Physical characterization of the rack effect and hydrogen bond networks in blue copper proteins

A summary of previous research is presented that indicates that the purpose of a blue copper protein's fold and hydrogen bond network, aka, the rack effect, enforce a copper(II) geometry around the copper(I) ion in the metal site. In several blue copper proteins, the C-terminal histidine ligand becomes protonated and detaches from the copper in the reduced forms. Mutants of amicyanin from Paracoccus denitrificans were made to alter the hydrogen bond network and quantify the rack effect by pK_a shifts.

The pK_a's of mutant amicyanins have been measured by pH-dependent electrochemistry. P94F and P94A mutations loosen the Northern loop, allowing the reduced copper to adopt a relaxed conformation: the ability to relax drives the reduction potentials up. The measured potentials are 265 (wild type), 380 (P94A), and 415 (P94F) mV vs. NHE. The measured pK_a's are 7.0 (wild type), 6.3 (P94A), and 5.0 (P94F). The additional hydrogen bond to the thiolate in the mutants is indicated by a red-shift in the blue copper absorption and an increase in the parallel hyperfine splitting in the EPR spectrum. This hydrogen bond is invoked as the cause for the increased stability of the C-terminal imidazole.

Melting curves give a measure of the thermal stability of the protein. A thermodynamic intermediate with pH-dependent reversibility is revealed. Comparisons with the electrochemistry and apoamicyanin suggest that the intermediate involves the region of the protein near the metal site. This region is destabilized in the P94F mutant; coupled with the evidence that the imidazole is stabilized under the same conditions confirms an original concept of the rack effect: a high energy configuration is stabilized at a cost to the rest of the protein.

Characterization of detectors and instrument systematics for the SPIDER CMB polarimeter

We know from the CMB and observations of large-scale structure that the universe is extremely flat, homogenous, and isotropic. The current favored mechanism for generating these characteristics is inflation, a theorized period of exponential expansion of the universe that occurred shortly after the Big Bang. Most theories of inflation generically predict a background of stochastic gravitational waves. These gravitational waves should leave their unique imprint on the polarization of the CMB via Thompson scattering. Scalar perturbations of the metric will cause a pattern of polarization with no curl (E-mode). Tensor perturbations (gravitational waves) will cause a unique pattern of polarization on the CMB that includes a curl component (B-mode). A measurement of the ratio of the tensor to scalar perturbations (r) tells us the energy scale of inflation. Recent measurements by the BICEP2 team detect the B-mode spectrum with a tensor-to-scalar ratio of r = 0.2 (+0.05, −0.07). An independent confirmation of this result is the next step towards understanding the inflationary universe.

This thesis describes my work on a balloon-borne polarimeter called SPIDER, which is designed to illuminate the physics of the early universe through measurements of the cosmic microwave background polarization. SPIDER consists of six single-frequency, on-axis refracting telescopes contained in a shared-vacuum liquid-helium cryostat. Its large format arrays of millimeter-wave detectors and tight control of systematics will give it unprecedented sensitivity. This thesis describes how the SPIDER detectors are characterized and calibrated for flight, as well as how the systematics requirements for the SPIDER system are simulated and measured.

Fundamental Studies of Carbon Oxygen Bond Activation in Nickel Diphosphine Ether Complexes. And, Metallomacrocycles as Ligands: Synthesis and Characterization of Aluminum-Bridged Bisglyoximato Complexes of Iron and Cobalt

In order to develop better catalysts for the cleavage of aryl-X bonds fundamental studies of the mechanism and individual steps of the mechanism have been investigated in detail. As the described studies are difficult at best in catalytic systems, model systems are frequently used. To study aryl-oxygen bond activation, a terphenyl diphosphine scaffold containing an ether moiety in the central arene was designed. The first three chapters of this dissertation focus on the studies of the nickel complexes supported by this diphosphine backbone and the research efforts in regards to aryl-oxygen bond activation.

Chapter 2 outlines the synthesis of a variety of diphosphine terphenyl ether ligand scaffolds. The metallation of these scaffolds with nickel is described. The reactivity of these nickel(0) systems is also outlined. The systems were found to typically undergo a reductive cleavage of the aryl oxygen bond. The mechanism was found to be a subsequent oxidative addition, β-H elimination, reductive elimination and (or) decarbonylation.

Chapter 3 presents kinetic studies of the aryl oxygen bond in the systems outlined in Chapter 2. Using a series of nickel(0) diphosphine terphenyl ether complexes the kinetics of aryl oxygen bond activation was studied. The activation parameters of oxidative addition for the model systems were determined. Little variation was observed in the rate and activation parameters of oxidative addition with varying electronics in the model system. The cause of the lack of variation is due to the ground state and oxidative addition transition state being affected similarly. Attempts were made to extend this study to catalytic systems.

Chapter 4 investigates aryl oxygen bond activation in the presence of additives. It was found that the addition of certain metal alkyls to the nickel(0) model system lead to an increase in the rate of aryl oxygen bond activation. The addition of excess Grignard reagent led to an order of magnitude increase in the rate of aryl oxygen bond activation. Similarly the addition of AlMe3 led to a three order of magnitude rate increase. Addition of AlMe₃ at -80 °C led to the formation of an intermediate which was identified by NOESY correlations as a system in which the AlMe3 is coordinated to the ether moiety of the backbone. The rates and activation parameters of aryl oxygen bond activation in the presence of AlMe3 were investigated.

The last two chapters involve the study of metalla-macrocycles as ligands. Chapter 5 details the synthesis of a variety of glyoxime backbones and diphenol precursors and their metallation with aluminum. The coordination chemistry of iron on the aluminum scaffolds was investigated. Varying the electronics of the aluminum macrocycle was found to affect the observed electrochemistry of the iron center.

Chapter 6 extends the studies of chapter 5 to cobalt complexes. The synthesis of cobalt dialuminum glyoxime metal complexes is described. The electrochemistry of the cobalt complexes was investigated. The electrochemistry was compared to the observed electrochemistry of a zinc analog to identify the redox activity of the ligand. In the presence of acid the cobalt complexes were found to electrochemically reduce protons to dihydrogen. The electronics of the ancillary aluminum ligands were found to affect the potential of proton reduction in the cobalt complexes. These potentials were compared to other diglyoximate complexes.

Structure-Guided Design and Biophysical Characterization of Novel Anti-HIV Reagents

Despite over 30 years of effort, an HIV-1 vaccine that elicits protective antibodies still does not exist. Recent clinical studies have identified that during natural infection about 20% of the population is capable of mounting a potent and protective antibody response. Closer inspection of these individuals reveal that a subset of these antibodies, recently termed potent VRC01-like (PVL), derive exclusively from a single human germline heavy chain gene. Induced clonal expansion of the B cell encoding this gene is the first step through which PVL antibodies may be elicited. Unfortunately, naturally occurring HIV gp120s fail to bind to this germline, and as a result cannot be used as the initial prime for a vaccine regimen. We have determined the crystal structure of an important germline antibody that is a promising target for vaccine design efforts, and have set out to engineer a more likely candidate using computationally-guided rational design.

In addition to prevention efforts on the side of vaccine design, recently characterized broadly neutralizing anti-HIV antibodies have excellent potential for use in gene therapy and passive immunotherapy. The separation distance between functional Fabs on an antibody is important due to the sparse distribution of envelop spikes on HIV compared to other viruses. We set out to build and characterize novel antibody architectures by incorporating structured linkers into the hinge region of an anti-HIV antibody b12. The goal was to observe whether these linkers increased the arm-span of the IgG dimer. When incorporated, flexible Gly4Ser repeats did not result in detectable extensions of the IgG antigen binding domains, by contrast to linkers including more rigid domains such as β2-microglobulin, Zn-α2-glycoprotein, and tetratricopeptide repeats (TPRs). This study adds an additional set of linkers with varying lengths and rigidities to the available linker repertoire, which may be useful for the modification and construction of antibodies and other fusion proteins.

Structural and Biophysical Characterization of Variants of the Mechanosensitive Channel of Large Conductance (MSCL)

The ability to sense mechanical force is vital to all organisms to interact with and respond to stimuli in their environment. Mechanosensation is critical to many physiological functions such as the senses of hearing and touch in animals, gravitropism in plants and osmoregulation in bacteria. Of these processes, the best understood at the molecular level involve bacterial mechanosensitive channels. Under hypo-osmotic stress, bacteria are able to alleviate turgor pressure through mechanosensitive channels that gate directly in response to tension in the membrane lipid bilayer. A key participant in this response is the mechanosensitive channel of large conductance (MscL), a non-selective channel with a high conductance of ~3 nS that gates at tensions close to the membrane lytic tension.

It has been appreciated since the original discovery by C. Kung that the small subunit size (~130 to 160 residues) and the high conductance necessitate that MscL forms a homo-oligomeric channel. Over the past 20 years of study, the proposed oligomeric state of MscL has ranged from monomer to hexamer. Oligomeric state has been shown to vary between MscL homologues and is influenced by lipid/detergent environment. In this thesis, we report the creation of a chimera library to systematically survey the correlation between MscL sequence and oligomeric state to identify the sequence determinants of oligomeric state. Our results demonstrate that although there is no combination of sequences uniquely associated with a given oligomeric state (or mixture of oligomeric states), there are significant correlations. In the quest to characterize the oligomeric state of MscL, an exciting discovery was made about the dynamic nature of the MscL complex. We found that in detergent solution, under mild heating conditions (37 °C – 60 °C), subunits of MscL can exchange between complexes, and the dynamics of this process are sensitive to the protein sequence.

Extensive efforts were made to produce high diffraction quality crystals of MscL for the determination of a high resolution X-ray crystal structure of a full length channel. The surface entropy reduction strategy was applied to the design of S. aureus MscL variants and while the strategy appears to have improved the crystallizability of S. aureus MscL, unfortunately the diffraction qualities of these crystals were not significantly improved. MscL chimeras were also screened for crystallization in various solubilization detergents, but also failed to yield high quality crystals.

MscL is a fascinating protein and continues to serve as a model system for the study of the structural and functional properties of mechanosensitive channels. Further characterization of the MscL chimera library will offer more insight into the characteristics of the channel. Of particular interest are the functional characterization of the chimeras and the exploration of the physiological relevance of intercomplex subunit exchange.

Engineering and Characterization of Cytochrome P450 Enzymes for Nitrogen-Atom Transfer Reactions

The creation of novel enzyme activity is a great challenge to protein engineers, but nature has done so repeatedly throughout the process of natural selection. I begin by outlining the multitude of distinct reactions catalyzed by a single enzyme class, cytochrome P450 monooxygenases. I discuss the ability of cytochrome P450 to generate reactive intermediates capable of diverse reactivity, suggesting this enzyme can also be used to generate novel reactive intermediates in the form of metal-carbenoid and nitrenoid species. I then show that cytochrome P450 from Bacillus megaterium (P450_BM3) and its isolated cofactor can catalyze metal-nitrenoid transfer in the form of intramolecular C–H bond amination. Mutations to the protein sequence can enhance the reactivity and selectivity of this transformation significantly beyond that of the free cofactor. Next, I demonstrate an intermolecular nitrene transfer reaction catalyzed by P450_BM3 in the form of sulfide imidation. Understanding that sulfur heteroatoms are strong nucleophiles, I show that increasing the sulfide nucleophilicity through substituents on the aryl sulfide ring can dramatically increase reaction productivity. To explore engineering nitrenoid transfer in P450BM3, active site mutagenesis is employed to tune the regioselectivity intramolecular C–H amination catalysts. The solution of the crystal structure of a highly selective variant demonstrates that hydrophobic residues in the active site strongly modulate reactivity and regioselectivity. Finally, I use a similar strategy to develop P450-based catalysts for intermolecular olefin aziridination, demonstrating that active site mutagenesis can greatly enhance this nitrene transfer reaction. The resulting variant can catalyze intermolecular aziridination with more than 1000 total turnovers and enantioselectivity of up to 99% ee.

Isolation of the murine interleukin 2 gene and characterization of its regulatory architecture

Interleukin 2 (IL2) is the primary growth hormone used by mature T cells and this lymphokine plays an important role in the magnification of cell-mediated immune responses. Under normal circumstances its expression is limited to antigen-activated type 1 helper T cells (T_H1) and the ability to transcribe this gene is often regarded as evidence for commitment to this developmental lineage. There is, however, abundant evidence than many non-T_H1 T cells, under appropriate conditions, possess the ability to express this gene. Of paramount interest in the study of T-cell development is the mechanisms by which differentiating thymocytes are endowed with particular combinations of cell surface proteins and response repertoires. For example, why do most helper T cells express the CD4 differentiation antigen?

As a first step in understanding these developmental processes the gene encoding IL2 was isolated from a mouse genomic library by probing with a conspecific IL2 cDNA. The sequence of the 5' flanking region from + 1 to -2800 was determined and compared to the previously reported human sequence. Extensive identity exists between +1 and -580 (86%) and sites previously shown to be crucial for the proper expression of the human gene are well conserved in both sequence location in the mouse counterpart.

Transient expression assays were used to evaluate the contribution of various genomic sequences to high-level gene expression mediated by a cloned IL2 promoter fragment. Differing lengths of 5' flanking DNA, all terminating in the 5' untranslated region, were linked to a reporter gene, bacterial chloramphenicol acetyltransferase (CAT) and enzyme activity was measured after introduction into IL2-producing cell lines. No CAT was ever detected without stimulation of the recipient cells. A cloned promoter fragment containing only 321 bp of upstream DNA was expressed well in both Jurkat and EL4.El cells. Addition of intragenic or downstream DNA to these 5' IL2-CAT constructs showed that no obvious regulatory regions resided there. However, increasing the extent of 5' DNA from -321 to -2800 revealed several positive and negative regulatory elements. One negative region that was well characterized resided between -750 and -1000 and consisted almost exclusively of alternating purine and pyrimidines. There is no sequence resembling this in the human gene now, but there is evidence that there may have once been.

No region, when deleted, could relax either the stringent induction-dependence on cell-type specificity displayed by this promoter. Reagents that modulated endogenous IL2 expression, such as cAMP, cyclosporin A, and IL1, affected expression of the 5' IL2-CAT constructs also. For a given reagent, expression from all expressible constructs was suppressed or enhanced to the same extent. This suggests that these modulators affect IL2 expression through perturbation of a central inductive signal rather than by summation of the effects of discrete, independently regulated, negative and positive transcription factors.

Characterization and modeling of premixed turbulent n-heptane flames in the thin reaction zone regime

n-heptane/air premixed turbulent flames in the high-Karlovitz portion of the thin reaction zone regime are characterized and modeled in this thesis using Direct Numerical Simulations (DNS) with detailed chemistry. In order to perform these simulations, a time-integration scheme that can efficiently handle the stiffness of the equations solved is developed first. A first simulation with unity Lewis number is considered in order to assess the effect of turbulence on the flame in the absence of differential diffusion. A second simulation with non-unity Lewis numbers is considered to study how turbulence affects differential diffusion. In the absence of differential diffusion, minimal departure from the 1D unstretched flame structure (species vs. temperature profiles) is observed. In the non-unity Lewis number case, the flame structure lies between that of 1D unstretched flames with "laminar" non-unity Lewis numbers and unity Lewis number. This is attributed to effective Lewis numbers resulting from intense turbulent mixing and a first model is proposed. The reaction zone is shown to be thin for both flames, yet large chemical source term fluctuations are observed. The fuel consumption rate is found to be only weakly correlated with stretch, although local extinctions in the non-unity Lewis number case are well correlated with high curvature. These results explain the apparent turbulent flame speeds. Other variables that better correlate with this fuel burning rate are identified through a coordinate transformation. It is shown that the unity Lewis number turbulent flames can be accurately described by a set of 1D (in progress variable space) flamelet equations parameterized by the dissipation rate of the progress variable. In the non-unity Lewis number flames, the flamelet equations suggest a dependence on a second parameter, the diffusion of the progress variable. A new tabulation approach is proposed for the simulation of such flames with these dimensionally-reduced manifolds.

Structural Characterization of Pro-inflammatory and Anti-inflammatory Immunoglobulin G Fc Proteins

Immunoglobulin G (IgG) is central in mediating host defense due to its ability to target and eliminate invading pathogens. The fragment antigen binding (Fab) regions are responsible for antigen recognition; however the effector responses are encoded on the Fc region of IgG. IgG Fc displays considerable glycan heterogeneity, accounting for its complex effector functions of inflammation, modulation and immune suppression. Intravenous immunoglobulin G (IVIG) is pooled serum IgG from multiple donors and is used to treat individuals with autoimmune and inflammatory disorders such as rheumatoid arthritis and Kawasaki’s disease, respectively. It contains all the subtypes of IgG (IgG1-4) and over 120 glycovariants due to variation of an Asparagine 297-linked glycan on the Fc. The species identified as the activating component of IVIG is sialylated IgG Fc. Comparisons of wild type Fc and sialylated Fc X-ray crystal structures suggests that sialylation causes an increase in conformational flexibility, which may be important for its anti-inflammatory properties.

Although glycan modifications can promote the anti-inflammatory properties of the Fc, there are amino acid substitutions that cause Fcs to initiate an enhanced immune response. Mutations in the Fc can cause up to a 100-fold increase in binding affinity to activating Fc gamma receptors located on immune cells, and have been shown to enhance antibody dependent cell-mediated cytotoxicity. This is important in developing therapeutic antibodies against cancer and infectious diseases. Structural studies of mutant Fcs in complex with activating receptors gave insight into new protein-protein interactions that lead to an enhanced binding affinity.

Together these studies show how dynamic and diverse the Fc region is and how both protein and carbohydrate modifications can alter structure, leading to IgG Fc’s switch from a pro-inflammatory to an anti-inflammatory protein.