipation for incorporation of multiple unnatural amino acids; 2) expressing a hard-to-express anticancer biotherapeutic and introducing a just-add-water system; 3) a biosensing ligand that interacts with nuclear hormone receptors. Emancipating sense codons toward a minimized genetic code is of significant interest to science and engineering. A promising approach to sense codon emancipation is the targeted in vitro removal of native tRNA. Here we introduce a new in-vitro or "cell-free" approach to emancipate sense codons via efficient and affordable degradation of endogenous tRNA using RNase-coated superparamagnetic beads. The presented method removes greater than 99% of tRNA in cell lysates, while preserving cell-free protein synthesis activity. The resulting tRNA-depleted lysate is compatible with in vitro-transcribed synthetic tRNA for the production of peptides and proteins. Biotherapeutics have many promising applications, such as anti-cancer treatments, immune suppression, and vaccines. However, due to their biological nature, some biotherapeutics can be challenging to rapidly express and screen for activity through traditional recombinant methods. In this work, we demonstrate the use of cell-free systems for the expression and direct screening of the difficult-to-express cytotoxic protein onconase. Using cell-free systems, onconase can be rapidly expressed in soluble, active form. Furthermore, the open nature of the reaction environment allows for direct and immediate downstream characterization without the need of purification. Also, we report the ability of a "just-add-water" lyophilized cell-fee system to produce onconase. Here we introduce a Rapid Adaptable Portable In-vitro Detection biosensor platform (RAPID) for detecting ligands that interact with nuclear hormone receptors (NHRs). The biosensor is based on an engineered, allosterically-activated fusion protein, which contains the ligand binding domain from a target NHR. The presented RAPID biosensor platform is significantly faster and less labor intensive than commonly available technologies, making it a promising tool for detecting environmental EDC contamination and screening potential NHR-targeted pharmaceuticals.

The thesis contains theoretical studies of the structure and dynamics in different complex systems. Depending on the systems and properties of interest we divide the thesis into five parts. In the first part, we study chemical dynamics in nanoconfined water. Here we primarily focus on the dielectric properties of dipolar fluids confined inside nano-containers of various sizes and shapes. We discover an extremely slow convergence of the static dielectric constant (ε) of water with the size of the nanospheres. Our studies reveal an ultrafast relaxation of the collective orientation of water which is absent in the Stockmayer fluid. We connect this anomaly to the substantially low value of the Kirkwood g-factor for spherically confined water. We extrapolate the values of ε to obtain its true value in the thermodynamic limit which corroborates well with the values in periodic systems. We perform molecular dynamics simulations with three different liquid-surface interactions to study the surface effects. The dielectric response of water under becomes anisotropic in non-spherical confinements, namely, cylindrical and slab geometries. Because of the difference in the dielectric boundary conditions along the different directions, the eigenvalues of the dielectric tensor becomes unequal. We derive the fluctuation formulae for the anisotropic dielectric constants. For the cylindrical geometry, we find that the axial component (εz) and the perpendicular component (εx/y) converge to the bulk value, with the diameter of the nanotube, in an opposite manner. εx/y shows relatively slower convergence starting from a lower value whereas εz shows faster convergence starting from a higher value. For the slab system, the parallel component (ε∥) does not show much deviation from the bulk value. On the contrary, the perpendicular component (ε⊥) exhibits extremely low values for smaller systems and shows a slow convergence toward bulk. Interestingly, in the slab geometry, the dielectric relaxation along the perpendicular direction becomes ultrafast with pronounced oscillations. Our results match well with recent dielectric microscopy experiments. We perform a constrained Ising model-based analysis and to understand the inwardly propagating destructions among correlations. In addition to the above, we study the heterogeneous dynamics of water inside nano-enclosures of different shapes. We investigate the position-dependent solvation of model ionic/dipolar probes and find that the slow components of solvation show highly non-monotonic behaviour with the distance of the probe from the surface. To study the static and dynamical heterogeneity, we obtain the non-Gaussian parameter [α2(t)] and non-linear density response function [χ4(t)]. α2(t) shows an anomalous long-time growth for non-spherical systems which we attribute to the slow dynamics of water along the non-periodic direction(s). In the second part, we focus on the chemical dynamics in small-sized (~μm) droplets that exhibit noticeably different chemistry than bulk water. Several chemical reactions show markedly enhanced rates in the droplet media. Here we present a generalized theoretical model and analytically solve the adjoint equations for two- and three-dimensional systems. We obtain exact expressions for the mean search time (MST) that is found to be proportional to R2/D [R=radius, D=relative diffusion constant]. We carry out Brownian dynamics simulations and show that the MST of reactive partner search in droplets is orders of magnitude smaller than that in the bulk. As the experiments often use an external electric field to charge the droplets, we study the effect of ions and electric fields on the bond dissociation energy. We find that the presence of an ion or electric field weakens the bond and enhances the intrinsic reaction rate. Our results describe the interplay between diffusion control and activation controlled processes. In the third part, we study the dynamics of interfacial water molecules in the biomolecular hydration layers. Here we first aim to resolve a long-standing controversy regarding the timescale of water dynamics in the protein hydration layer, that is, solvation dynamics and dielectric relaxation finds substantial slow relaxation whereas NMR experiments find retardation only by a factor of 2-3. To this goal, we obtain distributions of single-particle relaxation timescales and show that the average values obtained from experiments hide the true picture. To our surprise, we find the existence of both faster and slower than bulk water molecules in the hydration layer. We unravel the origin of disparate timescales (from sub-100 fs to hundreds of ps) observed in the solvation dynamics of natural probe tryptophan tagged to three different proteins- Lysozyme, Myoglobin, and sweet protein Monellin. We show that the neighbourhood charged residues and the intrinsic side-chain fluctuations contribute to the observed slow dynamics. We further decompose the response into protein core, side-chains, and water contributions to study the nature of coupling. We find surprising anti-correlation between the energy fluctuations of protein and water. Our simulation results support the widely discussed protein-solvent slaving picture developed from earlier Mössbauer spectroscopy experiments. We also study the origin of the power law decay observed in DNA solvation dynamics. We employ the Oosawa model, continuum model, mode coupling theory, and continuous time random walk based analyses to unearth the effect of counterion motions. We study the solvation dynamics of DNA bases and a minor groove bound probe. In the fourth part, we characterize the various protonated forms of metformin hydrochloride (MET) by employing computational and spectroscopic techniques. We develop an AMBER based force-field for three protonation states of MET and validate them against available experimental results. We use this force-field to study the interaction of MET with double-stranded B-DNAs. As there is evidence of anti-tumour and anti-cancer activity of MET, we aim to understand its mode of binding with DNA. We employ metadynamics based advanced sampling techniques to obtain the free energy landscape of the binding process. We find that MET prefers AT-rich minor grooves through non-intercalative mode of binding. We confirm these claims from fluorescence spectroscopy and circular dichroism experiments. In the fifth and last part of the thesis, we study the structure and aggregation of insulin oligomers (dimer and hexamer) in water and water-ethanol binary mixtures. Insulin is biologically active in its monomeric form but gets stored in the pancreas as hexamers. The inter-conversion between monomer and hexamer occurs via dimeric intermediates. Here we present the structural analysis of insulin hexamer in water and water-ethanol binary mixture by using atomistic molecular dynamics and X-ray crystallography. We find that the water molecules trapped inside the central hydrophilic cavity of hexamer play a central role in sustaining the robust barrel-shaped structure. The presence of ethanol (even in lower concentrations) deforms the hexameric assembly. Next, we study the energetics of the insulin dimer association/dissociation process. We obtain the free energy landscape from parallel tempering metadynamics simulations in a well-tempered ensemble with respect to two collective variables- inter-monomeric distance and the number of inter-monomeric contacts. We find that the activation barrier of dissociation drastically decreases in the presence of 5% and 10% (v/v) ethanol. We attribute this effect to the preferential solvation of the dimer forming hydrophobic surface of the monomers that results in the destruction of inter-monomeric hydrogen bonding. We analyze the evolving structures along the minimum energy pathway and establish the role of the solvent.

Water plays a critical role in the function and structure of biological systems. Current techniques to study biologically relevant events that span many length and time scales are limited by the prohibitive computational cost of including accurate effects from the aqueous environment. The aim of this work is to expand the reach of current molecular dynamics techniques by reducing the computational cost for achieving an accurate description of water and its effects on biomolecular systems. This work builds from the assumption that the “local” effect of water (e.g. the local orientational preferences and hydrogen bonding) can be effectively modelled considering only the atomistic detail in a very limited region. A recent adaptive resolution simulation technique (AdResS) has been developed to practically apply this idea; in this work it will be extended to systems of simple hydrophobic solutes to determine a characteristic length for which thermodynamic, structural, and dynamic properties are preserved near the solute. This characteristic length can then be used for simulation of biomolecular systems, specifically those involving protein dynamics in water. Before this can be done, current coarse grain models must be adapted to couple with a coarse grain model of water. This thesis is organized in to five chapters. The first will give an overview of water, and the current methodologies used to simulate water in biological systems. The second chapter will describe the AdResS technique and its application to simple test systems. The third chapter will show that this method can be used to accurately describe hydrophobic solutes in water. The fourth chapter describes the use of coarse grain models as a starting point for targeted search with all-atom models. The final chapter will describe attempts to couple a coarse grain model of a protein with a single-site model for water, and it’s implications for future multi-resolution studies.

This thesis deals with the understanding of the structure, dynamics and thermodynamics in complex systems (proteins, hydration layers, DNA, ice polymorphs) by employing computer simulations, theoretical analysis, and in some cases, collaborative experimental efforts. There are mainly 5 parts with 13 chapters. In Part I, we study the effects of solvent on the structure and stabilization of insulin hexamer. In Chapter 1 we discuss the structural features of the different oligomers of insulin. Chapter 2 deals with insulin hexamer in neat water. We find that a group of ~10 water molecules present in the cavity of insulin hexamer is crucial in stabilizing the structure of the biomolecular assembly. In Chapter 3, we study the effect of ethanol on the structure of insulin hexamer. Ethanol, by virtue of its amphiphilic nature, interacts with both hydrophilic and hydrophobic residues, thereby destroying the native state structure of insulin hexamer. In Part II, we study the interactions between several proteins and water. We start with a brief introduction of the different techniques used to study protein hydration layer (PHL) in Chapter 4. In Chapter 5, we find that the protein self-interaction energy fluctuations are strongly anti-correlated to the protein-water cross interaction energy fluctuations. The total energy spectrum of protein shows bimodal 1/f noise characteristics. We posit that water exerts control over protein dynamics via an exchange of energy between these two domains. In Chapter 6, from distributions of dynamical timescales, we find that PHL contains both fast and slow water molecules. Shell-wise decomposition of the PHL demonstrates a gradual increase of dielectric constant and decrease of specific heat from the protein surface to the bulk. In chapter 7 we study the heterogeneous solvation dynamics of protein. We find that the slow component in the solvation relaxation originates from side chain and hydration layer fluctuations. Charged neighbourhood of the probe results in slower dynamics. Cross-correlations between water and side-chains are anti-correlated, making the solvation faster. In Part III, we study the solvation dynamics of DNA. In Chapter 8 we briefly review the literature in this field. In Chapter 9 we employ multiple theoretical and simulation analyses to understand the origin of the long time power law behaviour in the solvation dynamics of DNA. We find that electrolytic friction and movement of ions along the DNA backbone could be responsible for this mysterious behaviour. Part IV deals with the different phases of water and their transitions. In Chapter 10, we discuss the phase diagram of water and its multiple regions. In Chapter 11 we compare the solid-liquid interfaces in TIP4P/ice and mW water models with Lennard-Jones argon. We find that ice-water interface is much sharper than its LJ counterpart, mainly due to sharp change in rotational entropy. We also study the growth rate of ice at different temperatures and compare it with experimental observations. We find that Wilson-Frenkel equation of crystal growth breaks down at higher temperatures. In Chapter 12, we observe the pressure induced crystal to glass transition in low density ice. High density crystalline ice show no transition. We find that hydrogen bond defects can be used as effective order parameters to study these phase transitions. At very high pressure (150 kbar), we observe the emergence of crystalline order from the disordered glassy phase. In Part V, we study of a small medicinally important molecule metformin. In Chapter 13, we develop a force field of this molecule and validate it with multiple experimental observations. We study the structural and dynamical features of metformin and develop a free energy landscape of DNA-metformin interactions. The last part of the thesis (Part VI) contains a single chapter (Chapter 14) which includes the concluding remarks and the future plans and research prospects derived from this thesis.
INSPIRE Fellowship, DST, India

The thesis presents detailed results of theoretical analyses based on extensive computer simulation studies with an aim to explore, quantify whenever possible, and understand the collective excitations, relaxation processes and temperature dependent phase separation kinetics in several binary mixtures. We also investigate the structure and dynamics of water in the vicinity of several biologically active proteins and small hydrophobes. Based on the phenomena studied the thesis has been divided into four major parts I. Collective excitations and ultrafast solvation dynamics in binary mixtures II. Non-equilibrium solvation dynamics in binary mixture: Composition dependence of non-linear relaxation III.Nanoscale heterogeneous phase separation kinetics in binary mixtures: Multistage dynamics IV.Spatial dependence of dielectric constant in protein-water systems and hydrophobic hydration driven self-assembly of hydrophobic molecules in water: Role of nucleation

碩士
國立宜蘭大學
生物技術研究所碩士班
97
Since the injection administration as the peptides and proteins delivery system, it imposes discomfort and inconvenience on patients, particularly for a long-term treatment. Therefore, the goal of this thesis is to find out an oral delivery system for improving the absorption of therapeutic protein drugs. Emulsion is one of strategies known delivery vehicles for those oral medicines. Water-in-oil -in-water (W/O/W) multiple emulsion has been known to be possible to protect protein drugs from enzyme degradation and enhance the absorption of hydrophilic drugs to systemic circulation. Therefore, we tried to use granulocyte colony-stimulating factor (G-CSF) as the indicator to test the emulsion formulation for the oral delivery system in the study. In this two-step emulsification procedure, the data of the droplet size (2 μm of diameter), encapsulation efficiency (40±5%), stability (at pH 1.2 for 2 hrs and 60°C for 5 days), permeability (into Caco-2 cell monolayer with 30 min) and in vitro cytotoxicity studies all showed that the emulsion formulation was indicated to promote the absorption of proteins drugs by oral administration. Thus, we used the E. coli expression system to express and purify the recombinant human G-CSF (rhG-CSF). And then, rhG-CSF was incorporated into the inner aqueous phase of the emulsion for the test of oral bioavailability. After 6 hrs of oral administration of various dosages of rhG-CSF in solution or in emulsion for 7 days, the proliferations of leukocytes in the two treatments with the highest dose of rhG-CSF (2,500 μg/kg/day) were significantly higher than the blank emulsion (p < 0.01). However, two of them, rhG-CSF in emulsion and not in emulsion, were not different. After 12 hrs of oral administration, mice, administrated by 50, 500, and 2500 μg/kg/day rhG-CSF emulsion, all presented a significant elevation in leukocyte numbers when compared to the blank emulsion (p < 0.01). Finally, it was indicated about the advantages of the W/O/W multiple emulsions with encapsulation efficiency, penetrating ability, and stability to resist acid pH (1.2) and high temperature (60°C). In the future, it could be potential for protein drugs delivery by oral administration.

碩士
東海大學
化學工程學系
92
Because of its extremely powerful resolution, reverse phase high pressure liquid chromatography (RP-HPLC) has been widely employed in the preparative- and large-scale of separation and purification for enormous proteins, peptides, and amino acids. But its application is hindered by the involvement of high concentration of organic solvents that might affect the protein stability and activity. Acetonitrile (ACN)-water system is the most commonly used mobile phase in RP-HPLC. Normally, proteins are eluted from RP-HPLC columns with relatively high concentrations of ACN(＞60%v/v), in which have to be removed by either buffer exchange or lyophilization in the following stages. For the chemical safety considerations and their influences on the separation membrane, chromatography resins, and vacuum evaporator system during lyophilization, people are sometimes hesitatied to apply the RP-HPLC technique in their purification processes. It has been demonstrated that the phase separation occurring under sub-zero temperature could remove the majority ACN. However, the sub-zero operation temperature may provoke sample freeze and the severe temperature shift will affect the protein properties. In this study, we combined the salt addition and cooling procedures to improve the efficiency at the above-zero temperature. We found that by the addition of property salts, such as potassium phosphate buffer (pH=7.0), phase separation of ACN-water could come about effectively at 4℃, whereas the protein purification process are normally carried out. We investigated the percentage of ACN removal and the recovery of protein from the ACN solution by altering the concentrations of salt, initial ACN, as well as sample protein, respectively. We also examined the effects of the proteins natures, such as proteins with different hydrophobicity, on the protein distribution between the ACN-rich phase and the water-rich phase. With an optimized condition for this salt-induced phase separation, our results demonstrated that phase separation efficiency of ACN-water system by the addition of small amount of proper salts(such as potassium phosphate buffer, pH=7.0 ) at 4℃could achieve the similar efficiency as that previously described by cooling to a sub-zero temperature. Meanwhile, the greater than 50% of ACN could be simply removed, while more than 90% of protein could be easily recovered. Therefore, the modified phase separation technique by coupling cooling and salt addition procedure offers us the more economic, more effective, and more reliable approach to achieve the buffer exchange, removing at concentrating protein extent for proteins, as well as maintaining protein stability and activity in general protein purification processes.