Electron transfer and protein engineering studies of the soluble methane monooxygenase from Methylococcus capsulatus (Bath)

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2003.Vita.Includes bibliographical references.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Chapter 1. Introduction: Electron Transfer in Biological Systems In many biological processes, including oxidative phosphorylation and photosynthesis, electron transfer reactions play vital roles. Electrons must be transported at catalytically relevant rates and with specificity to prevent indiscriminate electron transfer that would quickly bring cells to equilibrium. To meet these requirements, biological systems employ a panoply of organic and inorganic redox centers, most of which are sequestered within proteins. In addition to protecting a cofactor from undesirable reactions, the surrounding protein environment tunes its redox properties and mediates specific contacts with other molecules. This brief overview describes the types of redox centers used in biology, the application of electron transfer theory to physiological systems, the kinetic complexity introduced by interprotein interactions, and general mechanisms for regulating biological electron transfer. Chapter 2. Expression and Site-Directed Mutagenesis of the Reductase Component of Soluble Methane Monooxygenase from Methylococcus capsulatus (Bath) ... Chapter 3. Expression and Characterization of Ferredoxin and Flavin Adenine Dinucleotide-Binding Domains of the Reductase Component of Soluble Methane Monooxygenase from Methylococcus capsulatus (Bath) ... Chapter 4. Intermolecular Electron Transfer Reactions in Soluble Methane Monooxygenase from Methylococcus capsulatus (Bath): A Role for Hysteresis in Protein Function.(cont.) Chapter 5. Domain Engineering of the Reductase Component of Soluble Methane Monooxygenase from Methylococcus capsulatus (Bath) ... Chapter 6. Expression in Escherichia coli of the Hydroxylase Component of Soluble Methane Monooxygenase from Methylococcus capsulatus (Bath)by Jessica L. Blazyk.Ph.D

Component Interactions and Electron Transfer in Toluene/o-Xylene Monooxygenase

The multicomponent protein toluene/o-xylene monooxygenase (ToMO) activates molecular oxygen to oxidize aromatic hydrocarbons. Prior to dioxygen activation, two electrons are injected into each of two diiron(III) units of the hydroxylase, a process that involves three redox active proteins: the ToMO hydroxylase (ToMOH), Rieske protein (ToMOC), and an NADH oxidoreductase (ToMOF). In addition to these three proteins, a small regulatory protein is essential for catalysis (ToMOD). Through steady state and pre-steady state kinetics studies, we show that ToMOD attenuates electron transfer from ToMOC to ToMOH in a concentration-dependent manner. At substoichiometric concentrations, ToMOD increases the rate of turnover, which we interpret to be a consequence of opening a pathway for oxygen transport to the catalytic diiron center in ToMOH. Excess ToMOD inhibits steady state catalysis in a manner that depends on ToMOC concentration. Through rapid kinetic assays, we demonstrate that ToMOD attenuates formation of the ToMOC–ToMOH complex. These data, coupled with protein docking studies, support a competitive model in which ToMOD and ToMOC compete for the same binding site on the hydroxylase. These results are discussed in the context of other studies of additional proteins in the superfamily of bacterial multicomponent monooxygenases.National Institute of General Medical Sciences (U.S.) (5-R01-GM032134)United States. National Institutes of Health (T32GM008334

Control of substrate access to the active site in methane monooxygenase

Methanotrophs consume methane as their major carbon source and have an essential role in the global carbon cycle by limiting escape of this greenhouse gas to the atmosphere. These bacteria oxidize methane to methanol by soluble and particulate methane monooxygenases (MMOs). Soluble MMO contains three protein components, a 251-kilodalton hydroxylase (MMOH), a 38.6-kilodalton reductase (MMOR), and a 15.9-kilodalton regulatory protein (MMOB), required to couple electron consumption with substrate hydroxylation at the catalytic diiron centre of MMOH. Until now, the role of MMOB has remained ambiguous owing to a lack of atomic-level information about the MMOH–MMOB (hereafter termed H–B) complex. Here we remedy this deficiency by providing a crystal structure of H–B, which reveals the manner by which MMOB controls the conformation of residues in MMOH crucial for substrate access to the active site. MMOB docks at the α[subscript 2]β[subscript 2] interface of α[subscript 2]β[subscript 2]γ[subscript 2] MMOH, and triggers simultaneous conformational changes in the α-subunit that modulate oxygen and methane access as well as proton delivery to the diiron centre. Without such careful control by MMOB of these substrate routes to the diiron active site, the enzyme operates as an NADH oxidase rather than a monooxygenase. Biological catalysis involving small substrates is often accomplished in nature by large proteins and protein complexes. The structure presented in this work provides an elegant example of this principle.National Institute of General Medical Sciences (U.S.) (Grant GM 32114

Prediction of Antibacterial Activity from Physicochemical Properties of Antimicrobial Peptides

Consensus is gathering that antimicrobial peptides that exert their antibacterial action at the membrane level must reach a local concentration threshold to become active. Studies of peptide interaction with model membranes do identify such disruptive thresholds but demonstrations of the possible correlation of these with the in vivo onset of activity have only recently been proposed. In addition, such thresholds observed in model membranes occur at local peptide concentrations close to full membrane coverage. In this work we fully develop an interaction model of antimicrobial peptides with biological membranes; by exploring the consequences of the underlying partition formalism we arrive at a relationship that provides antibacterial activity prediction from two biophysical parameters: the affinity of the peptide to the membrane and the critical bound peptide to lipid ratio. A straightforward and robust method to implement this relationship, with potential application to high-throughput screening approaches, is presented and tested. In addition, disruptive thresholds in model membranes and the onset of antibacterial peptide activity are shown to occur over the same range of locally bound peptide concentrations (10 to 100 mM), which conciliates the two types of observations

Evolving concepts on the age-related changes in “muscle quality”

The deterioration of skeletal muscle with advancing age has long been anecdotally recognized and has been of scientific interest for more than 150 years. Over the past several decades, the scientific and medical communities have recognized that skeletal muscle dysfunction (e.g., muscle weakness, poor muscle coordination, etc.) is a debilitating and life-threatening condition in the elderly. For example, the age-associated loss of muscle strength is highly associated with both mortality and physical disability. It is well-accepted that voluntary muscle force production is not solely dependent upon muscle size, but rather results from a combination of neurologic and skeletal muscle factors, and that biologic properties of both of these systems are altered with aging. Accordingly, numerous scientists and clinicians have used the term “muscle quality” to describe the relationship between voluntary muscle strength and muscle size. In this review article, we discuss the age-associated changes in the neuromuscular system—starting at the level of the brain and proceeding down to the subcellular level of individual muscle fibers—that are potentially influential in the etiology of dynapenia (age-related loss of muscle strength and power)

Secondary Structure and Lipid Contact of a Peptide Antibiotic in Phospholipid Bilayers by REDOR

The chemical shifts of specific (13)C and (15)N labels distributed throughout KIAGKIA-KIAGKIA-KIAGKIA (K3), an amphiphilic 21-residue antimicrobial peptide, prove that the peptide is in an all α-helical conformation in the bilayers of multilamellar vesicles (MLVs) containing dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (1:1). Rotational-echo double-resonance (REDOR) (13)C{(31)P} and (15)N{(31)P} experiments on the same labeled MLVs show that on partitioning into the bilayer, the peptide chains remain in contact with lipid headgroups. The amphipathic lysine side chains of K3 in particular appear to play a key role in the electrostatic interactions with the acidic lipid headgroups. In addition to the extensive peptide-headgroup contact, (13)C{(19)F} REDOR experiments on MLVs containing specifically (19)F-labeled lipid tails suggest that a portion of the peptide is surrounded by a large number of lipid acyl chains. Complementary (31)P{(19)F} REDOR experiments on these MLVs show an enhanced headgroup-lipid tail contact resulting from the presence of K3. Despite these distortions, static (31)P NMR lineshapes indicate that the lamellar structure of the membrane is preserved