Biochemistry short questions

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Tryptophan?

L-Tryptophan is the unique protein amino acid (AA) bearing an indole ring: its biotransformation in living organisms contributes either to keeping this chemical group in cells and tissues or to breaking it, by generating in both cases a variety of bioactive molecules

In addition to protein turn-over, in humans the pathways of Trp indole derivatives cover the synthesis of the neurotransmitter/hormone serotonin (5-HT), the pineal gland melatonin (MLT), and the trace amine tryptamine.

The breakdown of the Trp indole ring defines instead the “kynurenine shunt” which produces cell-response adapters as L-kynurenine, kynurenic and quinolinic acids, or the coenzyme nicotinamide adenine dinucleotide (NAD+)

The presence of Trp residues in polypeptides, in fact, the Trp indole ring in –R residues gives unique properties to proteins and peptides promoting protein-protein, protein-peptide, or protein-biomolecule structural hydrophobic interactions.

The Trp indole ring is able to stabilize structures, domains, and interactions through Van der Waals forces while the indole-N shows propensity as a hydrogen bond donor evidencing a role of this AA also in protein binding and recognition.

A main consideration deriving from previous paragraphs is that Trp is precious for life: its biosynthetic pathway is in fact energetically expensive and requires the expression of several enzymes and substrates either for Trp operon in bacteria or for the shikimate and chorismate paths in plants.

This probably explains why L-Trp is an AA scarcely represented in the alimentary chain [21, 24–26] and its presence in animal cells and tissues must be tightly regulated.

2.Conformational entropy?

The factor that makes the greatest contribution to stabilization of the unfolded state is its conformational entropy.

It has been proposed that decreasing the conformational flexibility of the unfolded chain (by substitution with proline, or by replacement of glycine) should lead to an increase in the stability of the folded relative to the unfolded protein (Matthews et al, 1987) (See also disulphide bonds, below.)

Two such substitutions (A82P and G77A) in T4 lysozyme gave rise to only a small amount of stabilization (0.8 and 0.4 kcal/mol), possibly due to the counterbalancing removal of favourable interactions upon mutation.

(Matthews et al, 1987) A similar mutation that also eliminated a hydrogen bond and some hydrophobic interactions was destabilizing, suggesting that these factors outweighed the decrease in conformational stability (Dixon et al, 1992).

Watanabe et al (1994) have found a correlation between the number of proline residues in oligo-1,6-glucosidases from a number of bacterial species and their thermostability.

Structural analysis suggests that the optimal placement is at the N-cap of alpha helices and at the second position beta type I and beta type II turns.

When they substituted prolines at these positions in the homologous mesophilic enzymes they observed an increase in thermostability.

However, they made their substitutions based on recruitment from the more stable enzyme.

Structural analysis of at least one destabilized mutant (A. Day & A. Shaw unpublished) revealed the creation of a hydrophobic surface cavity upon mutation, which presumably counterbalanced any stabilizing effect of the conformational rigidity.

This suggests that, in the absence of any hints from nature in terms of homology, proline substitution for stabilization should be used with some care.

Watanabe et al ascribe their increase in stability to a decrease in conformational entropy of the unfolded state (c.f. the T4 lysozyme case described above by Matthews et al, 1987).

As their enzyme (and ours) is irreversibly inactivated upon heating, it is equally likely that any observed increase in stability is due, not to an overall stabilization of the protein, but to a slowing of the rate of unfolding. (See Kinetic Stability below.)

3. Liquid scintillation counting outline the principles? Including references.

Quantitative measurements of beta-radiation, alpha-radiation and low-energy gamma-radiation with traditional gas or solid state detectors may be difficult due to the self-absorption of the radiation in the sample itself and further absorption in the counter window.

Therefore, low-energy radiation is, in many cases, best detected and measured with liquid scintillation counting where absorption loss is absent.

Here, the sample and the detector (the liquid scintillator) is intimately mixed in a homogeneous solution.

In addition, this leads to nearly 100% counting geometry (only the liquid surfaces against the container wall and the air above the liquid account for a small loss in counting geometry).

A liquid scintillator consists of a solvent and a primary scintillant. In some cases a secondary scintillant is also added. The function of these substances is described below.

The solvent:

The primary function of the solvent is to solubilize the radioactive sample, preferentially into a homogeneous solution.

An equally important function is to absorb and transfer the energy deposited by the radioactive decay event to the primary scintillant.

The β-particles from the radioactive substance ionize and excite molecules in the solution, - first and foremost the solvent molecules because of its high concentration.

The excitation energy is transferred from one solvent molecule X to another.

Some of these excitation transfer chain reactions result in the encounter with a molecule of the primary scintillant Y. The excitation is transferred to this molecule:

The most common liquid scintillation solvents

Commonly used solvents are toluene and 1,4-dioxane. The first is used for organic solutions and the latter when the radioactivity is dissolved in an aqueous solution.

Many modern scintillation cocktails are based on the solvent pseudocumene because of lower vapour pressure and higher flash point than the two first.

Modern cocktails also contain surfactants of different kinds and in varying degree in order to facilitate solubilization of a large variety of solutions, both aqueous and organic.

The primary scintillator:

A scintillant exhibit properties which in many respects are just the opposite of those of solvents.

They absorb the energy of the solvent molecule and tend to decay rapidly mainly through the emission of light photons, thus having a high quantum fluorescent yield.

The wavelength of the light emitted should be compatible with the sensitive wavelength region of the photo-cathode in the PM-tube.

The concentration of the scintillant is in the area 3-5 g/L, i.e. a relatively dilute solution.

4.N-linked glycosylation?

Glycosylation types are classified according to the identity of the atom of the amino acid which binds the carbohydrate chain, i.e. C-linked, N-linked, O-linked or S-linked. N-, C- and S-glycosylation take place in the endoplasmic reticulum and/or the Golgi apparatus and only extracellular or secreted proteins are concerned.

In contrast, both intracellular and extracellular proteins can be O-glycosylated.

The sugar directly bound to the protein is termed the reducing sugar, as it reduces the amino acid it binds to, and the structure of an oligosaccharide is written from the non-reducing end to the reducing end.

We indicate the type of linkage to the protein and specify the nature of the reducing terminal sugar (if known) in brackets in the ‘Description’ field.

For monosaccharides, the name of the reducing terminal sugar appears alone, while for di- and polysaccharides the name is followed by three periods

N-linked glycosylation:

N-linked glycosylation refers to the attachment of oligosaccharides to a nitrogen atom, usually the N4 of asparagine residues. N-glycosylation occurs on secreted or membrane bound proteins, mainly in eukaryotes and archaea – most bacteria do not carry out this modification.

In eukaryotes, N-glycosylation begins as a co-translational event in the endoplasmic reticulum, where preassembled blocks of 14 sugars (including 2 N-acetylglucosamines, 9 mannoses and 3 glucoses) are first added to the nascent polypeptide chain.

After cleavage of 3 glucose and 1 mannose residues, the protein is transferred to the Golgi apparatus where the glycans lose a variable number of mannose residues and acquire a more complex structure during a process called ‘terminal glycosylation’.

There are 3 types of mature N-glycans:

high mannose (those that have escaped terminal glycosylation),

hybrid

complex (with different combinations of mannose, N-acetylglucosamine, N-acetylgalactosamine, fucose and sialic acid residues).

2. O-linked glycosylation:

O-linked glycosylation of secreted and membrane bound proteins is a post-translational event that takes place in the cis-Golgi compartment after N-glycosylation and folding of the protein. It refers to the attachment of glycans to serine and threonine, and, to a lesser extent, to hydroxyproline and hydroxylysine. O-linked glycans play important roles in protein localization and trafficking, protein solubility, antigenicity and cell-cell interactions.

O-linked glycans are built up in a stepwise fashion with sugars added incrementally. The most common type of O-glycosylation in secreted and membrane-bound mammalian proteins seems to be the addition of reducing terminal N-acetylgalactosamine (GalNAc). This type of O-linked glycan is also referred to as ‘mucin-type’ glycan. The reducing terminal GalNAc residue can be further extended with galactose (Gal), N-acetylglucosamine (GlcNAc) or GlcNAc and Gal resulting in 8 common core structures, which are often further decorated with the addition of up to three sialic acid residues.

5. Draw tryptophan at ph7 and discuss its importance in proteins?

Role in structure: Being hydrophobic, Tryptophan prefers to be buried in protein hydrophobic cores. The aromatic side chain can also mean that Tryptophan is involved in stackinginteractions with other aromatic side-chains.

Role in function: As it contains a non-carbon atom (nitrogen) in the aromatic ring system, Tryptophan is more reactive than Phenylalanine though it is less reactive than Tyrosine. The Tryptophan nitrogens can play a role in binding to non-protein atoms, but such instances are rare.

Like other aromatic amino acids, Tryptophan can be involved in interactions with non-protein ligands that themselves contain aromatic groups via stacking interactions. Tryptophan and other aromatic amino acids can be involved in binding to poly-proline containing peptides, for example, in SH3 or WW domains.

6. Describe major steps of receptor mediated endocytosis?

The movement of macromolecules such as proteins or polysaccharides into or out of the cell is called bulk transport. There are two types of bulk transport, exocytosis and endocytosis, and both require the expenditure of energy (ATP).

In exocytosis, materials are exported out of the cell via secretory vesicles. In this process, the Golgi complex packages macromolecules into transport vesicles that travel to and fuse with the plasma membrane. This fusion causes the vesicle to spill its contents out of the cell. Exocytosis is important in expulsion of waste materials out of the cell and in the secretion of cellular products such as digestive enzymes or hormones.

Endocytosis, on the other hand, is the process by which materials move into the cell. There are three types of endocytosis: phagocytosis, pinocytosis, and receptor-mediated endocytosis.

In phagocytosis or “cellular eating,” the cell’s plasma membrane surrounds a macromolecule or even an entire cell from the extracellular environment and buds off to form a food vacuole or phagosome. The newly-formed phagosome then fuses with a lysosome whose hydrolytic enzymes digest the “food” inside.

In pinocytosis or “cellular drinking,” the cell engulfs drops of fluid by pinching in and forming vesicles that are smaller than the phagosomes formed in phagocytosis. Like phagocytosis, pinocytosis is a non-specific process in which the cell takes in whatever solutes that are dissolved in the liquid it envelops.

Unlike phagocytosis and pinocytosis, receptor-mediated endocytosis is an extremely selective process of importing materials into the cell. This specificity is mediated by receptor proteins located on depressed areas of the cell membrane called coated pits.

The cytosolic surface of coated pits is covered by coat proteins. In receptor-mediated endocytosis, the cell will only take in an extracellular molecule if it binds to its specific receptor protein on the cell’s surface.

Once bound, the coated pit on which the bound receptor protein is located then invaginates, or pinches in, to form a coated vesicle. Similar to the digestive process in non-specific phagocytosis, this coated vesicle then fuses with a lysosome to digest the engulfed material and release it into the cytosol.

Mammalian cells use receptor-mediated endocytosis to take cholesterol into cells. Cholesterol in the blood is usually found in lipid-protein complexes called low-density lipoproteins (LDLs). LDLs bind to specific receptor proteins on the cell surface, thereby triggering their uptake by receptor-mediated endocytosis.

7. Thin layer chromotography principles?

8. Discuss the purpose why we include replicates in experimental strategies and why its important in enzymology?

9.List the different functional groups that can be covalantly added to proteins?

Many proteins are modified by the covalent linking of groups that can affect their function and/or localisation in the cell. Such covalent modifications occur after synthesis and folding of the polypeptide component. The main types of covalent modification and their functions are listed below.

Methylation/acetylation of amino acids at the N-terminal tails of histone proteins in eukaryotes can affect the structure of chromatin and ultimately gene expression. Prokaryotes also use methylation as a means of directly regulating protein activities. For example, the methylation of specific proteins controlling flagellar movement is an important mechanism for the regulation of bacterial chemotaxis.

Phosphorylation of proteins (catalysed by specific kinases) is a key regulatory mechanism in eukaryotic intracellular signalling and in metabolic pathways.

Lipidation of proteins (i.e. addition of lipid tails) targets them to cell membranes (plasma membrane and cell organelles).

Glycosylation is a feature of many extracellular proteins, whether secreted or on the cell surface, and may offer the protein some protection against proteases.

These modifications are catalysed by specific enzymes and can be reversed, permitting regulation of the protein's function. This reversibility is particularly significant with respect to protein phosphorylation

There are a lot of functional groups that we can encounter in proteins. The main functional groups are Amino group at the N-terminal and carboxyl group at the C- terminal. In addition to these two functional groups, you will see peptide bonds (a special amide bond) in between every amino acids of the polypeptide chain. Though a peptide bond is quite stable, it is also considered as a functional group (amide functional group).

10.Beta mercaptoethanol?

2-Mercaptoethanol (also β-mercaptoethanol, BME, 2BME, 2-ME or β-met) is the chemical compound, is used to reduce disulfide bonds and can act as a biological antioxidant by scavenging hydroxyl radicals (amongst others). It is widely used because the hydroxyl group confers solubility in water and lowers the volatility. Due to its diminished vapor pressure, its odor, while unpleasant, is less objectionable than related thiols.

2-Mercaptoethanol may be prepared by the action of hydrogen sulfide on ethylene oxide

Some proteins can be denatured by 2-mercaptoethanol, which cleaves the disulfide bonds that may form between thiolgroups of cysteine residues

By breaking the S-S bonds, both the tertiary structure and the quaternary structure of some proteins can be disrupted.[5]

Because of its ability to disrupt the structure of proteins, it was used in the analysis of proteins, for instance, to ensure that a protein solution contains monomeric protein molecules, instead of disulfide linked dimers or higher order oligomers

2-Mercaptoethanol and related reducing agents (e.g., DTT) are often included in enzymatic reactions to inhibit the oxidation of free sulfhydryl residues, and hence maintain protein activity.

It is used in several enzyme assays as a standard buffer.

2-Mercaptoethanol is used in some RNA isolation procedures to eliminate ribonuclease released during cell lysis.

Numerous disulfide bonds make ribonucleases very stable enzymes, so 2-mercaptoethanol is used to reduce these disulfide bonds and irreversibly denature the proteins. This prevents them from digesting the RNA during its extraction procedure

11.camelid antibodies?

he "Y" shape is iconic in the field of immunology. When it's drawn floating around a cell, you instinctively know it's an antibody. Well, each part of that Y is important, representing two heavy chains, joined by disulfide bonds, and two light chains, each joined to a heavy chain by a disulfide bond.

The "tips" of the Y are particularly key, as they contain the variable region of the antibody, including the antigen-binding site which gives the antibody specificity for particular antigens.

In 1989, Raymond Hamers' lab at Vrije Universiteit Brussel discovered something incredibly unique while investigating the immune system of dromedaries (a type of camel).

As the story goes, a couple of undergrads in the lab were bored of the simple experiments assigned to them and wanted something different to investigate.

Dr. Hamers had reserved some camel blood for a study on sleeping sickness, and asked the students if they were interested in trying to purify antibodies from an aliquot1.

The students did just that, and found that among the conventional antibodies, there was also a smaller type present.

Upon reviewing his students' data, Dr. Hamers thought their results were a mistake and was worried that these smaller antibodies were just fragments of normal antibodies or products of degradation from storing the blood so long.

Dr. Hamers' group had discovered heavy-chain antibodies, a type of antibody that is found in all members of the Camelidae family, including llamas, alpacas, and camels.

These antibodies lack light chains, and consist of two heavy chains attached to variable domains (variable heavy homodimers, VHH).

Because of this, heavy-chain antibodies are much smaller, about 95 kDa (while a typical IgG can range from 150-160 kDa).

The VHH alone, which is 15 kDa, is considered the smallest naturally derived antigen-binding fragment.

these 15 kDa Nanobodies perform just as well as classical mammalian antibodies with respect to target recognition and binding affinity4.

Thus, the potential benefits of using Nanobodies for both therapeutic and research applications are endless:

Faster accumulation as a therapeutic or imaging agent in tissue.

Higher stability due to its smaller size.

Lower toxicity due to more rapid clearance of unbound antibodies.

Additional routes of administration.

Increased manufacturing production efficiency and potentially lower costs.

12.List the advantages and disadvantages of bradford protein assay compared to determining protein concentration by A280?

Absorbance at 280 nm

How it works: Aromatic residues, like tyrosine and tryptophan, absorb UV light at 280 nm. So, if you have an extinction coefficent for your protein (e), you can measure the absorbance in a UV/Vis spectrometer and calculate the concentration of your protein using Beer’s law (A = elc, where l is the path length of the spectrometer). You can estimate the extinction coefficient of your protein based on the sequence using Expasy’s ProtParam tool. Because ProtParam only considers the linear sequence of your protein and doesn’t take into account the structure, which can affect the extinction coefficient, you’ll want to denature your protein before you measure the absorbance. (I like to denature proteins in 6 M guanidinium.)

Advantages: This technique is quick and doesn’t require any special reagents, except for the guanidinium, which you may have on hand anyway.

Disadvantages: This method relies on having an accurate extinction coefficient for your protein, which depends on the number of aromatic residues. If there aren’t a decent number of aromatic residues, your extinction coefficient will be quite low, and you will need a fairly concentrated sample to get a reasonable absorbance (generally an absorbance between 0.1 and 1.0 is considered within the “linear range”). Also, ProtParam warns that there may be at least a 10% error in the extinction coefficient if there are no tryptophans in your protein. Therefore, if your extinction coefficient is low, which is likely the case if there are no tryptophans in the sequence, a 10% error could significantly throw off your assessment of the final protein concentration.

Bradford Assay

How it works: The Bradford assay is a colorimetric assay based on the interaction between Coomassie brilliant blue (you know, the stuff you stain your gels with) and the arginine and aromatic residues in your protein. When the dye binds to these residues, its maximum absorption shifts from 470 nm to 595 nm. In general, you measure the absorbance of a series of known concentrations of a standard protein, generally BSA, and create a standard curve. You then use that standard curve to calculate the concentration of your protein sample based on its absorbance.

Advantages: This assay is quick, and the reagent is not affected by the presence of reducing agents, like DTT and ?-mercaptoethanol, that may be in your buffer.

Disadvantages: Basic conditions and detergents, such as SDS, can interfere with the dye’s ability to bind to the protein; however, there are detergent-compatible Bradford reagents.

Also, like the absorbance at 280 nm technique, the Bradford assay depends on the sequence of your protein. If your protein doesn’t contain a decent number of arginine and/or aromatic residues, then the dye will not bind to the protein as efficiently, resulting in an underestimation of your protein concentration.

Finally, this technique depends on comparing the absorbance of your protein to that of a standard protein. So, if your protein doesn’t react to the dye in a similar manner as your standard protein, your concentration can be off.

BCA Assay

How it works: The BCA assay is another colorimetric assay like the Bradford assay. It makes use of the biuret reaction, in which the protein backbone chelates Cu2+ ions and reduces them to Cu1+ ions. The Cu1+ ions then react with bicinchoninic acid (BCA) to form a purple-colored product that absorbs at 562 nm. The procedure is similar to that of the Bradford assay, in which you create a standard curve based on a series of known protein standards.

Advantages: Because the peptide backbone is involved in the reaction, the BCA assay is less sensitive to the types of amino acids in the protein. However, the reaction is influenced by cysteine, tyrosine, and tryptophan residues.

The reagent is not sensitive to detergents and denaturants, so it’s okay to have those in your buffer.

Disadvantages: The presence of reducing agents in your buffer can interfere with the dye, but there are reducing agent-compatible dyes available.

The reaction takes some time to proceed. Usually, the samples are incubated at 37°C for 15-30 min. Also, as in the Bradford assay, you determine your protein concentration by creating a standard curve from a known, standard protein. So again, if your protein doesn’t interact with the dye in a similar way as the standard protein, your concentration could be off.

So there you have it: a few things to consider when choosing a technique to measure protein concentration. Choosing the right technique for your protein will take a bit of trial and error, but having a good technique in your back pocket to accurately measure your protein concentration will save you a lot of time and energy and help you get more reproducible results.

13.Describe what happens in enzyme reactions when the enzyme is doubled and then quadrupled ?

14.Diagram illustrating the biosynthesis and insertion of membrane proteins?

15.Explain a cAMP is synthesised and broken down ?

Phosphodiesterase 4 (PDE4) is the predominant cAMP-degrading enzyme expressed in inflammatory cells.1

Numerous studies have shown that cAMP helps to regulate T cell function.29

It is thought that cAMP helps to maintain immune homeostasis by suppressing the release of proinflammatory mediators (eg, TNF-α, IL-17, and IFN-γ)1 and promoting the release of anti-inflammatory mediators (eg, IL-10)6by immune cells.

In vitro studies suggest that cAMP exerts these effects mainly through PKA.

Once activated by cAMP, the PKA translocates into the cell nucleus and activates a transcription factor called CREB (cAMP response element binding protein).

CREB plus a coactivator called CREB-binding protein (CBP) then bind to a cAMP response element (CRE) in the promoter region of a gene.

A decrease in PDE4 activity can lead to increased levels of cAMP, which in turn leads to increased transcription of genes that have CRE sites within their promoters, including the gene for IL-10, which is an anti-inflammatory mediator.

In contrast, cAMP elevation would tend to inhibit the expression of genes that are driven primarily by the transcription factor nuclear factor κ B (NF-κB), including the genes for some important pro-inflammatory mediators, because it is believed that the NF-κB p65 subunit must compete with CREB for CPB.

By decreasing the intracellular level of cAMP, PDE4 could prevent PKA from modulating pro- and anti-inflammatory mediators released by a cell, resulting in a pro-inflammatory response where anti-inflammatory mediators are down-regulated and pro-inflammatory mediators are upregulated.

Protein Kinase A

Like other protein kinases, protein kinase A (also known as the cyclic AMP-dependent protein kinase or A kinase) is an enzyme that covalently decorates proteins with phosphate groups.

The unique characteristic of protein kinase A is that its activity is regulated by fluctuating levels of cyclic AMP within cells (hence its alias as the cyclic AMP-dependent protein kinase).

This enzyme thus functions as the end effector for a variety of hormones that work through a cyclic AMP signalling pathway.

To put it another way, protein kinase A is ultimately responsible for essentially all of the cellular responses due to the cyclic AMP second messenger system.

Structure

The protein kinase A holoenzyme is a heterotetramer composed of two types of subunits:

Catalytic subunit: This subunit contains the enzyme's active site.

It also contains a domain that binds ATP (the source of phosphate) and a domain that binds the regulatory subunit.

Regulatory subunit: Two molecules of this subunit bind one another in an anti-parallel orientation to form a homodimer; for type I subunits (see below), this binding is covalent via disulfide bonds.

This subunit also has has two domains that bind cyclic AMP, a domain that interacts with a catalytic subunit, and a "auto-inhibitory" domain that serves as a substrate or pseudosubstrate for the catalytic subunit.

Regulatory subunits may also have biologic activity distinct from their role in modulating catalytic subunit activity.

Gamma tublin and location

Microtubule plays an imperative role in segregation of chromosome into daughter cells in meiosis.

In meiosis process, microtubule otherwise known as meiotic spindle, extends from the cellular periphery organelle such as centrosome towards the centre of the cell.

This growth of meiotic spindle from centrosome is made possible by Gamma-tubulin. Tubulin is the subunit of microtubule.

Gamma-tubulin is a subtype of tubullin and functions in microtubule assembly. In addition, there are two more members of the tubulin family, such as Alpha-tubulin and Beta-tubulin.

These two subtypes forms a dimer structure of tubulin, which are bound tightly together by non-covalent bonding. As a consequence, tubulin dimers stack on top of each other by non-covalent bonding, this ultimately forms the structure of microtubule which consists of 13 tubulin protofilaments[1].

However, neither Alpha-tubulin nor Beta-tubulin has made contribution to microtubule nucleation, which directly affects the outgrowth of microtubule, hence meiotic spindle.

This important function is solely filled by Gamma-Tubulin and associated proteins, collectively known as the Gamma-tubulin ring complex[4]. This page will be primarily focusing on the characteristics of Gamma-tubulin and its role in meiosis.

As a result, it is organised in the following orders. Firstly, history of Gamma-tubulin, followed by structure and function of Gamma-tubulin. Then, a discussion of Gamma-tubulin's involvement in meiosis.

Fourthly, current project on Gamma-tubulin. Lastly, a conclusion of Gamma-tubulin.Microtubule plays an imperative role in segregation of chromosome into daughter cells in meiosis

. In meiosis process, microtubule otherwise known as meiotic spindle, extends from the cellular periphery organelle such as centrosome towards the centre of the cell.

This growth of meiotic spindle from centrosome is made possible by Gamma-tubulin. Tubulin is the subunit of microtubule. Gamma-tubulin is a subtype of tubullin and functions in microtubule assembly.

In addition, there are two more members of the tubulin family, such as Alpha-tubulin and Beta-tubulin.

These two subtypes forms a dimer structure of tubulin, which are bound tightly together by non-covalent bonding. As a consequence, tubulin dimers stack on top of each other by non-covalent bonding, this ultimately forms the structure of microtubule which consists of 13 tubulin protofilaments[1].

However, neither Alpha-tubulin nor Beta-tubulin has made contribution to microtubule nucleation, which directly affects the outgrowth of microtubule, hence meiotic spindle.

This important function is solely filled by Gamma-Tubulin and associated proteins, collectively known as the Gamma-tubulin ring complex[4].

This page will be primarily focusing on the characteristics of Gamma-tubulin and its role in meiosis. As a result, it is organised in the following orders.

Firstly, history of Gamma-tubulin, followed by structure and function of Gamma-tubulin. Then, a discussion of Gamma-tubulin's involvement in meiosis. Fourthly, current project on Gamma-tubulin.

Lastly, a conclusion of Gamma-tubulin.

Gamma-tubulin functions significantly as an organising centre of microtubule assembly in eukaryotic cells. Gamma-tubulin accomplishes this role through the formation of Gamma-tubulin ring complex. This complex contains a number of copies of Gamma-tubulin as well as a number of other proteins that are involved in nucleation of microtubule[4]. Microtubule, or in the case of meiosis such as meiotic spindle, are formed by outgrowth from centrosome. Centrosomes contain hundreds of ring-shaped structures formed by Gamma-tubulin, and each of the tubulin performs as the nucleation site for the growth of one microtubule. In addition, the outgrowth of microtubule adapts orientation specificity[1]. That is, each Alpha-Beta tubulin dimers add to the Gamma-tubulin ring in a pre-mediated direction. As a consequence, the minus/Alpha end of each microtubule is embedded in the centrosome, and growth occurs at the plus/Beta end.

PI3 kinase

Phosphatidylinositol-4,5-bisphosphate 3-kinase (also called phosphatidylinositide 3-kinases, phosphatidylinositol-3-kinases, PI 3-kinases, PI(3)Ks, PI-3Ks or by the HUGO official stem symbol for the gene family, PI3K(s)) are a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which in turn are involved in cancer.

PI3Ks are a family of related intracellular signal transducer enzymes capable of phosphorylating the 3 position hydroxylgroup of the inositol ring of phosphatidylinositol (PtdIns).[2]

The pathway, with oncogene PIK3CA and tumor suppressor PTEN, is implicated in insensitivity of cancer tumors to insulin and IGF1, and in calorie restriction.[

PI 3-kinases have been linked to an extraordinarily diverse group of cellular functions, including cell growth, proliferation, differentiation, motility, survival and intracellular trafficking.

Many of these functions relate to the ability of class I PI 3-kinases to activate protein kinase B (PKB, aka Akt) as in the PI3K/AKT/mTOR pathway.

The p110δand p110γ isoforms regulate different aspects of immune responses. PI 3-kinases are also a key component of the insulin signaling pathway.

MAP kinases

Mitogen-activated protein kinases (MAPKs) are a highly conserved family of serine/threonine protein kinases involved in a variety of fundamental cellular processes such as proliferation, differentiation, motility, stress response, apoptosis, and survival.

Conventional MAPKs include the extracellular signal-regulated kinase 1 and 2 (Erk1/2 or p44/42), the c-Jun N-terminal kinases 1-3 (JNK1-3)/ stress activated protein kinases (SAPK1A, 1B, 1C), the p38 isoforms (p38α, β, γ, and δ), and Erk5.

The lesser-studied, atypical MAPKs include Nemo-like kinase (NLK), Erk3/4, and Erk7/8.

A broad range of extracellular stimuli including mitogens, cytokines, growth factors, and environmental stressors stimulate the activation of one or more MAPKK kinases (MAPKKKs) via receptor-dependent and -independent mechanisms.

MAPKKKs then phosphorylate and activate a downstream MAPK kinase (MAPKK), which in turn phosphorylates and activates MAPKs.

Activation of MAPKs leads to the phosphorylation and activation of specific MAPK-activated protein kinases (MAPKAPKs), such as members of the RSK, MSK, or MNK family, and MK2/3/5.

These MAPKAPKs function to amplify the signal and mediate the broad range of biological processes regulated by the different MAPKs.

While most MAPKKK, MAPKK, and MAPKs display a strong preference for one set of substrates, there is significant cross-talk in a stimulus and cell-type dependent manner.

Indole ring

Indole is an aromatic heterocyclic organic compound with formula C8H7N.

It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered pyrrole ring. Indole is widely distributed in the natural environment and can be produced by a variety of bacteria.

As an intercellular signal molecule, indole regulates various aspects of bacterial physiology, including spore formation, plasmid stability, resistance to drugs, biofilm formation, and virulence.[1]

The amino acid tryptophan is an indole derivative and the precursor of the neurotransmitter serotonin.[2]ndole is an aromatic heterocyclic organic compound with formula C8H7N.

It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered pyrrole ring. Indole is widely distributed in the natural environment and can be produced by a variety of bacteria.

As an intercellular signal molecule, indole regulates various aspects of bacterial physiology, including spore formation, plasmid stability, resistance to drugs, biofilm formation, and virulence.[1]

The amino acid tryptophan is an indole derivative and the precursor of the neurotransmitter serotonin.[2]

Indole is a solid at room temperature.

It occurs naturally in human feces and has an intense fecal odor. At very low concentrations, however, it has a flowery smell,[3] and is a constituent of many flower scents (such as orange blossoms) and perfumes. It also occurs in coal tar.

The corresponding substituent is called indolyl.

Indole undergoes electrophilic substitution, mainly at position 3 (see diagram in right margin). Substituted indoles are structural elements of (and for some compounds, the synthetic precursors for) the tryptophan-derived tryptamine alkaloids like the neurotransmitter serotonin, and melatonin. Other indolic compounds include the plant hormone auxin (indolyl-3-acetic acid, IAA), tryptophol, the anti-inflammatory drug indomethacin, the betablocker pindolol, and the naturally occurring hallucinogen dimethyltryptamine.

SH2 AND SH3 DOMAINS

Src homology (SH) domains 2 and 3, SH2 and SH3, are protein domains that bind to phosphotyrosine-containing sequence motifs and proline-rich motifs, respectively.

The SH2 domain was originally identified as a conserved amino acid sequence in the noncatalytic region of tyrosine kinases encoded by viral oncogenes such as v-src, v-yes, and v-fps (1) (see SRC Genes).

The most conserved sequence, however, was that of the catalytic region of these kinases, which was referred to as the Src homology 1 (SH1) domain.

The SH3 domain is also present in the noncatalytic region of many tyrosine kinases, but it was originally found as a sequence similarity between phosphatidylinositol-specific phospholipase C and a novel viral oncogene product, Crk (2).

Both SH2 and SH3 domains are now known to be present in a wide variety of signal transduction molecules and have been shown to mediate protein-protein interactions (3).

The SH2 domains consist of two a-helices, with a core of antiparallel b-sheet in between. The binding of phosphotyrosine-containing peptide to SH2 is bifurcated.

The primary interaction is between the phosphotyrosine and basic residues of the SH2 structure.

The second interaction, which confers the ligand specificity of each SH2 domain, is provided by the recognition of amino acid residues immediately carboxyl terminal of the phosphotyrosine.

The SH3 domains contain a five-stranded antiparallel b-sheet that forms two hydrophobic pockets.

SH3 ligands have the minimal consensus sequence X-Pro-Pro-X-Pro, where X tends to be an aliphatic residue.

Each X-Pro pair binds to one of the two hydrophobic pockets of SH3 in either orientation (ie, amino to carboxy terminal or carboxy to amino terminal).

Some SH2- and SH3-containing proteins are composed of only SH2 and SH3 domains.

These proteins are believed to function as phosphotyrosine-dependent adaptor proteins

PCR

The polymerase chain reaction (PCR) is the most powerful technique that has been developed recently in the area of recombinant DNA research and is having an impact on many areas of molecular cloning and genetics. With this technique a target sequence of DNA can be amplified a billion fold in several hours. This procedure has been applied to forensic analysis where minute samples of DNA was isolated from blood at a crime scene to determine if an individual was actually at the location of the crime. Most recently DNA from mummies and fossil tissue has been analyzed to determine ancient evolutionary relationships.

PCR is a DNA polymerase reaction and as with any polymerase reaction it requires a DNA template and a free 3'-OH. The template is provided by the DNA sample to be amplified and the free 3'-OH are provided by site-specific oligonucleotide primers. The primers are complementary to each of the ends of the sequence that is to be amplified. The three steps of the reaction are:

1. Denaturation - the DNA is heated usually to 95oC to render it single-stranded

2. Annealing - the two primers bind the appropriate complementary strand; the temperature for this step varies depending on the of size of the primer and its homology to the target DNA

3. Primer Extension - DNA polymerase extends the primer by its polymerase activity; this is done at a temperature optimal for the particular polymerase that is used; currently the most popular enzyme for this step is Taq polymerase, the DNA polymerase from the thermophilic ("heat-loving) bacteria Thermus aquaticus; the extension is performed at 72oC

NADH

Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all living cells.

The compound is a dinucleotide, because it consists of two nucleotides joined through their phosphate groups.

One nucleotide contains an adenine base and the other nicotinamide. Nicotinamide adenine dinucleotide exists in two forms: an oxidized and reduced form abbreviated as NAD+ and NADH respectively.

In metabolism, nicotinamide adenine dinucleotide is involved in redox reactions, carrying electrons from one reaction to another.

The coenzyme is, therefore, found in two forms in cells: NAD+ is an oxidizing agent – it accepts electrons from other molecules and becomes reduced.

This reaction forms NADH, which can then be used as a reducing agent to donate electrons. These electron transfer reactions are the main function of NAD.

However, it is also used in other cellular processes, most notably a substrate of enzymes that add or remove chemical groups from proteins, in posttranslational modifications.

Because of the importance of these functions, the enzymes involved in NAD metabolism are targets for drug discovery.

In organisms, NAD can be synthesized from simple building-blocks (de novo) from the amino acids tryptophan or aspartic acid.

In an alternative fashion, more complex components of the coenzymes are taken up from food as niacin.

Similar compounds are released by reactions that break down the structure of NAD. These preformed components then pass through a salvage pathway that recycles them back into the active form.

Some NAD is converted into nicotinamide adenine dinucleotide phosphate (NADP); the chemistry of this related coenzyme is similar to that of NAD, but it has different roles in metabolism.

Actin

Actin is a family of globular multi-functional proteins that form microfilaments. It is found in essentially all eukaryotic cells(the only known exception being nematode sperm), where it may be present at a concentration of over 100 μM; its mass is roughly 42-kDa, with a diameter of 4 to 7 nm.

An actin protein is the monomeric subunit of two types of filaments in cells: microfilaments, one of the three major components of the cytoskeleton, and thin filaments, part of the contractile apparatus in muscle cells. It can be present as either a free monomer called G-actin (globular) or as part of a linear polymer microfilament called F-actin (filamentous), both of which are essential for such important cellular functions as the mobility and contraction of cells during cell division.

Actin participates in many important cellular processes, including muscle contraction, cell motility, cell division and cytokinesis, vesicle and organelle movement, cell signaling, and the establishment and maintenance of cell junctions and cell shape. Many of these processes are mediated by extensive and intimate interactions of actin with cellular membranes.[2]In vertebrates, three main groups of actin isoforms, alpha, beta, and gamma have been identified. The alpha actins, found in muscle tissues, are a major constituent of the contractile apparatus. The beta and gamma actins coexist in most cell types as components of the cytoskeleton, and as mediators of internal cell motility. It is believed that the diverse range of structures formed by actin enabling it to fulfill such a large range of functions is regulated through the binding of tropomyosin along the filaments.[3]\

Cytochrome C

The cytochrome complex, or cyt c is a small hemeprotein found loosely associated with the inner membrane of the mitochondrion. It belongs to the cytochrome c family of proteins. Cytochrome c is highly water-soluble, unlike other cytochromes, and is an essential component of the electron transport chain, where it carries one electron. It is capable of undergoing oxidation and reduction, but does not bind oxygen. It transfers electrons between Complexes III (Coenzyme Q – Cyt C reductase) and IV (Cyt C oxidase). In humans, cytochrome c is encoded by the CYCS gene.[5][6]

There are four classes of cytochrome c:[14]

Class I includes the lowspin soluble cytochrome c of mitochondria and bacteria. It has the heme-attachment site towards the N terminus of histidine and the sixth ligand provided by a methionine residue towards the C terminus.

Class II includes the highspin cytochrome c'. It has the heme-attachment site close to the N terminus of histidine.

Class III comprises the low redox potential multiple-heme cytochromes. The heme c groups are structurally and functionally nonequivalent and present different redox potentials in the range 0 to −400 mV.

Class IV was originally created to hold the complex proteins that have other prosthetic groups as well as

Calmodulin (CaM) (an abbreviation for calcium-modulated protein) is a multifunctional intermediate calcium-binding messenger protein expressed in all eukaryotic cells.[1] It is an intracellular target of the secondary messenger Ca2+, and the binding of Ca2+ is required for the activation of calmodulin. Once bound to Ca2+, calmodulin acts as part of a calcium signal transduction pathway by modifying its interactions with various target proteins such as kinases or phosphatases.

Calmodulin is a small, highly conserved protein that is 148 amino acids long (16.7 KDa).

The protein has two approximately symmetrical globular domains each containing a pair of EF-hand motifs (the N- and C-domain) separated by a flexible linker region for a total of four Ca2+ binding sites.[5]

Each EF-hand motif allows calmodulin to sense intracellular calcium levels by binding one Ca2+ ion. Calcium ion binding regions are found in the following positions in the sequence of amino acids: 21-32, 57-68, 94-105 and 130-141; each region that calcium binds to is exactly 12 amino acids long.

These regions are located between two alpha helices in the EF-hand motifs, the first two regions (21-32 and 57-68) are on one side of the linker region the other two (94-105 and 130-141) are on the other side

Glucose is a simple sugar with the molecular formula C6H12O6, which means that it is a molecule that is made of six carbonatoms, twelve hydrogen atoms, and six oxygen atoms. Glucose circulates in the blood of animals as blood sugar. It is made during photosynthesis from water and carbon dioxide, using energy from sunlight. It is the most important source of energy for cellular respiration. Glucose is stored as a polymer, in plants as starch and in animals as glycogen.

Glucose is the most widely used aldohexose in living organisms.

One possible explanation for this is that glucose has a lower tendency than other aldohexoses to react nonspecifically with the amine groups of proteins.[8]

This reaction—glycation—impairs or destroys the function of many proteins.[8]

Glucose's low rate of glycation can be attributed to its having a more stable cyclic form compared to other aldohexoses, which means it spends less time than they do in its reactive open-chain form.[8]

The reason for glucose having the most stable cyclic form of all the aldohexoses is that its hydroxy groups (with the exception of the hydroxy group on the anomeric carbon of D-glucose) are in the equatorial position.

Many of the long-term complications of diabetes

Phosphatidylserine (abbreviated Ptd-L-Ser or PS) is a phospholipid and is a component of the cell membrane. It plays a key role in cell cycle signaling, specifically in relationship to apoptosis.

Phosphatidylserine is a phospholipid (more specifically a glycerophospholipid). It consists of two fatty acids attached in ester linkage to the first and second carbon of glycerol and serine attached through a phosphodiester linkage to the third carbon of the glycerol.[1]

Phosphatidylserine coming from plants and phosphatidylserine coming from animals differ in fatty acid composition

Cell signaling[edit]

Phosphatidylserine(s) are actively held facing the cytosolic (inner) side of the cell membrane by the enzyme flippase.

However, when a cell undergoes apoptosis, phosphatidylserine is no longer restricted to the cytosolic side by flippase.

Instead scramblase catalyzes the rapid exchange of phosphatidylserine between the two sides.

When the phosphatidylserines flip to the extracellular (outer) surface of the cell, they act as a signal for macrophages to engulf the cells.[3]

Coagulation[edit]

Phosphatidylserine plays a role in blood coagulation (also known as clotting).

When circulating platelets encounter the site of an injury, collagen and thrombin -mediated activation causes externalization of phosphatidylserine (PS) from the inner membrane layer, where it serves as a pro-coagulant surface.[4]

This surface acts to orient coagulation proteases, specifically tissue factor (TF) and factor VII, facilitating further proteolysis, activation of factor X, and ultimately generating thrombin.[4]

In the coagulation disorder Scott syndrome, the mechanism in platelets for transportation of PS from the inner platelet membrane surface to the outer membrane surface is defective.[5]

It is characterized as a mild bleeding disorder stemming from the patient's deficiency in thrombin synthesis

The hydrophobic effect is the observed tendency of nonpolar substances to aggregate in aqueous solution and exclude water molecules.[1][2]

The word hydrophobic literally means "water-fearing", and it describes the segregation of water and nonpolar substances, which maximizes hydrogen bonding between molecules of water and minimizes the area of contact between water and nonpolar molecules.

The hydrophobic effect is responsible for the separation of a mixture of oil and water into its two components.

It is also responsible for effects related to biology, including: cell membranes and vesicles formation, protein folding, insertion of membrane proteins into the nonpolar lipid environment and protein-small molecule associations.

Hence the hydrophobic effect is essential to life.[3][4][5][6] Substances for which this effect is observed are known as hydrophobes.

The hydrophilic groups prevent phase separation of the molecules by maintaining the hydrophobic groups in water through formation of strong hydrogen bonds with water molecules.

The driving force for this self-assembly is the hydrophobic effect.[citation needed]

A phosphatase is an enzyme that uses water to cleave a phosphoric acid monoester into a phosphate ion and an alcohol.

Because a phosphatase enzyme catalyzes the hydrolysis of its substrate, it is a subcategory of hydrolases.[1]

Phosphatase enzymes are essential to many biological functions, because phosphorylation (e.g. by protein kinases) and dephosphorylation (by phosphatases) serve diverse roles in cellular regulation and signaling.[2]

Whereas phosphatases remove phosphate groups from molecules, kinases catalyze the transfer of phosphate groups to molecules from ATP.

Together, kinases and phosphatases direct a form of post-translational modification that is essential to the cell's regulatory network.[3]

Phosphatase enzymes are not to be confused with phosphorylase enzymes, which catalyze the transfer of a phosphate group from hydrogen phosphate to an acceptor.

Due to their prevalence in cellular regulation, phosphatases are an area of interest for pharmaceutical research.

In contrast to kinases, phosphatase enzymes recognize and catalyze a wider array of substrates and reactions.

For example, in humans, Ser/Thr kinases outnumber Ser/Thr phosphatases by a factor of ten.[4]

To some extent, this disparity results from incomplete knowledge of the human phosphatome, that is, the complete set of phosphatases expressed in a cell, tissue, or organism.[3]

Many phosphatases have yet to be discovered, and for numerous known phosphatases, a substrate has yet to be identified.

However, among well-studied phosphatase/kinase pairs, phosphatases exhibit greater variety than their kinase counterparts in both form and function; this may result from the lesser degree of conservation among phosphatases

Adenosine triphosphate (ATP) is a complex organic chemical that participates in many processes.

Found in all forms of life, ATP is often referred to as the "molecular unit of currency" of intracellular energy transfer.[1]

When consumed in metabolic processes, it converts to either the di- or monophosphates, respectively ADP and AMP.

Other processes regenerate ATP such that the human body recycles its own body weight equivalent in ATP each day.[2] It is also a precursor to DNA and RNA.

From the perspective of biochemistry, ATP is classified as a nucleoside triphosphate, which indicates that it consists of three components, a nitrogenous base (adenine), the sugar ribose, and the triphosphate. It is used in cells as a coenzyme

Histones are one of the specific proteins involved in cell division and cancer.

Technically, the ball-like groups of histones are referred to as nucleosomes, but--for simplicity--the website will continue to refer to these groups as histones.

In more general terms, histones are the protein “balls” that DNA wraps around in order to help DNA coil itself and condense into a chromosome during interphase.

In fact, the image of nucleosomes (groups of histones) strung along a strand of DNA is often referred to as the "Beads on a String" model.

In the image to the left, you can see the histones, drawn as gray spheres that attach to the DNA and each other as the DNA condenses into a chromosome that can be easily divided and transported during cell division.

Ubiquitin is a small protein that exists in all eukaryotic cells.

It performs its myriad functions through conjugation to a large range of target proteins. A variety of different modifications can occur.

The ubiquitin protein itself consists of 76 amino acids and has a molecular mass of about 8.5 kDa. Key features include its C-terminal tail and the 7 lysine residues.

It is highly conserved among eukaryotic species: Human and yeast ubiquitin share 96% sequence identity.

Differently linked chains have specific effects on the protein to which they are attached, caused by differences in the conformations of the protein chains. K29-, K33-,[75]K63- and M1-linked chains have a fairly linear conformation; they are known as open-conformation chains. K6-, K11-, and K48-linked chains form closed conformations.

The ubiquitin molecules in open-conformation chains do not interact with each other, except for the covalent isopeptide bonds linking them together.

In contrast, the closed conformation chains have interfaces with interacting residues.

Altering the chain conformations exposes and conceals different parts of the ubiquitin protein, and the different linkages are recognized by proteins that are specific for the unique topologies that are intrinsic to the linkage.

Proteins can specifically bind to ubiquitin via ubiquitin-binding domains (UBDs). The distances between individual ubiquitin units in chains differ between lysine 63- and 48-linked chains.

The UBDs exploit this by having small spacers between ubiquitin-interacting motifs that bind lysine 48-linked chains (compact ubiquitin chains) and larger spacers for lysine 63-linked chains.

The machinery involved in recognising polyubiquitin chains can also differentiate between K63-linked chains and M1-linked chains, demonstrated by the fact that the latter can induce proteasomal degradation of the substrate.

When enzymes contain more than one active site, the binding of a substrate molecule to the first site may in­fluence substrate binding to a second site.

Binding of the second substrate may influence binding of a third, and so on. This phenomenon is called cooperativity.

The influence may be positive in that binding of the first substrate molecule facilitates binding of sub­sequent substrate molecules (called “positive coop­erativity”) or the influence may be negative in that binding of a second or subsequent substrate molecule occurs less readily than binding of the first (called “negative cooperativity”).

Cooperative effects are not restricted to enzymes but are observed with other proteins. We considered the cooperativity that ex­ists among the globin chains of hemoglobin, a cooperativity that facilitates successive binding of oxygen molecules to the alpha and beta globin chains (i.e., positive cooperativity).

Competitive inhibition is a form of enzyme inhibition where binding of an inhibitor prevents binding of the target molecule of the enzyme, also known as the substrate.[1]

This is accomplished by blocking the binding site of the substrate - the active site - by some means. The Vmax indicates the maximum velocity of the reaction, while the Kmis the amount of substrate needed to reach half of the Vmax. Km also plays a part in indicating the tendency of the substrate to bind the enzyme.[2]

Competitive inhibition can be overcome by adding more substrate to the reaction; therefore, increasing the chances of the enzyme and substrate binding.

As a result, this alters only the Km, leaving the Vmax the same.[3] This can be demonstrated using enzyme kinetics plots such as the Michaelis-Menten or the Lineweaver-Burk plot.

Once the inhibitor is bound to the enzyme, the slope will be affected, as the Km either increases or decreases from the original Km of the reaction.[4][5][6]

Most competitive inhibitors function by binding reversibly to the active site of the enzyme.[1]

As a result, many sources state that this is the defining feature of competitive inhibitors.[7]

This, however, is a misleading oversimplification, as there are many possible mechanisms by which an enzyme may bind either the inhibitor or the substrate but never both at the same time.[1]

For example, allosteric inhibitors may display competitive, non-competitive, or uncompetitive inhibition