Nitrogenase and hydrogenase are two critical iron-containing enzymes found in nature. Nitrogenase catalyzes the reduction of dinitrogen N 2) to ammonia, and hydrogenase catalyzes the evolution or consumption) of dihydrogen H2). A better understanding of these enzymes may have an impact on our ability to transition to more sustainable energy economy. Although X-ray crystallography has provided baseline structures of nitrogenase and hydrognease, and numerous spectroscopic studies and theoretical calculations have outlined catalytic mechanisms of these enzymes, the detailed mechanisms remain elusive. New spectroscopic techniques are needed. In this dissertation, three novel synchrotron radiation based nuclear resonant scattering techniques have been used to probe the metal sites of nitrogenase and hydrogenase. They are nuclear resonance vibrational spectroscopy NRVS), synchrotron radiation based perturbed angular correlations SRPAC), and nuclear forward scattering NFS, or synchrotron Mossbauer). Because of its unique selection rule, NRVS provides more detailed vibrational information about the Fe sites in metalloproteins than infrared and Raman spectroscopy. FeCO vibrations revealed by NRVS on a series of iron carbonyl complexes have been used to confirm a cis-FeCO)2 geometry in the iron center of Hmd hydrogenase. A possible water ligand has also been identified for the first time. In CO inhibited nitrogenase, NRVS revealed the relationship between the intensity of the strongest spectral feature at 188 cm-1 and the structural distortion of FeMo-co introduced by CO binding. NRVS also revealed a possible 7 iron structure for the VK cluster — a key FeMo-co precursor in the nitrogenase biosynthetic pathway. Selectively probing iron site vibrations in metalloproteins has been achieved for the first time by applying NRVS onto protein crystals from rubredoxin and MoFe protein of nitrogenase. SRPAC and NFS have been used for the first time to reveal nuclear hyperfine interactions on the metal sites of iron-sulfur proteins and related model complexes. These synchrotron radiation spectroscopic techniques demonstrate a great promise to obtain more detailed structural and electronic properties of the metal centers iron and nickel) in nitrogenase and hydrogenase than the conventional Mossbauer spectroscopy.

A new method and a modified method were developed to calculate folding energies of proteins based on the hydrogen deuterium exchange of globally protected amide protons. The modified method was electro spray ionization mass spectrometry ESI-MS) based and resembles the SUPREX method. This method is called the kinetic method. A novel method was also developed which is called the protein equilibrium population snapshot PEPS) method. Both these methods assume that the protein of interest follows a two state folding mechanism. The HX ESI-MS based kinetic method, which is the modified SUPREX method, measures hydrogen deuterium exchange at different guanidine hydrochloride GdHCl) concentrations as a function of time. This method utilizes ESI-MS, contrary to the usual MALDI-MS approach adopted by the SUPREX method. It measures the folding energies of proteins, which follow an EX2 type of exchange mechanism. Accurate folding energies were obtained for Ubiquitin as expected as it follows an EX2 exchange mechanism. Wild type staph nuclease and various quadruple staph nuclease mutants showed consistently higher values with good correlation. This can be attributed to deviation from EX2 exchange. Like SUPREX, this method is expected to work best for proteins, which follow EX2 exchange kinetics. The protein equilibrium population snapshot PEPS) method has been successfully used to accurately determine the folding energies of wild type staph nuclease and two of its mutants, which follow the EX1 mechanism of hydrogen deuterium exchange. This method measures the population distribution of open and closed states of a protein with hydrogen deuterium exchange as a function of denaturant concentration, under equilibrium. This method. when applied to Ubiquitin, which follows EX kinetics, also generated accurate folding energies comparable with literature values from NMR and fluorescence studies. So it is determined that the novel PEPS method is capable of measuring folding energies of a protein, regardless of the exchange mechanism is EX1 or EX2. This method is also based on fewer assumptions than the kinetic method.

X-ray Diffraction Microscopy XDM) has been gaining in popularity for nanoscale imaging of biological and material science samples. Its high penetration depth compared to electron microscopy) and its good dose efficiency compared to its lens-based X-ray alternative) make it uniquely suited for imaging whole biological specimens, where radiation damage is a concern. Despite these advantages, XDM is still far from being a routine imaging tool. This is due to the computational challenge of reconstructing an image from recorded diffraction intensities as well as difficult-to-satisfy experimental requirements. I address these challenges by improving on the computational methods and by implementing a more reliable experimental geometry for our existing diffraction microscope at the Advanced Light Source, Lawrence Berkeley Lab. First, a software library has been developed that streamlines the post-experiment processing of data and that improves on an important aspect of data analysis. Results will be shown that illustrate the collective improvement to the reconstruction process. A modified version of a tool commonly used to assess the consistency of reconstructions is proposed and criteria of its validity are derived. Results show that it has improved utility for judging reconstruction quality. Second, a scanning-type experimental setup has been implemented for our existing diffraction microscope. Several possible geometries are discussed and preliminary results from recent experimental data are shown.

This dissertation is composed of three parts. The first part is to argue the solvent effects on the solvatochromic shift of the n &rarr； pi* excitation of acetone in ambient and supercritical water fluid using a hybrid QM–CI/MM potential in MC simulations. The solute is described by the AM1 approach and water molecules are treated classically. Specially, the spontaneous polarization of the solvent due to the excitation of the solute was considered. The solvent effects on the blue shift of acetone in water fluids at various temperatures and solvent densities are examined. The second part is to investigate the role of dopa decarboxylase DDC) in the catalysis of converting anti-Parkinson drug L-dopa into dopamine. By means of combined QM/MM potentials in MD simulations, we first analyze the factors contributing to the tautomeric equilibrium of an intramolecular proton transfer in the external PLP–L-dopa aldimine the Michaelis complex). How the intrinsic properties, solvent effects as well as the enzyme environment control the shift of the equilibrium is discussed. Afterward, the free energy profiles for the decarboxylations of the external aldimines both in water and in DDC are calculated. The contributions of DDC to the rate enhancement of the reaction are elucidated. The reaction mechanism of L-dopa decarboxylation in DDC is proposed. The third part is to study the structural dynamics of lysine-specific demethylase LSD1) in complex with CoREST and protein-substrate interactions of LSD1 with histone H3 tail. MD simulations of LSD1&bull；CoREST complex bound to a 16 a.a. of the N-terminal H3-tail peptide H3-p16) were carried out using NAMD to study the conformational flexibility of the protein complex, especially the substantial oscillation of the TOWER domain. In addition, the simulations reveal some important protein-peptide and peptide-peptide interactions between LSD1 and H3-p16 that are absent in the crystal structure.

Amyloid deposition is a characteristic component of many human diseases, including Alzheimers disease, Parkinsons disease and type 2 diabetes. Human islet amyloid polypeptide IAPP also known as amylin) is the major protein component of the pancreatic islet amyloid associated with type 2 diabetes. There is considerable interest in developing inhibitors of amyloid formation, both because of their obvious therapeutic potential but also because they can provide powerful tools for mechanistic studies. There is a large body of work on inhibitors of the Alzheimer beta amyloid peptide Abeta), but less attention has been paid to the development of IAPP amyloid inhibitors. In this dissertation, peptide based inhibitors were rationally designed and a general strategy was developed whereby two moderate inhibitors of amyloid formation can be rationally selected via kinetic assays and combined to yield a highly effective inhibitor. Small molecule inhibitors were also developed. Rifampicin is reported to inhibit Abeta amyloid formation, but it does not prevent amyloid formation by IAPP and does not disaggregate preformed IAPP amyloid fibrils, instead it interferes with standard fluorescence based assays of amyloid formation. Simple sulphonated triphenyl methane derivatives are potent inhibitors of in vitro amyloid formation by IAPP. The tea-derived flavanol, –)-Epigallocatechin 3-Gallate, is an effective inhibitor of in vitro IAPP amyloid formation and disaggregates preformed amyloid fibrils derived from IAPP. IAPP is produced as a prohormone, proIAPP, and processed in the secretory granules of the pancreatic beta cells. Partially processed forms of proIAPP are found in amyloid deposits. It has been suggested that incomplete processing plays a role in amyloid formation by promoting interactions with sulfated proteoglycans of the extracellular matrix. Biophysical proof of principle evidences are provided for the role of proIAPP processing intermediate and sulfated proteoglycans in amyloid formation. Simple sulfonated triphenyl methyl derivatives inhibit amyloid formation by proIAPP processing intermediate and also inhibit glycosaminoglycans mediated amyloid formation by proIAPP processing intermediate. These studies may give a better understanding of the mechanism of amyloid formation and provide valuable insight into the development of effective therapeutic strategies for a wide range of amyloidogenic diseases.

Start codon recognition is a crucial event in initiation, committing the ribosome to beginning translation from a particular location； starting translation in the wrong location will fail to produce the desired protein and result in an incorrect and possibly toxic peptide. To investigate the mechanism of start codon recognition in eukaryotes, we used a reconstituted system of S. cerevisiae components. We show that base pairing between the initiator tRNA anticodon and the mRNA start codon results in a conformational change of the pre-initiation complex to a more stable state, likely the previously proposed “closed” state that arrests the movement of the complex on the mRNA. We provide evidence that a conformational change in the initiator tRNA is involved in transmitting to the rest of the complex this signal that matching anticodon/start codon base pairing has been achieved.

Steady-state and nanosecond time-resolved fluorescence of the nitro-2, 1, 3-benzoxadiazol-4-yl NBD) probe and line tension force were determined in phospholipids acyl-chain dynamics in giant liposomes made from a homologous series of phosphatidylcholines PC). The fluorescence spectroscopy of a NBD probe attached to the headgroup NBD PE) or the tail NBD PC) of the phospholipid were used to determine the rate of dithionite quenching in a homologous series of phospholipids. Similar experiments were performed in the gel and the fluid phases of the 1,2-dimyristoyl-sn-glycero-3-phosphocholine DMPC). Nanosecond time scale lifetimes and anisotropy measurements were obtained by a 470 nm LED pulse laser diode and time correlated single photon counting detection. The fluorescence lifetime and intensity of NBD PE and NBD PC in acyl-chain lengths from 12:0 PC to 20:0 PC were determined. The half life of dithionite quenching of NBD varied with both phospholipid chain length and the position of the fluorescent probes and of quenching in the gel, fluid and phase transition phases. Line tensions measured by laser ablation method in a homologous series of lipids, revealed acyl-chain length and phase state dependence. Studies were conducted on human Wilson disease protein. This is a copper transporting ATPase found in the copper secretory pathway. It possesses six cytosolic metal binding domains in the N-terminus. These domains are involved in the acquisition of copperI) from the metallochaperone HAH1. Insight into the stability of metal-binding domain four will be presented.

Accessory proteins regulate critical transmembrane signaling events. Dysregulated transmembrane signaling can result in disorders ranging from the benign, such as mild anemia, to the critical, such as neurofibromatosis. While the mechanisms of signaling regulation are diverse, common themes are present in the cellular context of regulation. Here, we explore two examples of transmembrane signaling regulation: the regulation of adhesive integrin interactions by Src kinase adaptor protein 2 SKAP2) in the context of erythropoiesis and the regulation of epidermal growth factor receptor EGFR) signaling by the tumor suppressor protein Merlin in the context of neurofibromatosis type II. Together, these studies emphasize the common context of transmembrane signaling regulation and explore the role of the dynamic membrane environment in this regulation. Our studies of SKAP2 in erythropoiesis identify a novel genetic cause of hypoproliferative anemia that is associated with decreased adhesive interactions in erythroblastic islands. Our studies support the role of SKAP2 as an integrin signaling modulator in erythropoiesis and highlight the importance of appropriate regulation of transmembrane adhesive interactions. In the context of tumor suppressor regulation, we found that ligand-bound EGFR is immobilized in the plasma membrane of confluent Merlin-expressing cells in a contact-, actin-, and signaling-dependent manner. Ligand-activated EGFR undergoes receptor-mediated internalization via either clathrin-mediated endocytosis CME) or non-clathrin endocytosis NCE). We found that Merlin specifically antagonizes NCE of EGFR independent of cell-cell contact by preventing distribution of the receptor to specific membrane microdomains. In addition, we demonstrated that Merlin-dependent immobilization of EGFR at the surface of confluent cells was signaling-dependent and occurred over a short time 100s), implicating membrane-proximal protein associations and conformational changes in Merlin as key partners in EGFR regulation. These studies of diverse transmembrane signaling events by SKAP2 and Merlin suggest a view of the local membrane environment that is dynamic yet still tightly regulated.