Structure Organization And Function Of The Human Body Biology Essay

Biology » Structure Organization And Function Of The Human Body Biology Essay

Cell are the structural and functional units of all living organisms. Some organisms, such as bacteria, are unicellular, consisting of a single cell. Other organisms, such as humans, are multicellular, or have many cells—an estimated 100,000,000,000,000 cells! Each cell is an amazing world unto itself: it can take in nutrients, convert these nutrients into energy, carry out specialized functions, and reproduce as necessary. Even more amazing is that each cell stores its own set of instructions for carrying out each of these activities.

Prokaryotic Cells - organisms that are lack of nuclear membrane, the membrane that surrounds the nucleus of a cell. Bacteria are the best known and most studied form of prokaryotic organisms, although the recent discovery of a second group of prokaryotes, called archaea, has provided evidence of a third cellular domain of life and new insights into the origin of life itself.

- prokaryotes are unicellular organisms that do not develop or differentiate into multicellular forms.

- are capable of inhabiting almost every place on the earth, from the deep ocean, to the edges of hot springs, to just about every surface of our bodies.

Prokaryotes are distinguished from eukaryotes on the basis of nuclear organization, specifically their lack of a nuclear membrane. Prokaryotes also lack any of the intracellular organelles and structures that are characteristic of eukaryotic cells. Most of the functions of organelles, such as mitochondria, chloroplasts, and the Golgi apparatus, are taken over by the prokaryotic plasma membrane. Prokaryotic cells have three architectural regions: appendages called flagella and pili—proteins attached to the cell surface; a cell envelope consisting of a capsule, a cell wall, and a plasma membrane; and a cytoplasmic region that contains the cell genome (DNA) and ribosomes and various sorts of inclusions.

Eukaryotes include fungi, animals, and plants as well as some unicellular organisms. Eukaryotic cells are about 10 times the size of a prokaryote and can be as much as 1000 times greater in volume. The major and extremely significant difference between prokaryotes and eukaryotes is that eukaryotic cells contain membrane-bound compartments in which specific metabolic activities take place. Most important among these is the presence of a nucleus, a membrane-delineated compartment that houses the eukaryotic cell’s DNA. It is this nucleus that gives the eukaryote—literally, true nucleus—its name.

The outer lining of a eukaryotic cell is called the plasma membrane. This membrane serves to separate and protect a cell from its surrounding environment and is made mostly from a double layer of proteins and lipids, fat-like molecules. Embedded within this membrane are a variety of other molecules that act as channels and pumps, moving different molecules into and out of the cell. A form of plasma membrane is also found in prokaryotes, but in this organism it is usually referred to as the cell membrane.

The cytoskeleton is an important, complex, and dynamic cell component. It acts to organize and maintain the cell's shape; anchors organelles in place; helps during endocytosis, the uptake of external materials by a cell; and moves parts of the cell in processes of growth and motility. There are a great number of proteins associated with the cytoskeleton, each controlling a cell’s structure by directing, bundling, and aligning filaments.

Inside the cell there is a large fluid-filled space called the cytoplasm, sometimes called the cytosol. In prokaryotes, this space is relatively free of compartments. In eukaryotes, the cytosol is the "soup" within which all of the cell's organelles reside. It is also the home of the cytoskeleton. The cytosol contains dissolved nutrients, helps break down waste products, and moves material around the cell through a process called cytoplasmic streaming. The nucleus often flows with the cytoplasm changing its shape as it moves. The cytoplasm also contains many salts and is an excellent conductor of electricity, creating the perfect environment for the mechanics of the cell. The function of the cytoplasm, and the organelles which reside in it, are critical for a cell's survival.

Two different kinds of genetic material exist: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Most organisms are made of DNA, but a few viruses have RNA as their genetic material. The biological information contained in an organism is encoded in its DNA or RNA sequence.

Prokaryotic genetic material is organized in a simple circular structure that rests in the cytoplasm. Eukaryotic genetic material is more complex and is divided into discrete units called genes. Human genetic material is made up of two distinct components: the nuclear genome and the mitochondrial genome. The nuclear genome is divided into 24 linear DNA molecules, each contained in a different chromosome. The mitochondrial genome is a circular DNA molecule separate from the nuclear DNA. Although the mitochondrial genome is very small, it codes for some very important proteins.

The human body contains many different organs, such as the heart, lung, and kidney, with each organ performing a different function. Cells also have a set of "little organs", called organelles, that are adapted and/or specialized for carrying out one or more vital functions. Organelles are found only in eukaryotes and are always surrounded by a protective membrane. It is important to know some basic facts about the following organelles.

The nucleus is the most conspicuous organelle found in a eukaryotic cell. It houses the cell's chromosomes and is the place where almost all DNA replication and RNA synthesis occur. The nucleus is spheroid in shape and separated from the cytoplasm by a membrane called the nuclear envelope. The nuclear envelope isolates and protects a cell's DNA from various molecules that could accidentally damage its structure or interfere with its processing. During processing, DNA is transcribed, or synthesized, into a special RNA, called mRNA. This mRNA is then transported out of the nucleus, where it is translated into a specific protein molecule. In prokaryotes, DNA processing takes place in the cytoplasm.

Ribosomes are found in both prokaryotes and eukaryotes. The ribosome is a large complex composed of many molecules, including RNAs and proteins, and is responsible for processing the genetic instructions carried by an mRNA. The process of converting an mRNA's genetic code into the exact sequence of amino acids that make up a protein is called translation. Protein synthesis is extremely important to all cells, and therefore a large number of ribosomes—sometimes hundreds or even thousands—can be found throughout a cell.

Ribosomes float freely in the cytoplasm or sometimes bind to another organelle called the endoplasmic reticulum. Ribosomes are composed of one large and one small subunit, each having a different function during protein synthesis.

2. Describe and distinguish between the cell and tissue organizations and systems.

Tissues are the collection of similar cells that group together to perform a specialized function. The four primary tissue types in the human body: epithelial tissue, connective tissue, muscle tissue and nerve tissue.

Epithelial Tissue - The cells are pack tightly together and form continuous sheets that serve as linings in different parts of the body.  It serves as membranes lining organs and helping to keep the body's organs separate, in place and protected.  Some examples of epithelial tissue are the outer layer of the skin, the inside of the mouth and stomach, and the tissue surrounding the body's organs.

Connective Tissue - There are many types of connective tissue in the body.  It adds support and structure to the body.  Most types of connective tissue contain fibrous strands of the protein collagen that add strength to connective tissue.  Some examples of connective tissue include the inner layers of skin, tendons, ligaments, cartilage, bone and fat tissue.  In addition to these more recognizable forms of connective tissue, blood is also considered a form of connective tissue.

Muscle Tissue - Muscle tissue is a specialized tissue that can contract.  Muscle tissue contains the specialized proteins actin and myosin that slide past one another and allow movement.  Examples of muscle tissue are contained in the muscles throughout your body.

Nerve Tissue - Nerve tissue contains two types of cells: neurons and glial cells.  Nerve tissue has the ability to generate and conduct electrical signals in the body.  These electrical messages are managed by nerve tissue in the brain and transmitted down the spinal cord to the body.

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Structure Function Studies Of Helicobacter Pylori Urease Biology Essay

Biology » Structure Function Studies Of Helicobacter Pylori Urease Biology Essay

Urease catalyzes the hydrolysis of urea into ammonia and carbon dioxide. The release ammonia neutralizes the gastric acid, and allows the colonization of Helicobacter pylori in human stomach. The apo-urease undergoes a post-translation carbamylation of an active-site lysine residue, followed by insertion of two nickel ions essential for metal catalysis to the active site. In H. pylori, four urease accessory proteins, UreE, UreF, UreG, and UreH, are essential to the maturation process of urease. It is postulated that the apo-urease either bind a pre-formed UreG/UreF/UreH complex, or the individual urease accessory proteins sequentially to form a pre-activation complex. The Ni-binding protein UreE then interacts with the UreG of the complex, and triggers the GTP-dependent activation of urease.

How these urease accessory proteins interact with each other and with the urease to form the activation complex is poorly understood, partly because of the lack of high-resolution structures available for these urease accessory proteins. Until recently, the only urease accessory protein whose structure is available is UreE. We have recently determined the crystal structure of UreF, and have obtained a preliminary structure of the UreF/UreH complex. The novel structural information allows us to use the protein engineering approach to address the following questions: (1) Is the dimerization of H. pylori UreF/UreH complex essential to the maturation of urease? (2) Is the interaction between UreF and UreH essential to the maturation of urease? (3) Where is the interacting surface on the UreF/UreH complex that are responsible for binding UreG and the urease? As we have already obtained crystals of the UreG/UreF/UreH complex that diffract to good resolution, we propose to determine the crystal structure of this ternary complex. Through this work, the structures of all urease accessory proteins involved in urease maturation will be available, and together with the mutagenesis data, we will have a better understanding of how the urease accessory proteins associate with the urease to form the pre-activation complex for the maturation of urease.

Infection of Helicobacter pylori induces inflammation in the human stomach, and causes gastric or duodenal ulcers. High activity of urease is one of the factors that facilitate the colonization of H. pylori in the stomach. Urease catalyzes the hydrolysis of urea into ammonia, which neutralizes the gastric acid and allows the pathogen to survive in the human stomach.

In the active site of urease, there is a carbamylated lysine residue that are involved in binding two nickel ions essential to the metal catalysis of the enzyme. In H. pylori, the maturation of urease (i.e. the carbamylation of the active site lysine residue and the insertion of nickel ions) is assisted by four urease accessory proteins, namely, UreE, UreF, UreG, and UreH (UreH is the H. pylori ortholog of UreD found in other species). The current model for urease maturation suggests that the urease binds UreF, UreG and UreH to form a pre-activation complex, which then interacts with the Ni-binding protein UreE to trigger the GTP-dependent activation of urease.

How the urease accessory proteins interact with each other and with the urease for the maturation process is poorly understood. This proposal aims to address a number of important questions concerning the structure-function of urease accessory proteins UreF and UreH:

(1) Is the dimerization of H. pylori UreF/UreH complex essential to the maturation of urease?

We have determined the crystal structure of H. pylori UreF and UreF/UreH complex. Both of them exist as dimers in the crystal structure. Moreover, we showed that H. pylori UreF/UreH complex exists as a 2:2 dimer in solution. Based on the crystal structures, we will introduce mutations in the dimeric interface to break the dimerization of H. pylori UreF/UreH complex, and test if these mutations affect the in vivo maturation of urease.

(2) Is the interaction between UreF and UreH essential to the maturation of urease?

Our preliminary data showed that the truncation of the C-terminal residues of UreF breaks the UreF/UreH complex, and abolishes the activation of urease. To further investigate the functional importance of UreF/UreH interaction in urease maturation, we will introduce mutations in the interface of the UreF/UreH complex, and test if these mutations affect the formation of UreF/UreH complex, and in vivo maturation of urease.

(3) How does the UreF/UreH complex interact with UreG and the urease?

The UreF/UreH is also known to form bigger complexes with UreG, and with the urease. How UreF/UreH complex associates with UreG and the urease to form the activation complex for the maturation of urease is poorly understood. Based on the crystal structure of the UreF/UreH complex we solved, we propose to perform scanning mutagenesis to map the surface on UreF and UreH for interaction with UreG and with the urease. The mutants’ ability to form complex with UreG and the urease will be correlated with their ability to activate urease in vivo.

(4) What is the structure of UreG/UreF/UreH complex?

We have already obtained crystals of UreG/UreF/UreH complex. We propose to solve the high-resolution structure of the complex by X-ray crystallography. The structure will provide the first high-resolution structure of how these urease accessory proteins interact with each other.

Through this work, we will determine the crystal structure of UreF/UreH/UreG complex. Together with the mutagenesis data and the in vivo urease activation assay, our proposed work will contribute a significant step towards a better understanding on the structure-function relationship of these urease accessory proteins.

Objectives

To test whether the dimerization of the H. pylori UreF/UreH complex is essential to the maturation of urease

To investigate the functional interaction between UreF and UreH by mutagenesis.

To map the interaction surface on the UreF/UreH complex for binding UreG and the urease by scanning mutagenesis.

To determine the structure of UreG/UreF/UreH complex by X-ray crystallography

Activity of urease is one of the factors that facilitate colonization of Helicobacter pylori in the human stomach. Urease is a nickel-containing enzyme that hydrolyzes urea into ammonia and carbamic acid, which decomposes spontaneously into carbonic acid and ammonia [1].

NH2-CO-NH2 + H2O ? NH3 + NH2-COOH

NH2-COOH + H2O ? NH3 + H2CO3

The ammonia neutralizes the gastric acid, and allows the pathogen to survive in the human stomach.

The structure of urease from various species have been determined [2-5]. Urease is composed of ?, ? and ? subunits. In H. pylori, the ureA gene encodes the b and g subunits as a fusion protein, and the ureB gene encodes the a-subunit. In the active site of urease, a carbamylated lysine residue is involved in binding two nickel ions, which are essential to the catalysis of urea hydrolysis. The maturation of urease involves the carbamylation of the lysine residue and the insertion of nickel ions to the active site. In H. pylori, the urease accessory proteins that are involved in urease maturation are: UreE, UreF, UreG and UreH [1]. UreH is the H. pylori ortholog of UreD found in other species. In this proposal, we use the notation of ‘UreH(D)’ when we refer in general to the homologous UreH or UreD proteins, and use ‘UreH’ when we refer specifically to the protein UreH in H. pylori.

UreF and UreH(D) play pivotal roles in the formation of activation complex for the maturation of urease. UreF was reported to form complex with UreH(D) [6-9], and the two proteins interact with UreG to form the heterotrimeric complex UreG/UreF/UreH(D) [9, 10]. UreG is a SIMIBI class GTPase, which is homologous to the hydrogenase maturation factor HypB [11]. The apo-urease can form complex with UreG/UreF/UreH(D), or its components of UreH(D) and UreF/UreH(D) [9, 10, 12, 13]. It has been shown that apo-urease can be activated in vitro by just adding excess amount of carbon dioxide and nickel ion [14]. The in vitro activation of urease is increased when in complex with UreF/UreH(D) and UreG/UreF/UreH(D) [13, 15]. Chemical cross-linking experiments suggest that binding of UreF/UreH(D) may induce conformational changes of the urease [16], which may allow the diffusion of nickel ion and carbon dioxide into the active site to promote activation of urease [17].

The current model for in vivo urease maturation proposed by Hausinger’s group is illustrated in Fig. 1 [1]. The apo-urease interacts with UreG, UreF and UreH(D) to form a pre-activation complex. UreE, a dimeric nickel-binding protein, then interacts with UreG of the complex, and triggers the GTP-dependent activation of urease [15, 18, 19].

The formation of activation complex for the maturation of urease involves protein-protein interaction among the urease accessory proteins and the urease. However, structure-function studies of how these urease accessory proteins interact with each other was only poorly understood. One obstacle was that expression of UreH(D) alone in E. coli resulted in the formation of inclusion bodies. Recently, Hausinger’s group has successfully expressed soluble K. aerogenes UreD in fusion with the maltose binding protein (MBP-UreD), which allows for the first time in vitro characterization of UreH(D). They showed that UreH(D) can interact with UreF in ~ 1:1 binding ratio, but only weakly with UreG [9].

Until recently, the only urease accessory protein whose structure is available is UreE [18, 20-22]. The structure of UreF was recently determined by Chirgadze’s group [23], and in parallel, by our group [24]. The work proposed here will fill the much-needed gap of knowledge on the structure-function studies of urease accessory proteins.

1. We have determined the crystal structure of H. pylori UreF. Our group has determined the crystal structure of H. pylori UreF using the MAD method with Se-Met labeled protein [24]. The structure of the native UreF, refined to 1.85 Å resolution by us, is similar to the structure of Se-Met derivative reported independently by Lam et al. [23]. UreF is an all-alpha protein consisting of 10 helices. It forms dimers in the crystal structure (Fig. 2). The dimeric interface is formed by docking of helix-1 to the helix-8 and helix-9 of the opposite UreF molecule.

2. We have established an efficient protocol to express and purify UreF/UreH complex. As mentioned above, one obstacle for the structure-function studies of the UreF/UreH complex was that expression of UreH alone resulted in insoluble inclusion bodies (Fig. 3). We have successfully solved this problem by co-expressing UreH with GST-UreF in E. coli (Fig. 3). After affinity chromatography purification and removal of the GST-fusion tag, the UreF/UreH complex can be purified in large quantity (~10 mg per liter of bacterial culture).

3. We have established assays to correlate in vitro protein-protein interactions with in vivo maturation of urease. We showed that when co-expressed together, UreF and UreH form a soluble complex that can be pull-down by GST affinity column (Fig. 4A, lane 2). We noticed that the C-terminal residues of UreF were protected from degradation upon complex formation with UreH (Fig. 4A, lane 1 & 2). We showed that truncation of the C-terminal residues of UreF (UreF-DC20) disrupted the formation of a soluble UreF/UreH complex (Fig. 4A, lane 3). We have also established an assay to test the in vivo maturation of urease (Fig. 5), and showed that the mutation (UreF-DC20) that disrupted the interaction between UreF and UreH also abolished in vivo maturation of urease. By GST pull-down, we demonstrated that the UreF/UreH complex interacts with UreG (Fig. 4B, lane 4), and with the urease (Fig. 4B, lane 4). These preliminary data demonstrated that feasibility of the proposed structure-function studies.

4. We have obtained the preliminary crystal structure of H. pylori UreF/UreH complex. With the purified UreF/UreH complex, we were lucky to obtain crystals of the complex that diffract to high resolution (Fig. 6A). Diffraction data was collected to 2.5Å resolution. We phased the structure by molecular replacement using the structure of UreF as a search template. Our preliminary structure of UreF/UreH complex showed that the UreF/UreH complex forms a 2:2 dimer in the crystal structure (Fig. 6B). We anticipate that the refinement of the UreF/UreH complex structure will be finished very shortly, and the structure will provide a rational based for the mutagenesis studies proposed in this study.

5. We have showed that the UreF/UreH complex form dimers in solution. To test if the UreF and UreF/UreH form dimers in solution, we have loaded purified samples of UreF and UreF/UreH to an analytical size-exclusion-chromatography column coupled to a static light scattering detector (Fig. 7). The apparent M.W. for UreF was 43 kDa, which is in between the theoretical M.W. of a monomeric (28 kDa) and a dimeric (56 kDa) form of UreF. The results suggest that UreF alone does have a tendency to form dimers, and the dimeric form of UreF is in exchange with the monomeric form in solution. On the other hand, the formation of dimer is more-or-less complete in the UreF/UreH complex. The apparent M.W. measured for UreF/UreH complex was 116 kDa, which is consistent with the theoretical M.W. of 116 kDa for a 2:2 dimer of UreF/UreH complex.

6. We have established an efficient protocol to express and purify UreG/UreF/UreH complex, and obtained crystals of the complex. We have found that the most efficient way to obtain the H. pylori UreG/UreF/UreH complex is to co-express UreG, GST-UreF and UreH together in E. coli. The ternary complex can be easily purified by affinity chromatography followed by removal of GST-fusion tag by protease digestion. In our hand, the yield of UreG/UreF/UreH complex is ~5mg per liter of bacterial culture. More encouraging is that we have successfully obtained crystals of UreG/UreF/UreH that diffracted to a reasonable resolution of ~3Å (Fig. 8). These preliminary data strongly suggest that the proposed structure determination of the ternary complex of UreG/UreF/UreH by X-ray crystallography is highly feasible.

Track Record of PI

The PI has extensive experience on structure determination by both NMR and X-ray crystallography, and using protein engineering to probe the structure-function of proteins. In addition to the structure determination of UreF and UreF/UreH complex discussed above, he has solved the solution structure of barstar, an inhibitor of barnase, and studied its dynamics behavior by NMR spectroscopy [25, 26]. He also studied the effect of mutations on the stability and structural perturbation on the DNA-binding domain of the tumor suppressor p53 by NMR spectroscopy [27, 28]. He has used an approach that combines evidence from NMR experiments and molecular dynamics simulation to study the folding pathway and the denatured states of barnase and chymotrypsin inhibitor-2 [29-31]. Supported by previous GRF grants, he solved the solution [32] and crystal [33] structure of ribosomal protein L30e from Thermococcus celer, the crystal structures of a thermophilic acylphosphatase from Pyrococcus horikoshii to 1.5Å [34], and human acylphosphatase to 1.45Å [35], an orange fluorescent protein from Cnidaria tube anemone to 2.0Å [36], seabream antiquitin to 2.8Å [37], the crystal structure of trichosanthin in complex with the C-terminal residues of ribosomal stalk protein P2 to 2.2Å [38], and the solution structure of the N-terminal dimerization of P2 [39]. We believe that, with our strong background in structural biology and the solid preliminary data, we are in a leading position to determine the structure of the UreG/UreF/UreH ternary complex, and to study the how the urease accessory proteins interact with each other for the maturation of urease.

Our preliminary data suggest that the H. pylori UreF/UreH complex forms a 2:2 dimer in solution. Both the crystal structure of H. pylori UreF, and the preliminary structure of UreF/UreH complex suggest that the dimerization is likely to be mediated by UreF. It is presently not known whether the dimerization is a unique property of H. pylori UreF - for example H. pylori and K. aerogenes UreF only share 19% sequence identity. Interestingly, the quaternary structure of H. pylori urease is different from ureases from other bacterial species. Unlike the urease (UreABC) from K. aerogenes that forms a trimeric complex (UreABC)3, the H. pylori urease (UreAB) forms a tetramer of trimers ((UreAB)3)4. Nevertheless, that H. pylori UreF/UreH complex exists as a dimer in solution and in crystal structure raises an interesting question - is the dimerization of H. pylori UreF/UreH complex essential to the maturation of urease?

To address this question, we will introduce mutations that are designed to break the dimerization of UreF and UreF/UreH complex. As shown in Fig. 2, the dimeric interface is formed by docking of helix-1 to the helix-8 and helix-9 of the opposite UreF molecule. A closer look at the dimeric interface of the crystal structure of UreF reveals a number of interactions that may be importance to the dimerization of UreF (Fig. 9). For example, to break the hydrogen bonding network among Q37, Q205 and Q212, we will replace the Gln residue with either alanine or asparagine to create triple mutants of Q37A/Q205A/Q212A and Q37N/Q205N/Q212N. We anticipate that both truncation of and shortening of the amide chain should break the hydrogen bond network. To disrupt the hydrophobic interaction around F33, we will substitute the Phe residue with alanine (F33A) or with a polar residue (e.g. F33R). Substitution of polar residue like arginine at Phe-33 should highly disfavor dimerization because the high desolvation penalty will prevent the polar residue to be buried upon dimerization. If necessary, we will create quadruple mutants (e.g. Q37A/Q205A/Q212A/F33A) to ensure disruption of UreF dimerization.

3.1.1 GST pull-down assay for UreF/UreH interaction. First, we test if these mutants will affect the formation of soluble UreF/UreH complex by GST pull-down assay (Fig. 4A). UreH will be co-expressed with mutants of UreF fused with GST-tag, and the bacterial lysate will be loaded to a GSTrap column (GE Healthcare). After extensive washing with binding buffer (20 mM Tris pH7.5, 0.2M NaCl, 5mM DTT), the proteins will be eluted with 10mM glutathione.

As these mutations are located at the dimerization interface, which are far away from the UreF/UreH interface, we anticipate that they will not affect UreF/UreH interaction.

3.1.2 Size-exclusion-chromatography/static-light-scattering (SEC/LS). We will test if these mutants affect dimerization of UreF by SEC/LS. Purified samples of UreF mutants and its complex with UreH complex will be loaded to an analytical Superdex 200 column connected to an online miniDawn light scattering detector and an Optilab DSP refractometer (Wyatt Technologies). The light scattering data will be analyzed using the ASTRA software provided by the manufacturer to obtain the molecular weight of the protein samples.

If the mutations break the dimerization, we anticipate that the measured molecular weight will be 28 kDa for UreF, and 58 kDa for UreF/UreH complex.

3.1.3 In vivo maturation of urease. We will test if test if these mutants affect in vivo maturation of urease. We have established an assay for in vivo maturation of urease (Fig. 5). We have cloned the H. pylori urease operon, ureABIEFGH, into the pRSETA vector to create the pHpA2H vector. We will introduce the mutations into the ureF gene in the pHpA2H vector. E. coli will be transformed with wild-type and mutant pHpA2H vectors, or the negative control plasmids (pHpAB and the empty vectors). The bacterial cells will be grown in the presence of 0.5 mM nickel sulfate, and were induced overnight with 0.4 mM IPTG. After cell lysis by sonication, urease activity of the bacterial lysate will be assayed in 50 mM HEPES buffer at pH 7.5 with 50 mM urea substrate, and will be measured by the amount of ammonia released using the method described in ref. [40].

If the dimerization of UreF and UreF/UreH is essential to the maturation of urease, the mutations that break the dimerization will also abolish the maturation of urease. On the other hand, if the maturation of urease is not affected by these mutations, it is likely that the dimerization is not essential to the urease maturation.

Our preliminary data showed that removal of the C-terminal residues of UreF breaks the UreF/UreH complex, and abolishes the maturation of urease. The availability of a preliminary structure of UreF/UreH complex allows us to introduce site-directed mutations that are designed to break the UreF/UreH interaction, and to further investigate the functional importance of UreF/UreH interaction. Our structure showed that upon complex formation, the C-terminal residues of UreF become structured and form an extra helix (helix-11) that dock to a binding cavity of UreH. Three hydrophobic residues V235, I239, and M242 on helix-11 are buried to a hydrophobic pocket of UreH.

To further investigate the functional importance of UreF/UreH interaction in urease maturation, we will create alanine and hydrophobic-to-polar (e.g. V?N) substitutions at V235, I239 and M242, which are designed to break the UreF/UreH complex formation. We will test if these mutations affect the formation of the UreF/UreH complex by the GST pull-down assay described in 3.1.1, and if they affect maturation of urease as described in 3.1.3. If the interaction between UreF and UreH is essential to the maturation of urease, we anticipate the mutations that break the UreF/UreH interaction will also abolish the maturation of urease.

The UreF/UreH is also known to form bigger complexes with UreG, and with the urease (UreA/UreB). How UreF/UreH complex associates with UreG, and the urease to form the pre-activation complex (UreA/UreB-UreG/UreF/UreH) for the maturation of urease is poorly understood. It has been reported that UreG does not interact directly to the urease, suggesting the UreF/UreH complex serves as a bridge that recruits UreG to the activation complex.

Our group has recently collected 2.5Å diffraction data for the H. pylori UreF/UreH, and has obtained a preliminary structure of the complex, which allows us to identify surface residues of UreF and UreH. To map the interacting surface of UreF/UreH complex for binding of UreG and the urease (UreA/UreB), we propose to perform alanine-scanning mutagenesis of surface residues on UreF and UreH. We will first focus on relatively more conserved surface residues (For UreF: P44, I45, Y48, S51, E55, Y72, E119, R121, Y183, K195, Q201, Q205, H244, E245, R250, L251, S254. For UreH: D60, G61, T78, K84, P111, I115, F177, E140, R146, E151, R213). We will also introduce multiple substitutions at these residue positions, if they are close in space according to the preliminary structure of UreF/UreH complex. We will first test if these mutations affect UreF/UreH interaction as described in 3.1.1. If so, we will exclude those mutants from the library.

After we have created the mutant library of UreF/UreH complex, we will test the mutants' ability to form complex with UreG, and with the urease (UreA/UreB). In brief, mutants of the GST-UreF/UreH complex will be co-expressed in E. coli. Our preliminary data suggest that the GST fusion tag will not interfere with binding of UreG or UreA/UreB (Fig. 4B). The bacterial lysate of GST-UreF/UreH (or its mutants) will be mixed with bacterial lysate expressing UreG or UreA/UreB, and then loaded to a GSTrap column for the pull-down assay. For those mutations that break the interaction, we will also perform the reciprocal pull-down in which the GST-tag is fused to the UreH, UreG, UreA or UreB. This is to confirm that the breakage of interaction is due to the mutations, but not due to a nearby GST-tag.

To address the question if the interaction between UreF/UreH and UreF (or UreA/UreB) is essential to maturation of urease, we will test the ability of the UreF/UreH mutants to activation urease in vivo as described in 3.1.3. If the interaction is essential to urease maturation, we anticipate the mutations that break the interaction will also abolish the urease maturation.

3.4.1 Expression and purification of UreG/UreF/UreH complex - We have established an efficient expression purification protocols for the ternary UreG/UreF/UreH complex. His-GST-tagged UreF, UreG and UreH will be co-expressed together in E. coli BL21(DE3) strain using the expression plasmids pET-Duet-HisGST-UreF/UreG and pRSF-UreH. After affinity chromatography purification, the His-GST fusion tag will be removed by the PreScission Protease (GE Healthcare). The protein complex will be further purified by gel filtration. Typical yield of the UreG/UreF/UreH complex is ~ 5mg per liter of bacterial culture.

3.4.2 Optimization of crystallization conditions - Preliminary screening of crystallization conditions was performed. We have already obtained crystals of the UreG/UreF/UreH that diffract to ~3Å (Fig. 8). We will further optimize the crystallization condition by grid-searching the pH and precipitant concentrations, and addition of additives or detergents. Quality of the diffraction data will be used to guide optimization of the crystallization conditions. We will also optimize the cryo-protection procedures (e.g. the choice of cryo-protectants and their concentration) to improve the quality of diffraction data collected. When necessary, we have access to synchrontron beam line at Diamond Light Source, Oxford, through collaboration with Dr. Yu-Wai Chen (King's College London).

3.4.3 Phase determination - We will first attempt to phase the structure by molecular replacement. At the time of writing this proposal, we are refining the structure of H. pylori UreF/UreH complex. We will use the UreF/UreH complex structure as a search template to solve the phase of the UreG/UreF/UreH complex by molecular replacement. In parallel, we will also prepare selenium-methionine labeled sample of UreG/UreF/UreH for multi-wavelength anomalous diffraction (MAD) phasing by expressing the protein complex in minimal medium containing Se-Met as described in Doublie [41]. The H. pylori UreG, UreF, and UreH proteins contain 9, 10, and 8 methionine out of 199, 254, and 265 residues, which should provide enough phasing power for MAD phasing. The PI's group has previously established the expression protocols for Se-Met labeling for H. pylori UreF, and determined its structure by MAD phasing. We have access to synchrontron beam line at Diamond Light Source for collection of MAD data.

3.4.4 Model building and refinement - Models will be built interactively by the program COOT [42], and refined using PHENIX [43]. The progress of refinement will be monitored by Rfree- and R-factors. Quality of the crystal structure will be validated by the program MOLPROBITY [44].

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Management Structure And Human Resources Marketing Essay

NYTimes.com’s 3D Video news section is a new way to report information that needs to go beyond print, photography, and interactive flash graphics. Using 3D technology, the 3D Video news section will be a tab on the NYTimes.com website that will enable users to play 3D video of events like important speeches, natural catastrophes, and milestone events.

Formed as a venture by the New York Times Company, the 3D video section will be integrated into the existing managerial structure of NYTimes.com, and will be an integral part of the newsroom.

With the blockbuster success of 3D movies such as Avatar, and with 3D technology becoming increasingly ubiquitous, all that was needed to make 3D news a reality is fast Internet speeds. Ultrabroadband, which operates at 200 times current Internet speeds at about 1 gigabyte per second, is expected to hit the market by 2012 – giving customers unparalleled fast access to the Internet. To exploit this technology, news needs to move and adapt, and considering the dire state of traditional news media today – 3D news could be the answer.

This plan will equip the New York Times newsroom with 3D cameras and editing software, and train reporters in their usage. It will enable reporters to take their stories to the next level, and add a new dimension to digital news, which has now become the preferred way most consumers get their news.

3D video news will be the best generator of revenue for the New York Times to date, which has been suffering from lowered subscription costs leading to lower advertising rates. 3D video news will increase viewership, target advertising, and command larger than ever CPMs, making NYTimes.com the preferred way everybody gets their news. NYTimes.com will have the first mover advantage over all competitors, and can roll out content as soon as ultrabroadband hits the small business and consumer market.

It will also solve problems of unfaithful readership, by becoming the first news organization to provide news beyond print and photography. It will utilize existing journalistic skills to tell a story, but through an entirely new technology. Under the New York Times brand name, which is trusted and reputable, 3D video will keep customers faithful, and attract new ones.

The Company

The New York Times is a daily newspaper that began publishing in 1851. It is circulated nationwide through all 50 states, including the District of Columbia. It is also circulated worldwide  .

The Times has a website, NYTimes.com, that commands a large market. In 2009, NYTimes.com was attracting 17.9 million unique users per month. Content is also distributed through social media (Twitter, Facebook) and mobile applications available for most smart phones.

For NYTimes.com, the primary revenue source is advertising. Earlier this year, the company also announced that starting 2011, NYTimes.com would no longer be free to use. Using micropayments, where users pay for content piece by piece. The more somebody uses the site, the more he or she will be paying. According to Arthur O. Sulzberger Jr., the publisher of the New York Times, the company is relying on reader loyalty to ensure future success. However, no further details about how exactly the company will be implementing micropayments were offered. A few articles will be free to use, he said, till users hit a pay wall and have to begin shelling out money.

NYTimes.com’s primary competitors are other news sites like Yahoo! News and CNN.com. Internationally, its sister website, global.nytimes.com, which combines content from the New York Times and the International Herald Tribune, has to compete against news websites like Reuters and the BBC.

Digital News: A Summary

The Internet has had enormous impact on traditional news companies. In 2008, it surpassed all media except television as the preferred way that consumers get their news, according to the Pew Research Center  . The jump towards online news was enormous. In 2007, only 24 percent of Americans said they were getting news primarily from the Internet. In 2008, this number rose dramatically to 40 percent. 2008 was by and large considered the milestone, and just the beginning, of the dramatic shift towards digital news consumption. Delivery of news through digital methods had become the norm by 2009. According to the Project for Excellence in Journalism  , six out of 10 Americans were getting their news online by then.

What is interesting about the digital shift is that social media or blogs are not ready to take audiences away from traditional news organizations. Though more people are consuming news online, they are doing so using online versions of newspapers. The Project for Excellence in Journalism says that this trend could change, especially with the younger generation. In a survey by Nielsen, the younger demographics were likely to point to news aggregators like Google as being the primary way they get their news. They are more like to be “grazers” – get the headline, the author, and the first few sentences of the story, and then leave it at that. The challenge for traditional news organizations is to get users to the website, instead of letting them leave before stepping foot inside  .

Audience Behavior

How people perceive news, especially online, has changed dramatically. They are no longer passive consumers. Indeed, the news that is most read online is usually one that triggers some sort of participation. This has come about mostly due to social media sites like Facebook and Twitter that allow audience participation, and the increased portability of news due to users who get their news via cellphone or portable computers.

The problem for traditional news sites is that audiences are “grazers.” Though half of all audience traffic for news is tied to legacy news organizations like the New York Times and CNN, nobody is spending much time at those websites. According to Nielsen  , the average news user spends only 3 minutes and 4 seconds on a news website per session. But visitors to the New York Times website spends at least a minute longer there than on news aggregator sites like Google. The challenge is to keep people’s attention, and this business plan will do that.

Figure 1: Top 20 sites by sector (Source: PEJ’s State of the Media 2010)

According to Nielsen, NYTimes.com was the fifth most visited site in 2009, commanding 18.5 million unique visitors.

Economics

Advertising, as always, remains the problem in the digital shift. Though some companies experimented with pay walls, there were increased signs that consumers are not ready to pay for news.

However, the one place where news companies are trying to increase advertising is videos.

Figure 2: Online Ad Spending by format in 2009 (Source: eMarketer, “U.S. Ad Spending Turns a Corner”, December 11, 2009)

As shown in the figure above, the only category that grew other than search was video advertising, which reached $1.02 billion in 2009, up 40.2% from $732 million in 2008. Because advertising has shown that it alone will not be a sufficient source of revenue, news organizations are increasingly looking at alternative revenue streams. The most obvious one is charging users for content, either through a pay wall (made successful by the Wall Street Journal) or micropayments. As discussed above, The New York Times announced earlier this year that it will be charging users to read it online using micropayments.

How can traditional news companies make the shift to digital but still keep high revenue?

Opportunities

How to solve the “grazing” problem.

According to the PEJ report, consumers look at many different sites to get their news  . Establishing a brand and ensuring users remain loyal to that brand is one of the best ways a news site can ensure success. Users are discriminating to some degree: if NYTimes.com is able to gain loyalty for a specific “type” of news, and be the best at it, they can guarantee that people will return. Therefore, our proposal of using UBB to incorporate 3D videos of events will make NYTimes.com “the place to go” for a specific kind of news.

Ensuring interactivity is key to success.

News sites can no longer afford to keep information flowing uni-directionally. News is a social currency to most people. NYTimes.com needs to retain interactivity in order to keep customers. In our 3D video scheme, we would also have a real-time chat box in which users can submit comments, feedback, and carry on conversations about the content they are watching. At the same time, if they also have video footage or photos of the event in question, they can submit these. Their footage will also be available to those watch the content on NYTimes.

People don’t want to pay. What can we do?

Most of the research by PEJ indicates that there are a few situations in which people are willing to pay. As the Wall Street Journal model shows, people pay for news that is difficult to get through other means, like finance news. People will also pay for high quality, high value content, which is why iTunes is successful. Our 3D video plan will be high quality, and 3D – difficult to get through other means.

Advertising

Video advertising has shown enormous gains in the past year. Our plan utilizes video, which commands a higher CPM than other forms of advertising methods like banners or pop-ups.

Competitors/Threats

Currently, no news organization utilizes 3D news delivery. Because of the relative newness of the technology, our plan estimates that there will not be any competition from other news organizations. Of course, as 3D technology gains a foothold, other news organizations will seek to do what our plan does: to integrate the technology in the delivery of the news online.

Our biggest competitor in 3D video news will most likely be the Wall Street Journal, which also competes with the New York Times for consumers. However, at this time, WSJ.com has indicated no desire to go 3D.

The only other news organization which is experimenting with 3D technology is Sky News, based in the UK. Sky News debuted its “Second Life Newsroom”, which enables customers to “visit” the Sky News newsroom through Second Life – they can be presenters, create and anchor their own shows, and so on. Sky has also indicated that they are considering the possibility of presenting news and events through 3D. 7,000 people did the Hajj pilgrimage and “went” to Mecca via Second Life in 2007.

However, Sky News is not doing what our plan does: utilize the resources of the New York Times to give our reporters 3D capabilities through training and technology to bring consumers event coverage in 3D. Therefore, we do not see any competition in the short term. We will have the first mover’s advantage – and therefore build customer loyalty before other news organizations. We will also have the technological advantage – make our mistakes and learn from them – before everyone else.

Competitive Advantage

Not only are we moving first, we are also able to use the enormous clout of the New York Times Company to leverage our position. We can exploit the expertise of the current New York Times staff – both editorial and technological – and therefore gain a considerable advantage.

We can also enter into partnerships with pioneers in the 3D technology field, like Sony Corp., which produces 3D capable cameras (stereoscopic cameras). According to Sony’s Chief Technology Officer, Gary Podorowsky, Sony 3D cameras are the best in the market. If we combine the resources of the New York Times and Sony, we could benefit from huge economies of scale, putting us at an advantage over small companies that may try to produce 3D video.

Because 3D content is expensive to produce, we have a massive advantage over competitors because we are working with the New York Times. Financing will be easier, partnerships will be simpler, and we can benefit from the clout the organization has. Furthermore, we will be producing all content in-house – giving us full control over what we choose to produce and when.

One of the major weaknesses that ESPN has with reference to their plan of broadcasting the Soccer World Cup in 3D is that there are only 85 live sporting events over the entire time period. There will be plenty of “black space” on the channel when no games are being played. We do not have that problem – we produce as and when it is necessary to produce content, and we will only produce necessary content that consumers want.

Weaknesses

The biggest weakness of our product is the 3D glasses, which would have to be worn to watch the 3D videos on the website. However, we believe that with 3D technology becoming a little bit more mainstream, people will be willing to wear those glasses.

If you already will own a 3D television set, will be watching the 2010 soccer World Cup in 3D on ESPN  , or be watching the new 3D network that Sony and Discovery are planning to launch together, 3D glasses will be as much a fixture in the home as anything else.

The other problem is the massive reorganization of reporting and journalism that will be needed to make this plan a reality. The costs of retraining and equipment will be discussed more below, but at the outset, incorporating 3D video into NYTimes.com will need reporters to rethink the way they shoot and produce video.

Our plan operates under the umbrella of the New York Times, and specifically under NYTimes.com. The existing management structure of NYTimes.com will be kept the same, with an extra arm added to oversee the 3D video section on the website.

The extra arm will be divided between “editorial” and “technology” teams. The editorial team, comprised of 8-10 journalists with extensive digital media skills, will be responsible for organizing the video components. This includes deciding which events to cover, how best to create the video, and also working with NYTimes.com’s other reporters to create hybrid projects.

For example, if the New York City Marathon is being covered, there will be of course a print piece accompanying the video, along with photographs, and perhaps an interactive graphic showing the race route. Therefore, the Editorial team will be working closely with the NYTimes editors and reporters to create the project.

The technology team is focused more on training and the technical aspects of the video production. Comprised of 13-15 people, the team will be responsible for teaching camera work to both the video reporters and the regular reporters, producing the video, and will be working very closely with the editorial team.

Because this is a very creative enterprise, both teams would be acting mostly independently, but supervised by a manager who will co-ordinate between both teams and determine overall strategy. That manager will also be the main liaison to the web editor of NYTimes.com, as well as the managing editor for the New York Times.

Apart from this staff of about 26 people, resources for sales, marketing, distribution, and so on will be shared with the NYTimes.com’s existing pool of people.

Content Production

Content production in 3D technology is of course the biggest obstacle in this business plan. It is difficult to shoot in 3D, and requires expensive equipment and extensive training.

There is also not a lot of content out there. Since our plan relies exclusively on in-house content, production needs to be fast, and plentiful. Therefore, retraining in 3D technology and editorial decisions need to be fast – so content will be plentiful on the website. The entire point of digital news is that people constantly want to see fresh new content, and there is a grave danger that stale content will be left up for weeks without new videos coming in.

Eye Fatigue Problems

The issue of eye fatigue is definitely going to be a factor. Though this is not relevant to people who watch 3D movies, makers of 3D television sets are now grappling with the headaches, eye fatigue, and other health problems the technology brings up. Though this may not affect us as much – because we aim to have short, 5-10 minute videos, not 24-hours-a-day broadcasts – this is a technical issue that will be still factored in when creating the videos and deciding how long they will be.

The glasses

Many have questioned 3D technology because people will not want to wear 3D glasses all the time. This is another factor that will be considered when dealing with 3D videos on NYTimes.com. Though this is again more of an obstacle for longer, full-length broadcasts of 3D content, our plan needs to figure out how to get the glasses to subscribers.

Starting 2011, the New York Times website will no longer be free to use. Therefore, when consumers subscribe, a box of 3D glasses will be sent to them. Glasses are cheap to produce, and distribution costs will be negligible. As long as every household has a few pairs, the technical issues behind how people will access the videos will be solved.

Technology

Reporters will be equipped with a Sony 3D camera. Sony announced earlier this year it was building a range of consumer-friendly and easy-to-use cameras to film in 3D  . Though exact retail costs were not disclosed by Sony, a look at other similar technology by Panasonic indicates a possible price point of $21,000 per camera  . At the beginning, we can invest in 5 cameras, and as more reporters are comfortable with using the technology, buy more.

1

5

$105,000

2

8

$168,000

3

12

$252,000

Editing software can be bought at a one-time cost of about $50,000.

Training

Training in shooting 3D will require experts to be brought in to teach shooting and camera work. We estimate a one-time cost of $25,000 for this. Once the editorial and tech staff know how to use this, they can train other reporters.

Sources

At the outset, the financing will be done through the New York Times Company, which will pay all the costs associated with buying equipment and retraining.

After this, the 3D video arm will be largely independent, and use advertising to pay the rest of the costs. Online video advertising is clearly a better money spinner than traditional advertising. Video advertising commands a more profitable CPM than banner ads or other forms of online advertising. At the beginning of the video, an advertisement will be played. Throughout the video, advertisers will be able to put logos in the corner, with an option of interjecting advertisements in the middle of the video, depending on the length of the broadcast.

Recently, DoubleClick compared the effectiveness of video advertisements versus static advertisements. The study found that video ads received click-through-ratios between 4 and 7 times that of static banner ads  . With video ads, it is also easier to measure interaction rates with users, which means advertising can be targeted better.

The above figure shows the results of a survey by eMarketer and the Interactive Advertising Bureau that looked at CPMs commanded by different types of advertising. It showed a much higher CPM in video ads than any other ad format. The average CPM was $43. The CPM commanded by video ads range from $40 to $50. This is a huge lead over banner ads which are sold by publishers, which command only a $10 to $20 CPM.

The success of advertising on Hulu, which has advertisements at the beginning of clips, as well as between, is a good testament to how profitable this plan will be. Hulu sells ads at a slightly lower CPM, around $25 per 1,000 views. However, because Hulu’s content is not original, this is to be expected. Our plan has original content, which can certainly command a higher CPM.

It is also important to note the domino effect this will have on CPMs in other areas of NYTimes.com. The more people that view videos are also likely to stick around and look at other sections of the website, which means a higher traffic overall and more revenue overall. According to the New York Times’s most recent earnings report, digital advertising revenues grew 18 percent in the first quarter of the year, a positive sign that this is the right time to expand on the digital front.

The majority of the consumer base for NYTimes.com’s 3D video news sections will come largely from the people who use the web to get their news.

Online news users tend to be younger than print news readers, with 29 percent of them under 30 years old. 50 percent of them are employed full time. This data, gathered from an online news survey by PEJ and the Internet and American Life Project, is presented below.

According to Quantcast, NYTimes.com is evenly read by males and females, with a large proportion having a post-graduate degree. This means that our product is targeted towards the “young” and technologically savvier populations. They are also the ones more likely to be ready to experiment with the new technology, and more ads can be targeted to them as they have more disposable income.

Where to advertise?

This service needs to go viral. Word-of-mouth advertising has been shown to create “buzz” around a product. Therefore, advertisements on sites like Mashable, Facebook, and Twitter would be the best way to get the word out. YouTube is another good way to go viral. Videos with a fun, edgy feel about this new service will be put on YouTube and more likely to be viewed by the target demographic.

Of course, in house advertising would be a good way to get existing readers to try this out for themselves. Ads in the print edition of the newspaper and on the website would make this very effective. To target further, ads in the sections of the newspapers read by young adult would be most effective.

For the following projections, we are assuming an initial viewership of 20 percent of the current number of readers of NYTimes.com. For month 1, 3.5 million users will watch the video. There will be one video per month in months 1-5, and four videos per month for months 6-12. Every month, we are estimating viewership to rise slightly, by 100,000 per month. The CPM commanded is $43. There are two ads per video.

1

3.5

$0.30

2

3.6

$0.31

3

3.7

$0.32

4

3.8

$0.33

5

3.9

$0.34

6

4

$1.38

7

4.1

$1.41

8

4.2

$1.44

9

4.3

$1.48

10

4.4

$1.51

11

4.5

$1.55

12

4.6

$1.58

Costs in the first month are under $200,000. The service will be profitable right from its inception.

There is no doubt that news organizations need resuscitation. Small breakthroughs have been made possible by digital news. However, the problem is that companies like the New York Times do not offer anything vastly new on their website, save a few graphics and interactive designs.

This business plan solves that, making people want to pay (through micropayments) for a niche product that cannot be found anywhere else. The product is unique, but relatively easy to implement. It requires minimal investment in technology and retraining, but because it operates under the trusted and reputable brand name of the New York Times, has an existing consumer base that ensures its profitability.

It commands high CPMs from advertisers, and will both keep existing customers but also “steal” them away from competitors because we have something they do not: coverage of events and stories in an unprecedented way that transports you right to the scene. Whether it is a historic speech or a devastating earthquake, readers of the New York Times can be there and experience it themselves, knowing all the while that they can trust that they are looking at content that is produced with the highest journalistic standards in mind.

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