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.

Development and Application of Embedding Methods for the Simulation of Large Chemical Systems

The high computational cost of correlated wavefunction theory (WFT) calculations has motivated the development of numerous methods to partition the description of large chemical systems into smaller subsystem calculations. For example, WFT-in-DFT embedding methods facilitate the partitioning of a system into two subsystems: a subsystem A that is treated using an accurate WFT method, and a subsystem B that is treated using a more efficient Kohn-Sham density functional theory (KS-DFT) method. Representation of the interactions between subsystems is non-trivial, and often requires the use of approximate kinetic energy functionals or computationally challenging optimized effective potential calculations; however, it has recently been shown that these challenges can be eliminated through the use of a projection operator. This dissertation describes the development and application of embedding methods that enable accurate and efficient calculation of the properties of large chemical systems.

Chapter 1 introduces a method for efficiently performing projection-based WFT-in-DFT embedding calculations on large systems. This is accomplished by using a truncated basis set representation of the subsystem A wavefunction. We show that naive truncation of the basis set associated with subsystem A can lead to large numerical artifacts, and present an approach for systematically controlling these artifacts.

Chapter 2 describes the application of the projection-based embedding method to investigate the oxidative stability of lithium-ion batteries. We study the oxidation potentials of mixtures of ethylene carbonate (EC) and dimethyl carbonate (DMC) by using the projection-based embedding method to calculate the vertical ionization energy (IE) of individual molecules at the CCSD(T) level of theory, while explicitly accounting for the solvent using DFT. Interestingly, we reveal that large contributions to the solvation properties of DMC originate from quadrupolar interactions, resulting in a much larger solvent reorganization energy than that predicted using simple dielectric continuum models. Demonstration that the solvation properties of EC and DMC are governed by fundamentally different intermolecular interactions provides insight into key aspects of lithium-ion batteries, with relevance to electrolyte decomposition processes, solid-electrolyte interphase formation, and the local solvation environment of lithium cations.

Strategic ecosystem studies of large slow flowing lowland rivers. Progress report: April - October 1989

This report covers the period April to September, 1989. During this period sampling of invertebrates has concentrated on planktonic animals and those associated with one of the dominant macrophytes in the system, Nuphar lutea, the yellow water lily, since these are particularly important in the diets of larval and juvenile cyprinid fish. A proportion of samples has been partly analysed and some preliminary data are presented here.

The coarse fishes of Britain

The need for research upon the coarse fishes of Britain, questions of the breeding and rearing of large numbers of young fish, more especially roach, was established back in 1937. The investigation which was begun in 1939 at Barrington in Cambridgeshire was therefore, with the full approval of the National Federation of Anglers, devoted to the general life histories of as many species as possible. Now, five years later, the results of the investigation are presented in the hope that they will provide a basis of knowledge upon which sound policies can be devised for the improvement of fish stocks and the increase of sport. In this report the scientific data, which will be published in full elsewhere, have been condensed as much as possible, but nothing of importance has been omitted and nothing has been concealed.

Design and analysis of a kind of large flattened mode optical fibre

In this paper, a refractive index pro. le design enabling us to obtain a. at modal field around the fibre centre is investigated. The theoretical approach for designing such multilayer large flattened mode (LFM) optical fibres is presented. A comparison is made between the properties of a three-layer LFM structure and a standard step-index pro. le with the same core size. The obtained results indicate that the effective area of the LFM fibre is about twice as large as that of the standard step-index fibre, but the LFM fibre has less effective ability to filter out the higher order modes than the standard step-index fibre with the same bending radius.

Optimization of thermal environment during crystal growth of large-sized Nd : YAG by Temperature Gradient Technique

A finite-element model is employed to analysis the thermal environments in Temperature Gradient Technique (TGT) furnace during the growth of large-sized Nd:YAG crystal. The obtained results show that when the crucible is located at the lower position inside of the heater, a flatter solid-liquid interface is established, which makes it easier to obtain the core-free Nd:YAG crystal. Meanwhile, the lower crucible position can induce higher axial temperature gradient, which is beneficial to the release of latent heat. (c) 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.