Assignment Code: BBCCT-105/TMA/2022

Course Code: BBCCT-105

Assignment Name: Proteins

Year: 2022

Verification Status: Verified by Professor

Maximum Marks: 100

Answer all the questions given below.

Q1. Classify amino acids based on their nutritional importance and metabolic fate. (10M)

Ans) Proteins are also known as informational macromolecules since they are the molecular instruments that are used to express genetic information. Proteins make up the majority of a cell's mass (together with water). All proteins, whether simple or complex, are made up of smaller units called amino acids. To form the basic structure of proteins, these amino acids are covalently bonded in linear sequences by peptide bonds. Multiple combinations of the same twenty amino acids formed various proteins in cells. These amino acids are thought of as letters on which the structure of proteins is written. Proteins come in a variety of sizes, ranging from tiny peptides with a few amino acids to enormous polymers with molecular weights measured in kilodaltons (kDa).

The following is a classification of amino acids based on their nutritional value and metabolic fate:

Nutritional Classification of Amino Acids

Essential Amino Acids:

Essential amino acids are amino acids that are not generated by the body yet are critically important. As a result, these amino acids must be obtained through diet. They are required for the human body's optimal growth and upkeep. Ten amino acids out of a total of twenty are considered necessary. Leucine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine are some of these amino acids.

Semi-essential Amino Acids: Two amino acids, arginine, and histidine, are generated in adults but not in children, making them semi-essential amino acids.

Non-essential Amino Acids: Non-essential amino acids are those that are produced by the body. To address biological needs, the body synthesises glycine, alanine, serine, cysteine, aspartate, asparagine, glutamate, glutamine, tyrosine, and proline.

Metabolic Fate-Based Classification

Amino acids serve a variety of functions in our bodies. The majority of them are employed as protein building components. Amino acids are not able to be stored in the body. As a result, any amino acid that is not necessary for immediate biosynthetic demands is deaminated in the body. Amino acids' carbon skeleton is used as a source of metabolic energy or transformed to fatty acids via acetyl CoA. As a result, amino acids can be categorised based on their metabolic fate, as their carbon skeletons act as precursors for the creation of glucogenic and ketogenic intermediates.

Glucogenic Amino Acids: Glucogenic amino acids are those that undergo metabolic breakdown and supply their carbon skeleton as a precursor for the synthesis of glucose, glycogen, and other sugars. Alanine, Arginine, Asparagine, Aspartate, Cysteine, Glutamate, Glycine, Histidine, Methionine, Proline, Serine, and Valine are all Glucogenic amino acids.

Ketogenic amino acids are those that create acetyl CoA or acetoacetyl CoA as a result of their metabolic breakdown (i.e., they don't produce metabolites that can be converted to glucose). The amino acids leucine and lysine are both ketogenic.

Glucogenic and Ketogenic Amino Acids: During metabolism, a portion of the carbon skeleton of amino acids in this category is converted to acetyl-CoA or acetoacetyl-CoA (ketogenic route), while the rest is converted to glucose. Partially ketogenic and partially glucogenic amino acids include lysine, isoleucine, phenylalanine, tyrosine, and tryptophan.

Q2. Describe non mechanical methods of cell disruption with suitable examples. (5M)

Ans) The first type of non-mechanical cell disruption method includes treatment with acid or alkali or with organic solvent, hence known as chemical method.

Chemical Method

Under this method there are three subtypes like using:

1. Acid/alkali/ detergent,

2. Enzymatic,

3. Osmotic shock.

Treating with Alkali: This method involves suspending cells or tissues in an alkaline solution with a pH range of 10 to 12. In a matter of minutes, this alkaline solution digests the cell membrane, allowing cytosol to flow out of the cell. Organic solvents such as alcohols, dimethyl sulfoxide, methyl ethyl ketone, or toluene could be used in different situations. These solvents also operate on the cell's exterior membrane, dissolving it and allowing the cytosol and other cellular components to escape.

Cell Disruption Method using Chemical Treatment

Treating with Detergent

Adding detergents to the cell membrane is another approach to disrupt it. Detergents are a type of molecule with special features that allow them to manipulate (disrupt or form) hydrophobic–hydrophilic relationships in biological samples. Detergents are used in biological research to lyse cells (release soluble proteins), solubilize membrane proteins and lipids, control protein crystallisation, avoid nonspecific binding in affinity purification and immunoassay techniques, and as electrophoresis additives.

Detergents such as Triton X 100, sodium dodecyl sulphate (SDS), and ethyl dimethyl ammonium bromide are routinely used to disturb cells. Non-ionic detergent Triton x 100 can solubilize membrane proteins. An anionic detergent, sodium dodecyl sulphate, denatures proteins by disrupting non-covalent protein links, causing them to lose their native conformation and structure.

As a result, this detergent is employed in electrophoresis to disrupt protein-protein interactions and separate proteins into distinct subunits using SDS page electrophoresis. Ethyl dimethyl ammonium bromide, a cationic detergent, acts on the cell membrane, particularly on lipopolysaccharides and phospholipids, creating minute pores that cause osmotic pressure differences and allow the cell to rupture.

Disintegration of Bio membrane after Treatment with Detergent

Enzymatic Treatment

Enzymatic cell disruption is the second type of non-mechanical cell disruption.

Cellulases are enzymes that break down the cellulose in plant cell walls. This characteristic of the enzyme allows it to be applied to the surface of a plant cell and incubated for a period of time. The partially digested cell wall can be seen after the proper incubation time. This permits the cell wall to disintegrate, allowing internal subcellular organelles and other macromolecules, such as proteins, to exit the plant cell.

Pectinases, xylanases, and chitinases are enzymes that are utilised to break down the cell walls of yeast and fungi. Another good example of a gram-positive bacterium cell wall digesting enzyme is lysozyme. The bacterial cell wall's -1,4-glycosidic linkages are hydrolysed by lysozyme. The zymolase enzyme is also utilised to break down the Yeast cell wall.

Effect of Enzyme on Biological Membrane of a Cell

However, chemical digestion has several drawbacks, such as the cost of downstream processing (biological product recovery) to neutralise the action of enzymes or chemicals following chemical digestion, which is an unnecessary expense. Furthermore, because enzymes are biocatalysts, they may act on other products or molecules in the cell, causing those cells and molecules to break, resulting in a loss in overall product quantity, which will reduce the quantity of the end desired product.

Q3. With the help of schematic diagram explain separation of proteins using dialysis. (5M)

Ans) Dialysis is a process in which two solutions with different concentrations are separated by a semipermeable membrane, and solute molecules from the higher concentration diffuse across the semipermeable barrier towards the lower concentration. Semipermeable membranes have tiny gaps that allow molecules to "diffusion." A tiny bag or tube of this semipermeable membrane is offered. As a result, diffusion is the driving force behind this method.

Flow of Solute Molecules across the Semipermeable Membrane

After ammonium sulphate precipitation, dialysis is used to remove salt from the protein production. By taking advantage of the protein's size, it further purifies the proteins from crude extracts. Because the size of the protein molecules is significantly larger than the size of the salt molecules present in the crude extract, salt ions will leak through the dialysis membrane, allowing the protein concentration to rise.

"Pour the crude protein extract into a semipermeable membrane tube, knot both ends of the tube, and suspend in a large volume of buffer solution contained in a beaker (with a magnetic bead) with an acceptable ionic strength," says the protocol. To allow mild movement in the buffer solution, place the beaker on top of the magnetic stirrer plate.

Because of the osmotic discrepancies between the crude extract (high concentration) inside the membrane and the buffer solution (low concentration) outside the membrane, salt particles (solute) can pass through the membrane until they reach equilibrium with the salt particles in the buffer (outside the membrane). Proteins of a big size stay within the membrane.

Membranes ranging from 5 to 50 KDa are available with various "molecular weight-cut off (MCO)" values, and these membranes are often constructed of cellulose and cellulose esters. Membranes with varying pore diameters and brands are available from a variety of manufacturers on the market.

Dialysis of Crude Protein

Q4. List four important applications of each of the following:

Gel filtration chromatography

Ans) The important applications of gel filtration chromatography are:

Biomolecules such as polysaccharides, proteins, enzymes, hormones, antibiotics, and nucleic acids are separated and purified.

1. Protein desalting and phenol removal from nucleic acid preparations.

2. Determining the relative molecular mass of the protein solution and condensing it.

3. Studies on protein binding.

Affinity chromatography.

The important applications of affinity chromatography are:

1. Proteins, enzymes, receptor proteins, nucleic acids, and antibodies are purified. Making homogeneous cells out of a variety of cells.

2. oligo dT (deoxythymidyllic acid) bound on agarose matrix was used to isolate mRNA from total RNA preparation (tRNA, rRNA, and mRNA).

3. Metal binding proteins are separated.

4. Another use of this approach is the manufacture of immobilised enzymes and their use in industry.

Ion exchange chromatography

The important applications of ion exchange chromatography are:

1. Biological components such as amino acids, peptides, proteins, carbohydrates, and nucleic acids are separated.

2. Vitamins, enzymes, isoenzymes, and immunoglobulins are separated (anti bodies).

3. Polypeptides and polynucleotides are separated. Chargaff used this technique extensively to establish "Chargaff's rule" (refer unit-14 of BBCCT- 101).

4. Separation of products made with recombinant DNA technology, such as growth factors.

5. Blood components such as albumin and IgG are separated.

6. Ions (Ca2+, Mg2+) are removed from drinking water (water purifying units).

7. This is how the amino acid auto analyser, which is revolutionising protein chemistry, works.

Gas chromatography

The important applications of gas chromatography are:

1. Unknown substances are detected by comparing them to standards.

2. Analysis of volatile chemicals such as fatty acids, with a focus on food tampering.

3. Food adulteration detection and quality assurance

4. Pesticides in agriculture and dairy products are investigated.

5. It is used to identify blood alcohol, plasma lipids, and urine steroids in clinical biochemistry.

Q5. Write the principle of electrophoresis. Explain SDS page electrophoresis using a neatly label diagram. (10M)

Ans) The separation of charged particles or molecules in the presence of an electric field is the basis of electrophoresis. Ions move towards oppositely charged electrodes in an applied electric field, i.e., anions (-ve) move towards positively charged anode and cations (+ve) move towards negatively charged cathode, although uncharged carbohydrates can be separated following the derivatization process.

SDS-PAGE

Protein separation is largely determined by the nature of the proteins to be separated. We will look at a few electrophoresis methods that are widely used in this article. SDS-PAGE is PAGE with the addition of SDS [CH3-(CH2)10-CH2OSO- 3Na+]. Hydrophobic interactions, disulphide bridges, and hydrogen bonds stabilise the structure of proteins. Breaking the above linkages is required to investigate the individual polypeptides of a protein. To separate proteins into their component peptides, solubilizing chemicals such as urea, SDS, and -mercaptoethanol are used. Urea breaks hydrogen bonds, while SDS and -mercaptoethanol break hydrophobic and disulphide bridges, respectively.

Before beginning the experiment, the protein sample to be separated is boiled for 5 minutes in a sample buffer containing bromophenol blue, SDS, and -mercaptoethanol, which totally denatures the protein and gives the polypeptide chains a net negative charge. Bromophenol blue is used as a tracking dye to keep track of how long the electrophoretic run takes. SDS-PAGE consists of two gels: a separating gel (10-15 percent acrylamide, 10 cm) and a stacking gel (10-15 percent acrylamide, 10 cm) (2-4 percent acrylamide, 1 cm). After the gel solution is poured and the comb is removed after polymerization, a plastic comb is put between glass plates and the gel solution is poured. This creates loading wells for the application of samples. After allowing the separating gel to polymerize, 1 cc of stacking gel is put between the glass plates. Despite the fact that the gel is created between two glass plates that are clamped together and separated by spacer arms.

In the presence of an applied electric field, a preboiled protein sample was injected into the wells of the stacking gel and allowed to separate. Proteins begin to migrate towards the anode, with smaller proteins moving quickly and bigger proteins moving more slowly due to molecular sieve qualities. As the blue dye hits the bottom of the gel, the current is switched off. The gel was then scraped from the glass plates and dyed with the specified stainer for 2-3 hours. Then destainer solution rinsed overnight (7 percent acetic acid). This removes unbound background dye from the gel, leaving stained blue protein bands that are plainly visible.

(a) Schematic Representation of PAGE

Image Showing Power Pack, Electrophoretic Unit and Accessories used in PAGE

Gel with Separated Protein Bands after SDS-PAGE

Q6. Give a detailed account on protein sequencing. (10M)

Ans) Protein sequencing is a collection of methodologies, tactics, and procedures used to determine a protein's amino acid sequence. A protein's amino acid sequence (also known as main structure) is the order of amino acids in the linear chain of the protein.

Protein Sequencing: Sanger Method

Sanger had to first identify the free amino groups in insulin. He came up with a reagent called dinitrofluorobenzene (FDNB or DNFB, often known as Sanger's reagent) that reacted with amino groups in proteins to generate an acid-stable dinitrophenyl (DNP) derivative.

Reaction of 2, 4-dinitrofluorobenzene (DNFB) with polypeptides and the resultant product of DNP polypeptide

The free DNP amino acid derivatives were extracted and compared to standards generated from known amino acids after the DNP protein was treated with acid to break the polypeptide backbone. Sanger deduced that insulin was made up of two peptide chains, one (chain A) with a glycine residue at the amino terminus and the other (chain B) with a phenylalanine at the amino terminus.

Following that, it was discovered that chain A had twenty amino acids and chain B had thirty-one. Individual chains were then disassembled into smaller parts: The polypeptide backbone was cleaved with acid, the cysteine disulphide linkages were broken with Performic acid, and the polypeptide was hydrolysed at precise locations on the chain with proteolytic enzymes. The reaction products were isolated and their sequence was established.

Sanger's approach has undergone various refinements and modifications, and there is now a mode. In this approach, most proteins may be sequenced in a matter of hours or days utilising only a small quantity of protein (microgram). The diagram below depicts a high-level overview of protein sequencing.

Overview of Protein Sequencing by Sanger’s Method

Q7. Write the working principle of mass spectrometry and its applications. (5M)

Ans) When compared to other spectroscopic techniques, mass spectrometry has certain unique concepts. When a charged particle passes through a magnetic field, it is deflected along a circular path with a radius proportional to the mass to charge ratio [m (mass) /e (charge)]. To generate a radical cation known as the molecular ion, a high-energy stream of electrons is employed to displace an electron from the molecule. This molecular ion is also fractured and broken down into smaller ions. The ions are concentrated into a beam, accelerated towards the magnetic field, then deflected along circular routes based on their masses. By altering the magnetic field, the ions can be concentrated on the detector and recorded.

The most significant footstep in the mass spectrometric examination of a molecule is the construction of gas phase ions. The most excellent example is electronic ionization:

This molecular ion is known for fragmenting molecules. It can split into a radical or an ion with an even number of electrons, or a molecule and a new radical cation, because it is a radical cation with an odd number of electrons. The significant disparities between these two sorts of ions require an explanation. These ions are presented in an appropriate form in this regard:

These two types of ions have a wide range of chemical properties. Every primary product ion formed from the molecular ion has the potential to continue degeneration and, as a result, to cause further degeneration. On the basis of their mass-to-charge ratio, mass spectrometers separate individual ions, which are separated in proportion to their abundance.

Mass Spectrometry in Action: Mechanisms of Action (How it Works)

An comparable component can be found in all mass spectrometry techniques. The different types of mass spectrometry are determined by the nature of these components. However, each mass spectrometry tool's mechanistic role is performed in a distinct manner. Three functionalities are required of a mass spectrometer:

Ions are created by either removing or adding an electron from a molecule to produce a positively charged cation or an anion. The image below depicts the formation of a cation and anion from a molecule M:

Generation of Cation with Loss of Electron and Anion with Gain of Electron from a Molecule (M)

The electron (e-) carries the negative charge in the above operation. When the negative charge on a molecule is removed, the molecule becomes a positive ion, also known as a cation. When a molecule accepts an electron, it becomes a negatively charged ion or anion.

Ion separation: Ions (mostly cations) are separated based on their mass to charge ratio (m/z), where M represents mass and Z represents charge. Ion separation is accomplished by accelerating them with an electric field and deflecting them with an electromagnet.

Ion Detection: In the separated population of ions, qualitative (identification of a specific ion) and quantitative (quantity or amount of a specific ion) analysis are carried out. Following the detection of ions, the resulting data is collected for processing, with the end result being actionable information. The picture below depicted the outline of the mass spectrometer's three functions.

All the Three Functions (Ionization, Separation and Detection) of Mass Spectrometer Connected to Each Other and Produce the Ultimate Actionable Information

Q8. Illustrate the oxygen binding curve of haemoglobin and myoglobin. (5M)

Ans) The oxygen binding curves for myoglobin show a significant increase as pO2 levels rise. The half-saturation of the binding sites (denoted as P50) is at the relatively low value of 2 torr, indicating that oxygen binds with great affinity to myoglobin (mm Hg). In comparison, the oxygen binding curve for haemoglobin in red blood cells exhibits a number of distinct characteristics. This curve is completely different from myoglobin's oxygen binding curve. Because of their S-like shape, such curves are referred to as sigmoid curves.

When compared to myoglobin, the oxygen binding curve for haemoglobin (P50 =26 torr) reveals that oxygen binding to haemoglobin is poor. The sigmoid binding curve for haemoglobin reveals a unique oxygen binding property. This graph indicates that oxygen binding at one site within haemoglobin promotes oxygen binding at the remaining empty sites.

(A) Oxygen Binding Curve [Fractional Saturation Vs Partial Pressure of Oxygen (PO2)] for Myoglobin Showed the Hyperbolic Characteristics, (B) Oxygen Binding Curve [Fractional Saturation Vs Partial Pressure of Oxygen (PO2)] for Haemoglobin Showed the Sigmoid Characteristics

The P50 represents the partial pressure at which myoglobin or haemoglobin is 50 percent saturated with oxygen. This is a conventional measure of haemoglobin affinity for oxygen.

(A) Myoglobin Oxygen Binding Curve Represented the 50% Occupancy (P50) (B) Haemoglobin Oxygen Binding Curve Showed the P50 with Sigmoid Characteristics

Q9. Describe the role of chaperones in protein folding. (5M)

Ans) Chaperones help polypeptides fold into their proper natural conformation by accompanying and assisting them. They also help misfolded polypeptides fold correctly or generate a microenvironment in which folding can take place. Chaperones can be present in all cellular compartments, including those of bacteria and humans. In both prokaryotes and eukaryotes, they are highly homologous and evolutionarily conserved. In all organisms, the process that assists protein folding is the same. However, we must emphasise that a chaperone is not required for every protein. The majority of proteins fold on their own, while just a small percentage of newly created proteins require assistance.

In the cytosol and mitochondrial matrix, there are two types of chaperones. Hsp 70 is aided in its action by Hsp 40 and other proteins, and it requires ATP to function.

Chaperones have the following responsibilities:

1. Prevent incorrect aggregation by maintaining the native structure of the protein.

2. Denaturation of proteins and nascent polypeptides must be avoided.

3. Certain proteins that must remain unfolded until they are translocated across the organelle's membrane are prevented from folding.

4. Facilitate the formation of oligomeric structures from folded subunits in the quaternary state.

5. Repair any damage caused by misfolding.

6. Present misfolded proteins to the proteolytic machinery in charge of signalling molecule conformational control.

The chaperones must recognise proteins in their non-native forms in order to perform these activities.

Chaperonins are multi-subunit barrel-shaped or cylindrical structures made up of 7,8, or 9 polypeptide units in general, whereas molecular chaperones are single big molecules. Chaperonins are two thick rings that form a barrel when they are positioned one on top of the other.

TriC (a eukaryotic chaperonin), for example, has 8 subunits per ring, but GroEL (a mitochondrial, chloroplast, and bacterial chaperonin) has 7 subunits per ring. These chaperonins are supported by co-chaperonins and, like chaperons, require ATP to operate. When the chaperonin barrel connects to the polypeptide to be folded, it should be in a tight condition, and when the folded polypeptide is released, it should be in a relaxed state.

Q10. What is a database? Give a detailed account on biological databases. (10M)

Ans) The Protein database contains sequences from a variety of sources, including annotated coding sections from GenBank, RefSeq, and TPA, as well as records from SwissProt, PIR, PRF, and PDB. The primary determinants of biological structure and function are protein sequences.

Because of genome sequencing and other large-scale ongoing research endeavours, there is a vast amount of knowledge on protein and DNA sequences available today. We have databases, which are collections of structured, searchable, and up-to-date data, to use the created data for further research. There are literally hundreds of public, freely available databases in bioinformatics. The information is frequently stored in databases and given a unique identifier. The contents of the majority of these are not source data; they were pulled from other databases by a process of filtering, converting, and manual correction and annotation.

Types of Database

There are many distinct database types, depending on the information being saved and its nature (sequences or structures). Sequence databases and structural databases are databases that deal with sequences and structures, respectively.

Depending on the nature of the sequences, there are two types of sequence databases:

1. DNA sequences (nucleic acids)

2. Protein sequences (amino acids)

Primary Protein Sequence Databases

PIR: The Protein Sequence Database was developed in the early 1960's. It is located at the National Biomedical Research Foundation (NBRF). Since 1988 it has been maintained by PIR-International.

Swiss-Prot: In 1986, Swiss-Prot was founded. SIB (Swiss Institute of Bioinformatics) and EMBL collaborate to keep it up to date. Many more sites, including other sequencing databases, are linked to Swiss-Prot. The Swiss-Prot database includes descriptions of the protein's function, post-translational modifications, domains and locations, secondary structure, quaternary structure, relationships to other proteins, disorders linked with protein deficiencies, variations, and many more.

It is only necessary to give a good integration between nucleic acid sequences, protein sequences, and protein tertiary structures, as well as with specialised data collections, in order to deliver high-quality annotation. The Swiss-Prot database, as a non-redundant database, aims to preserve minimal redundancy by indicating conflicts between distinct sequencing reports in the feature table of the related record.

TrEMBL: TrEMBL is a Swiss-Prot computer-annotated supplement that contains all EMBL nucleotide sequence entries that have not yet been merged into Swiss-Prot. Swiss-Prot accession numbers have been assigned to all TrEMBL entries that should eventually be updated to the standard Swiss-Prot quality.

GenPept: GenPept is an add-on to the GenBank database of nucleotide sequences. Its entries are translations of GenBank entries' coding sections. They have very few annotations. The GenBank entry or entries referenced by the accession number in the GenPept entry must be consulted for complete annotations.

NRL3D: PIR is the company that creates and maintains NRL 3D. It includes sequences from the Protein Data Bank (PDB). Secondary structure, active site, binding site, and changed site annotations, as well as information of the experimental method, resolution, and R-factor, are all included in the entries. It's worth noting that NRL 3D makes the PDB's sequence data available for both text and sequence searches. It also contains cross-reference data for use with other PIR Protein Sequence Databases.

Secondary Protein Sequence Databases

PROSITE: PROSITE was the first secondary database to be created. Many protein families are represented in this database. Sequence domains or motifs are used to further divide and group the families. Proteins and protein domains from the same family share functional characteristics and are descended from a common ancestor. This serves as the foundation for categorisation.

The following are biologically important areas or residues that make up a signature:

1. Sites of biological catalysis

2. Sites of attachment for prosthetic groups

3. Amino acids implicated in metal ion binding

4. Disulphide linkages involve cysteine.

5. Aspects of a molecule's binding (ATP/ADP, GTP/GDP, Ca, DNA)

These patterns also make it easier for computer tools to determine which protein family a new sequence belongs to quickly and accurately. ScanProsite, MotifScan, InterProScan, ps scan, pftools, and PRATT are some of PROSITE's computational tools.

Protein Data Bank: The PDB is an American database founded in 1971 at Brookhaven National Laboratories on Long Island, New York, by the late Walter Hamilton. It is now handled by Rutgers University's Research Collaboratory for Structural Bioinformatics. It is housed in the National Institute of Standards and Technology in Maryland and the San Diego Supercomputer Center in New Jersey, California. The Protein Data Bank is a three-dimensional database of protein, nucleic acid, and carbohydrate structures. X-ray crystallography and NMR techniques are used to generate the majority of the data in the PDB.

PUBMED: PubMed is a database made public by the National Center for Biotechnology Information (NCBI). The National Center for Biotechnology Information at the National Library of Medicines (NLM), which is part of the US National Institute of Health, created the Entrez Retrieval System (NIH). PubMed, Nucleotide and Protein Sequences, Protein Structures, Complete Genomes, Taxonomy, OMIM, and many other NCBI services employ the Entrez text-based search and retrieval system. PubMed is a database of biomedical literature citations. LinkOut allows users to access full-text articles from journal websites as well as other connected Web resources. PubMed also gives access to the other Entrez molecular biology resources as well as links to them.

Q11. Describe three important cell signalling events. (5M)

Ans) Cells receive signals from the outside world on a regular basis in order to accomplish processes like cell division and differentiation. Signalling pathways involve a variety of secondary messengers. These signalling molecules are released by cells and travel through the bloodstream to reach their intended targets.

There are 3 important events in cell signalling:

1. Reception: The signal molecule is received by a cell, and the ligand attaches to a receptor protein on the cell's surface.

2. Transduction: After the signal molecule binds to the receptor protein, the receptor protein undergoes a conformational change, which initiates the transduction process. Each relay molecule in the signal transduction pathway affects the next molecule during transduction.

3. Response: The cell's biological function/reaction is demonstrated in the response stage of the signalling process.

Q12. Differentiate between haemoglobin and myoglobin. (5M)

Ans) The differences between haemoglobin and myoglobin are:

Q13. What is immunoglobulin? Describe the general structure of immunoglobulin using IgG as an example. (10M)

Ans) Antibodies, or immunoglobulins, are glycoprotein molecules generated by plasma cells (white blood cells). They are an important aspect of the immune response because they recognise and attach to certain antigens, such as bacteria or viruses, and help to destroy them. Antibodies are proteins that provide immunity and defence. The actomyosin complex, on the other hand, is vital for human growth and movement.

Antibodies are globulin proteins that are tailored for an organism's immunity. Many body fluids, such as saliva, milk, tears, urine, and respiratory tract mucus, can be used to isolate them. They are the ones with the highest concentrations and may be easily extracted in large amounts from blood serum. Emil von Behring first recognised them as protective agents in serum in 1890. Heidelberger proved that antibodies are proteins in 1930.

When serum is separated by electrophoresis, it forms four major fractions. Three fractions of globulin protein and one fraction of serum albumin Antibody activity is observed mostly in fractions containing (gamma) globulins. Immunoglobulins (Ig) or antibodies are globulins that have an immunological role. These are globulin proteins, which are a type of glycoprotein that plays a role in immunity. Five different immunoglobulin classes (isotypes) have been found. Actin and myosin, two muscle proteins, will be discussed in the second half of the unit. The role of these protein filaments in muscle contraction would be a major emphasis.

Immunoglobulin G

When compared to other classes of antibodies, IgG antibodies have an average molecular weight of 160 kDa, are relatively tiny in size, and are more numerous in serum (about 80%). IgG is typically found in protective tissue gaps and body surfaces due to its tiny size, which allows it to quickly exit from blood arteries compared to other forms of antibodies. There are four subclasses of IgG in humans, each with its own amino acid sequence in the -chain constant region and number of interchain-disulphide linkages.

Structure and Isotypes of Immunoglobulin G molecule

Q14. Explain sliding filament model of muscle contraction. (5M)

Ans) The mechanism of myosin and actin myofilaments sliding over each other is known as the cross-bridge cycle. When the head sections of myosin myofilaments bind and release quickly while travelling along the actin myofilament, muscle contraction occurs. The user achieves a high energy state by binding ATP. ATP hydrolysis into ADP and inorganic phosphate occurs in the presence of the enzyme ATP (Pi).

The energy released by ATP hydrolysis aids in the transformation of the myosin head into an angular cock shape. This structure is now ready to bind with Actin protein, if it is available. ADP and Pi remain bound to myosin, keeping it in a high-energy state. The muscular contraction cycle is activated when calcium ions connect to Troponin protein, which helps to open up active actin binding sites. Once the actin binding sites have been opened, the high-energy myosin quickly fills the gap, forming a cross bridge. Myosin binding to actin aids Pi release and causes myosin to alter conformation to a low-energy state.

The sliding filament model, in which the expansion and contraction occur inside the I and H-bands, is the name for this type of movement. The A-band does not contract or expand because the myofilaments themselves do not contract or expand. Because ATP dissolves the myosin-actin cross-bridge and frees the myosin for the next contraction, it is essential for muscle contraction. Muscles contract in a sequence of binding and releasing between the sarcomere's two thin and thick strands. Myosin produces a power stroke by dragging actin filaments to the side of M-line using ATPs. Sarcomere shortening and muscle contraction occur at a period when actin filaments are dragged to a distance of only 10nm.

The myosin reaches its low energy state for another round of muscle contraction once this power stroke mechanism is completed. The cross bridge, once constructed, remains intact even when it is in a low-energy state. ATP is reattached to myosin in a fresh round of muscle contraction, bringing it to a high energy state and allowing the cross-bridge cycle to begin again, resulting in more muscle contraction. As a result, ATP is required to keep the muscle flexed; hence, in the absence of ATP, the muscle will remain relaxed.