Use of pseudoephedrine as a practical chiral auxiliary for asymmetric synthesis

The use of pseudoephedrine as a practical chiral auxiliary for asymmetric synthesis is describe. Both enantiomers of pseudoephedrine are inexpensive commodity chemicals and can be N-acylated in high yields to form tertiary amides. In the presence of lithium chloride, the enolates of the corresponding pseudoephedrine amides undergo highly diastereoselective a1kylations with a wide range of alkyl halides to afford α-substituted products in high yields. These products can then be transformed in a single operation into highly enantiomerically enriched carboxylic acids, alcohols, and aldehydes. Lithium amidotrihydroborate (LAB) is shown to be a powerful reductant for the selective reduction of tertiary amides in general and pseudoephedrine amides in particular to form primary alcohols.

On the Mumford-Tate conjecture for Abelian varieties with reduction conditions

In this thesis we study Galois representations corresponding to abelian varieties with certain reduction conditions. We show that these conditions force the image of the representations to be "big," so that the Mumford-Tate conjecture (:= MT) holds. We also prove that the set of abelian varieties satisfying these conditions is dense in a corresponding moduli space.

The main results of the thesis are the following two theorems.

Theorem A: Let A be an absolutely simple abelian variety, End° (A) = k : imaginary quadratic field, g = dim(A). Assume either dim(A) ≤ 4, or A has bad reduction at some prime ϕ, with the dimension of the toric part of the reduction equal to 2r, and gcd(r,g) = 1, and (r,g) ≠ (15,56) or (m -1, m(m+1)/2). Then MT holds.

Theorem B: Let M be the moduli space of abelian varieties with fixed polarization, level structure and a k-action. It is defined over a number field F. The subset of M(Q) corresponding to absolutely simple abelian varieties with a prescribed stable reduction at a large enough prime ϕ of F is dense in M(C) in the complex topology. In particular, the set of simple abelian varieties having bad reductions with fixed dimension of the toric parts is dense.

Besides this we also established the following results:

(1) MT holds for some other classes of abelian varieties with similar reduction conditions. For example, if A is an abelian variety with End° (A) = Q and the dimension of the toric part of its reduction is prime to dim( A), then MT holds.

(2) MT holds for Ribet-type abelian varieties.

(3) The Hodge and the Tate conjectures are equivalent for abelian 4-folds.

(4) MT holds for abelian 4-folds of type II, III, IV (Theorem 5.0(2)) and some 4-folds of type I.

(5) For some abelian varieties either MT or the Hodge conjecture holds.

Reactions of small molecules facilitated by metal-acceptor interactions

The role of metal-acceptor interactions arising from M–BR3 and M–PR3 bonding is discussed with respect to reactions between first-row transition metals and N2, H2, and CO. Thermally robust, S = 1/2 (TPB)Co(H2) and (TPB)Co(N2) complexes (TPB = tris(2- (diisopropylphosphino)phenyl)borane) are described and the energetics of N2 and H2 binding are measured. The H2 and N2 ligands are bound more weakly in the (TPB)Co complexes than in related (SiP3)M(L) complexes (SiP3 = tris(2- (diisopropylphosphino)phenyl)silyl). Comparisons within and between these two ligand platforms allow for the factors that affect N2 (and H2) binding and activation to be delineated. The characterization and reactivity of (DPB)Fe complexes (DPB = bis(2- (diisopropylphosphino)phenyl)phenylborane) in the context of N2 functionalization and E–H bond addition (E = H, C, N, Si) are described. This platform allows for the one-pot transformation of free N2 to an Fe hydrazido(-) complex via an Fe aminoimide intermediate. The principles learned from the N2 chemistry using (DPB)Fe are applied to CO reduction on the same system. The preparation of (DPB)Fe(CO)2 is described as well as its reductive functionalization to generate an unprecedented Fe dicarbyne. The bonding in this highly covalent complex is discussed in detail. Initial studies of the reactivity of the Fe dicarbyne reveal that a CO-derived olefin is released upon hydrogenation. Alternative approaches to uncovering unusual reactivity using metal- acceptor interactions are described in Chapters 5 and 6, including initial studies on a new π-accepting tridentate diphosphinosulfinyl ligand and strategies for designing ligands that undergo site-selective metallation to generate heterobimetallic complexes.

Geometric quantization and foliation reduction

A standard question in the study of geometric quantization is whether symplectic reduction interacts nicely with the quantized theory, and in particular whether “quantization commutes with reduction.” Guillemin and Sternberg first proposed this question, and answered it in the affirmative for the case of a free action of a compact Lie group on a compact Kähler manifold. Subsequent work has focused mainly on extending their proof to non-free actions and non-Kähler manifolds. For realistic physical examples, however, it is desirable to have a proof which also applies to non-compact symplectic manifolds.

In this thesis we give a proof of the quantization-reduction problem for general symplectic manifolds. This is accomplished by working in a particular wavefunction representation, associated with a polarization that is in some sense compatible with reduction. While the polarized sections described by Guillemin and Sternberg are nonzero on a dense subset of the Kähler manifold, the ones considered here are distributional, having support only on regions of the phase space associated with certain quantized, or “admissible”, values of momentum.

We first propose a reduction procedure for the prequantum geometric structures that “covers” symplectic reduction, and demonstrate how both symplectic and prequantum reduction can be viewed as examples of foliation reduction. Consistency of prequantum reduction imposes the above-mentioned admissibility conditions on the quantized momenta, which can be seen as analogues of the Bohr-Wilson-Sommerfeld conditions for completely integrable systems.

We then describe our reduction-compatible polarization, and demonstrate a one-to-one correspondence between polarized sections on the unreduced and reduced spaces.

Finally, we describe a factorization of the reduced prequantum bundle, suggested by the structure of the underlying reduced symplectic manifold. This in turn induces a factorization of the space of polarized sections that agrees with its usual decomposition by irreducible representations, and so proves that quantization and reduction do indeed commute in this context.

A significant omission from the proof is the construction of an inner product on the space of polarized sections, and a discussion of its behavior under reduction. In the concluding chapter of the thesis, we suggest some ideas for future work in this direction.

Oxidative chemisorption of SO_2 on γ - Al_2O_3 and deposition of H_2-permselective SiO_2 films

The interaction of SO_2 with γ - Al_2O_3 and the deposition of H_2 permselective SiO_2 films have been investigated. The adsorption and oxidative adsorption of SO_2 on γ - Al_2O_3 have been examined at temperatures 500-700°C by Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). At temperatures above 500°C most of SO_2 adsorbed on the strong sites on alumina. The adsorbed SO_2 species was characterized by an IR band at 1065 cm^(-1). The equilibrium coverage and initial rate of adsorption decreased with temperature suggesting a two-step adsorption. When γ - Al_2O_3 was contacted with a mixture of SO_2 and O_2, adsorption of SO_2 and oxidation of the adsorbed SO_2 to a surface sulfate characterized by broad IR bands at 1070 cm^(-1), 1390 cm^(-1) took place. The results of a series of TGA experiments under different atmospheres strongly suggest that surface SO_2 and surface sulfate involve the same active sites such that SO_2 adsorption is inhibited by already formed sulfate. The results also indicate a broad range of site strengths.

The desorption of adsorbed SO_2 and the reductive desorption of oxidatively adsorbed SO_2 have been investigated by microreactor experiments and thermogravimetric analysis (TGA). Temperature programmed reduction (TPR) of adsorbed SO_2 showed that SO_2 was desorbed without significant reaction with H_2 when H_2 concentration was low while considerable reaction occurred when 100% H_2 was used. SO_2 adsorbed on the strong sites on alumina was reduced to sulfur and H_2S. The isothermal reduction experiments of oxidatively adsorbed SO_2 reveal that the rate of reduction is very slow below 550°C even with 100% H_2. The reduction product is mainly composed of SO_2. TPR experiments of oxidatively adsorbed SO_2 showed that H_2S arose from a sulfate strongly chemisorbed on the surface.

Films of amorphous SiO_2 were deposited within the walls of porous Vycor tubes by SiH_4 oxidation in an opposing reactants geometry : SiH_4 was passed inside the tube while O_2 was passed outside the tube. The two reactants diffused opposite to each other and reacted within a narrow front inside the tube wall to form a thin SiO_2 film. Once the pores were plugged the reactants could not reach each other and the reaction stopped. At 450°C and 0.1 and 0.33 atm of SiH_4 and O_2, the reaction was complete within 15 minutes. The thickness of the SiO_2 film was estimated to be about 0.1 µm. Measurements of H_2 and N_2 permeation rates showed that the SiO_2 film was highly selective to H_2 permeation. The H_2:N_2 flux at 450°C varied between 2000-3000.

Thin SiO_2 films were heat treated in different gas mixtures to determine their stability in functioning as high-temperature hydrogen-permselective membranes. The films were heat-treated at 450-700°C in dry N_2, dry O_2, N_2-H_2O, and O_2-H_2O mixtures. The permeation rates of H_2 and N_2 changed depending on the original conditions of film formation as well as on the heat treatment. Heating in dry N_2 slowly reduced the permeation rates of both H_2 and N_2. Heating in a N_2-H_2O atmosphere led to a steeper decline of H_2 permeability. But the permeation rate of N_2 increased or decreased according to whether the film deposition had been carried out in the absence or presence of H_2O vapor, respectively. Thermal treatment in O_2 caused rapid decline of the permeation rates of H_2 and N_2 in films that were deposited under dry conditions. The decline was moderate in films deposited under wet conditions.

Selective hydroxylation of small alkanes by the particulate methane monooxygenase

The particulate methane monooxygenase (pMMO) catalyzes the oxidation of methane to methanol under ambient temperatures and pressures. Other small alkanes and alkenes are also substrates of this enzyme. We measured and compared the initial rate constants of oxidation of small alkanes (C1 to C5) catalyzed by pMMO. Both primary and secondary alcohols were formed from oxidation of n-butane and n-pentane. The alcohols produced from alkane oxidation can be further oxidized, probably by pMMO, to aldehydes and ketones. The apparent regioselectivity for n-butane and n-pentane is 100% 2-alcohols because the formation of primary alcohols is slower than further oxidation of these alcohols. The hydroxylation at the secondary carbons is highly stereoselective: (R)-alcohols are preferentially formed. The enantiomeric excess increases slightly with decreasing reaction temperature. The steric course of hydroxylation on primary carbons was also studied by using isotopically substituted ethane: (S)- or (R)-CH_3-CHDT, and (S)- or (R)-CD_3- CHDT and the reactions were found to proceed with 100% retention of configuration. A primary isotopic effect of k_H/k_D=5.0 was observed in these experiments.

Carbon-carbon bond forming reactions from bis(carbene)-platinum(II) complexes and olefin polymerization and oligomerization using group 4 post-metallocene complexes

A long-standing challenge in transition metal catalysis is selective C–C bond coupling of simple feedstocks, such as carbon monoxide, ethylene or propylene, to yield value-added products. This work describes efforts toward selective C–C bond formation using early- and late-transition metals, which may have important implications for the production of fuels and plastics, as well as many other commodity chemicals.

The industrial Fischer-Tropsch (F-T) process converts synthesis gas (syngas, a mixture of CO + H₂) into a complex mixture of hydrocarbons and oxygenates. Well-defined homogeneous catalysts for F-T may provide greater product selectivity for fuel-range liquid hydrocarbons compared to traditional heterogeneous catalysts. The first part of this work involved the preparation of late-transition metal complexes for use in syngas conversion. We investigated C–C bond forming reactions via carbene coupling using bis(carbene)platinum(II) compounds, which are models for putative metal–carbene intermediates in F-T chemistry. It was found that C–C bond formation could be induced by either (1) chemical reduction of or (2) exogenous phosphine coordination to the platinum(II) starting complexes. These two mild methods afforded different products, constitutional isomers, suggesting that at least two different mechanisms are possible for C–C bond formation from carbene intermediates. These results are encouraging for the development of a multicomponent homogeneous catalysis system for the generation of higher hydrocarbons.

A second avenue of research focused on the design and synthesis of post-metallocene catalysts for olefin polymerization. The polymerization chemistry of a new class of group 4 complexes supported by asymmetric anilide(pyridine)phenolate (NNO) pincer ligands was explored. Unlike typical early transition metal polymerization catalysts, NNO-ligated catalysts produce nearly regiorandom polypropylene, with as many as 30-40 mol % of insertions being 2,1-inserted (versus 1,2-inserted), compared to <1 mol % in most metallocene systems. A survey of model Ti polymerization catalysts suggests that catalyst modification pathways that could affect regioselectivity, such as C–H activation of the anilide ring, cleavage of the amine R-group, or monomer insertion into metal–ligand bonds are unlikely. A parallel investigation of a Ti–amido(pyridine)phenolate polymerization catalyst, which features a five- rather than a six-membered Ti–N chelate ring, but maintained a dianionic NNO motif, revealed that simply maintaining this motif was not enough to produce regioirregular polypropylene; in fact, these experiments seem to indicate that only an intact anilide(pyridine)phenolate ligated-complex will lead to regioirregular polypropylene. As yet, the underlying causes for the unique regioselectivity of anilide(pyridine)phenolate polymerization catalysts remains unknown. Further exploration of NNO-ligated polymerization catalysts could lead to the controlled synthesis of new types of polymer architectures.

Finally, we investigated the reactivity of a known Ti–phenoxy(imine) (Ti-FI) catalyst that has been shown to be very active for ethylene homotrimerization in an effort to upgrade simple feedstocks to liquid hydrocarbon fuels through co-oligomerization of heavy and light olefins. We demonstrated that the Ti-FI catalyst can homo-oligomerize 1-hexene to C₁₂ and C₁₈ alkenes through olefin dimerization and trimerization, respectively. Future work will include kinetic studies to determine monomer selectivity by investigating the relative rates of insertion of light olefins (e.g., ethylene) vs. higher α-olefins, as well as a more detailed mechanistic study of olefin trimerization. Our ultimate goal is to exploit this catalyst in a multi-catalyst system for conversion of simple alkenes into hydrocarbon fuels.

Multiscale model reduction methods for deterministic and stochastic partial differential equations

Partial differential equations (PDEs) with multiscale coefficients are very difficult to solve due to the wide range of scales in the solutions. In the thesis, we propose some efficient numerical methods for both deterministic and stochastic PDEs based on the model reduction technique.

For the deterministic PDEs, the main purpose of our method is to derive an effective equation for the multiscale problem. An essential ingredient is to decompose the harmonic coordinate into a smooth part and a highly oscillatory part of which the magnitude is small. Such a decomposition plays a key role in our construction of the effective equation. We show that the solution to the effective equation is smooth, and could be resolved on a regular coarse mesh grid. Furthermore, we provide error analysis and show that the solution to the effective equation plus a correction term is close to the original multiscale solution.

For the stochastic PDEs, we propose the model reduction based data-driven stochastic method and multilevel Monte Carlo method. In the multiquery, setting and on the assumption that the ratio of the smallest scale and largest scale is not too small, we propose the multiscale data-driven stochastic method. We construct a data-driven stochastic basis and solve the coupled deterministic PDEs to obtain the solutions. For the tougher problems, we propose the multiscale multilevel Monte Carlo method. We apply the multilevel scheme to the effective equations and assemble the stiffness matrices efficiently on each coarse mesh grid. In both methods, the $\KL$ expansion plays an important role in extracting the main parts of some stochastic quantities.

For both the deterministic and stochastic PDEs, numerical results are presented to demonstrate the accuracy and robustness of the methods. We also show the computational time cost reduction in the numerical examples.

Selective data gathering in community sensor networks

Smartphones and other powerful sensor-equipped consumer devices make it possible to sense the physical world at an unprecedented scale. Nearly 2 million Android and iOS devices are activated every day, each carrying numerous sensors and a high-speed internet connection. Whereas traditional sensor networks have typically deployed a fixed number of devices to sense a particular phenomena, community networks can grow as additional participants choose to install apps and join the network. In principle, this allows networks of thousands or millions of sensors to be created quickly and at low cost. However, making reliable inferences about the world using so many community sensors involves several challenges, including scalability, data quality, mobility, and user privacy.

This thesis focuses on how learning at both the sensor- and network-level can provide scalable techniques for data collection and event detection. First, this thesis considers the abstract problem of distributed algorithms for data collection, and proposes a distributed, online approach to selecting which set of sensors should be queried. In addition to providing theoretical guarantees for submodular objective functions, the approach is also compatible with local rules or heuristics for detecting and transmitting potentially valuable observations. Next, the thesis presents a decentralized algorithm for spatial event detection, and describes its use detecting strong earthquakes within the Caltech Community Seismic Network. Despite the fact that strong earthquakes are rare and complex events, and that community sensors can be very noisy, our decentralized anomaly detection approach obtains theoretical guarantees for event detection performance while simultaneously limiting the rate of false alarms.

Z-selective olefin metathesis using chelating ruthenium alkylidene catalysts

Publications about olefin metathesis will generally discuss how the discovery and development of well-defined catalysts to carry out this unique transformation have revolutionized many fields, from natural product and materials chemistry, to green chemistry and biology. However, until recently, an entire manifestation of this methodology had been inaccessible. Except for a few select examples, metathesis catalysts favor the thermodynamic trans- or E-olefin products in cross metathesis (CM), macrocyclic ring closing metathesis (mRCM), ring opening metathesis polymerization (ROMP), and many other types of reactions. Judicious choice of substrates had allowed for the direct synthesis of cis- or Z-olefins or species that could be converted upon further reaction, however the catalyst controlled synthesis of Z-olefins was not possible until very recently.

Research into the structure and stability of metallacyclobutane intermediates has led to the proposal of models to impart Z-selectivity in metathesis reactions. Having the ability to influence the orientation of metallacyclobutane substituents to cause productive formation of Z- double bonds using steric and electronic effects was highly desired. The first successful realization of this concept was by Schrock and Hoveyda et al. who synthesized monoaryloxide pyrolidine (MAP) complexes of tungsten and molybdenum that promoted Z-selective CM. The Z-selectivity of these catalysts was attributed to the difference in the size of the two axial ligands. This size difference influences the orientation of the substituents on the forming/incipient metallacyclobutane intermediate to a cis-geometry and leads to productive formation of Z-olefins. These catalysts have shown great utility in the synthesis of complicated natural product precursors and stereoregular polymers. More recently, ruthenium catalysts capable of promoting Z-selective metathesis have been reported by our group and others. This thesis will discuss the development of ruthenium-based NHC chelated Z-selective catalysts, studies probing their unique metathesis mechanism, and synthetic applications that have been investigated thus far.

Chapter 1 will focus on studies into the stability of NHC chelated complexes and the synthesis of new and improved stable chelating architectures. Chapter 2 will discuss applications of the highly active and Z-selective developed in Chapter 1, including the formation of lepidopteran female sex pheromones using olefin cross metathesis and highly Z- and highly E-macrocycles using macrocyclic ring closing metathesis and Z-selective ethenolysis. Chapter 3 will explore studies into the unique mechanism of olefin metathesis reactions catalyzed by these NHC chelated, highly Z-selective catalysts, explaining observed trends by investigating the stability of relevant, substituted metallacyclobutane intermediates.

A fundamental approach to phase noise reduction in hybrid Si/III-V lasers

Spontaneous emission into the lasing mode fundamentally limits laser linewidths. Reducing cavity losses provides two benefits to linewidth: (1) fewer excited carriers are needed to reach threshold, resulting in less phase-corrupting spontaneous emission into the laser mode, and (2) more photons are stored in the laser cavity, such that each individual spontaneous emission event disturbs the phase of the field less. Strong optical absorption in III-V materials causes high losses, preventing currently-available semiconductor lasers from achieving ultra-narrow linewidths. This absorption is a natural consequence of the compromise between efficient electrical and efficient optical performance in a semiconductor laser. Some of the III-V layers must be heavily doped in order to funnel excited carriers into the active region, which has the side effect of making the material strongly absorbing.

This thesis presents a new technique, called modal engineering, to remove modal energy from the lossy region and store it in an adjacent low-loss material, thereby reducing overall optical absorption. A quantum mechanical analysis of modal engineering shows that modal gain and spontaneous emission rate into the laser mode are both proportional to the normalized intensity of that mode at the active region. If optical absorption near the active region dominates the total losses of the laser cavity, shifting modal energy from the lossy region to the low-loss region will reduce modal gain, total loss, and the spontaneous emission rate into the mode by the same factor, so that linewidth decreases while the threshold inversion remains constant. The total spontaneous emission rate into all other modes is unchanged.

Modal engineering is demonstrated using the Si/III-V platform, in which light is generated in the III-V material and stored in the low-loss silicon material. The silicon is patterned as a high-Q resonator to minimize all sources of loss. Fabricated lasers employing modal engineering to concentrate light in silicon demonstrate linewidths at least 5 times smaller than lasers without modal engineering at the same pump level above threshold, while maintaining the same thresholds.

Iridium and rhodium analogues of the Shilov cycle catalyst; and the investigation and applications of the Reduction-Coupled Oxo Activation (ROA) mechanistic motif towards alkane upgrading

This dissertation will cover several disparate topics, with the overarching theme centering on the investigation of organometallic C-H activation and hydrocarbon transformation and upgrading. Chapters 2 and 3 discuss iridium and rhodium analogues of the Shilov cycle catalyst for methane to methanol oxidation, and Chapter 4 on the recently discovered ROA mechanistic motif in catalysts for various alkane partial oxidation reactions. In addition, Chapter 5 discusses the mechanism of nickel pyridine bisoxazoline Negishi catalysts for asymmetric and stereoconvergent C-C coupling, and the appendices discuss smaller projects on rhodium H/D exchange catalysts and DFT method benchmarking.

Development of tantalum phenoxy-imine compounds for selective ethylene oligomerization

The development of catalysts that selectively oligomerize light olefins for uses in polymers and fuels remains of interest to the petrochemical and materials industry. For this purpose, two tantalum compounds, (FI)TaMe2Cl2 and (FI)TaMe4, implementing a previously reported phenoxy-imine (FI) ligand framework, have been synthesized and characterized with NMR spectroscopy and X-ray crystallography. When tested for ethylene oligomerization catalysis, (FI)TaMe2Cl2 was found to dimerize ethylene when activated with Et2Zn or EtMgCl, and (FI)TaMe4 dimerized ethylene when activated with B(C6F5)3, both at room temperature.

Development of Metalloenzyme Dioxygen Reduction Cathodes

The prime thrust of this dissertation is to advance the development of fuel cell dioxygen reduction cathodes that employ some variant of multicopper oxidase enzymes as the catalyst. The low earth-abundance of platinum metal and its correspondingly high market cost has prompted a general search amongst chemists and materials scientists for reasonable alternatives to this metal for facilitating catalytic dioxygen reduction chemistry. The multicopper oxidases (MCOs), which constitute a class of enzyme that naturally catalyze the reaction O₂ + 4H⁺ + 4e^- → 2H₂O, provide a promising set of biochemical contenders for fuel cell cathode catalysts. In MCOs, a substrate reduces a copper atom at the type 1 site, where charge is then transferred to a trinuclear copper cluster consisting of a mononuclear type 2 or “normal copper” site and a binuclear type 3 copper site. Following the reduction of all four copper atoms in the enzyme, dioxygen is then reduced to water in two two-electron steps, upon binding to the trinuclear copper cluster. We identified an MCO, a laccase from the hyperthermophilic bacterium Thermus thermophilus strain HB27, as a promising candidate for cathodic fuel cell catalysis. This protein demonstrates resilience at high temperatures, exhibiting no denaturing transition at temperatures high as 95°C, conditions relevant to typical polymer electrolyte fuel cell operation.

In Chapter I of this thesis, we discuss initial efforts to physically characterize the enzyme when operating as a heterogeneous cathode catalyst. Following this, in Chapter II we then outline the development of a model capable of describing the observed electrochemical behavior of this enzyme when operating on porous carbon electrodes. Developing a rigorous mathematical framework with which to describe this system had the potential to improve our understanding of MCO electrokinetics, while also providing a level of predictive power that might guide any future efforts to fabricate MCO cathodes with optimized electrochemical performance. In Chapter III we detail efforts to reduce electrode overpotentials through site-directed mutagenesis of the inner and outer-sphere ligands of the Cu sites in laccase, using electrochemical methods and electronic spectroscopy to try and understand the resultant behavior of our mutant constructs. Finally, in Chapter IV, we examine future work concerning the fabrication of enhanced MCO cathodes, exploring the possibility of new cathode materials and advanced enzyme deposition techniques.

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.