Chapter-Protein Structure and Engineering

(d)

Explanation: Each cell and tissue type has different protein specific to it, like collagen in muscles, haemoglobin in erythrocytes, albumin in egg etc.

(a)

Explanation: Certain rogue proteins called ‘prions’ which are incorrectly shaped can cause diseases such as mad cow disease. These proteins seem to involve an alteration in protein structure.

(c)

Explanation: The absence of an enzyme protein called adenosine deaminase can cause a baby to be born with the diseases SCID (severe combined immunodeficiency disease); such babies cannot survive.

(b)

Explanation: Amino acids are joined together in proteins by peptide bonds. A peptide bond forms between the carboxyl group of one amino acid and the amino group of the adjacent amino acid.

(c)

Explanation: In 1955, Dr. Frederick Sanger developed the method of protein sequencing and for the first time he showed that proteins are linear polymers of amino acids.

(a)

Explanation: Dr. Frederick Sanger determined the complete amino acid sequence of insulin and proved that proteins have definite structure.

(d)

Explanation: In 1958, Frederick Sanger received Nobel Prize for determining the sequence of Insulin. In 1980, he received his second Nobel Prize for sequencing of DNA.

(b)

Explanation: Frederick Sanger determined the complete amino acid sequence of insulin and proved that proteins have definite structure. He used about 300 mg of pure insulin provided by Eli Lily to sequence the protein structure.

(a)

Explanation: G.N. Ramachandran (also gave Ramachandran plot for protein conformation) along with Gopinath Kartha proposed the triple helical structure of collagen in 1954 using X-ray diffraction.

(c)

Explanation: The polypeptides are held in position by hydrogen bonds. In both α–helices and β–pleated sheets the C=O of one amino acid bonds to the H-N of an adjacent amino acid, like this, C=O - - - H-N.

(d)

Explanation: Quaternary structure results when two or more polypeptide chains, called subunits, combine to produce large protein structures. Haemoglobin, which carries oxygen in the blood is an example of quaternary protein structure.

(d)

Explanation: Quaternary structure results when two or more polypeptide chains, called subunits, combine to produce large protein structures. A haemoglobin molecule is formed from four separate polypeptide chains.

(c)

Explanation: Ionic bond is a bond in which one or more electrons from one atom are removed and attached to another atom, resulting in positive and negative ions, which attract each other.

(b)

Explanation: An ionic bond (or electrostatic interactions) is a type of chemical bond based on electrostatic forces between two oppositely charged ions. The bond strength of ionic bonds is vastly reduced in water due to the dielectric strength of water.

(d)

Explanation: This is because water molecules are polar molecules so attracted to each other and are cohesive. Oil molecules are non polar so have no charged regions on them. This shows that they are neither repelled or attracted to each other. The attractiveness of the water molecules for each other then has the effect of squeezing the oil drops together and forming a larger drop.

(b)

Explanation: Hydrogen bonds are formed when a hydrogen atom is covalently bonded to one electronegative atom (usually oxygen (O) or nitrogen (N) and is simultaneously attracted to another electronegative atom. In water, each molecule is potentially bonded to four other water molecules through hydrogen bonds.

(a)

Explanation: Chymotrypsin is produced in the pancreas as the inactive form called ‘Zymogen’.

(b)

Explanation: Chymotrypsin is produced in the pancreas as the inactive form called ‘Zymogen’ which is activated only when required in the duodenum, which is its site of activity.

(c)

Explanation: Chymotrypsin is produced in the pancreas as the inactive form called ‘Zymogen’ which is activated only when required in the duodenum, which is its site of activity. It was the first enzyme available commercially in crystalline form.

(d)

Explanation: It is made up of a linear chain of 245 amino acids which folds to form a globular structure. The main catalytic force for Chymotrypsin is the catalytic pocket, which is the set of three amino acids residues.

(a)

Explanation: Sickle-cell anaemia is a severe hereditary form of anaemia. In this disease mutated haemoglobin distorts red blood cells into a crescent shape, causing the cells to become stuck in capillaries.The fragile, sickle-shaped cells deliver less oxygen to the body's tissues, and can break into pieces that disrupt blood flow.

(b)

Explanation: Sickle-cell anaemia is a severe hereditary form of anaemia. The sickle-cell disease occurs when the sixth amino acid, glutamic acid is replaced by valine to change its structure and function.

(a)

Explanation: In 1949, Linus Pauling found that sickle cell haemoglobin is different from the normal haemoglobin protein in their electrophoretic mobility due to difference in charges.

(c)

Explanation: The Laboratory of Molecular Biology (LMB) is a research institute in Cambridge, UK, which was at the forefront of the revolution in molecular biology which occurred in the 1950-60s, since then it remains a major medical research laboratory.

(d)

Explanation: Max Perutz (Nobel prize Winner for 3D structure of haemoglobin) came to Cambridge to study for a PhD, joining the X-ray crystallographic group led by JD Bernal, considered mainly the founder of LMB. Here he started his life-long work on hemoglobin.

(c)

Explanation: The data generated by Protein fingerprinting are so large that only computers can predict these; which leads to the birth of Bioinformatics.

(b)

Explanation: Paper chromatography is done using Butanol: Water: Acetic Acid in the ration of 4:5:1. Depending on their partition coefficient, the peptides will separate on the basis of their hydrophilicity and hydrophobicity.

(c)

Explanation: GRAS (Generally Regarded as Safe) listed microbes are non-pathogenic, non-toxic and generally should not produce antibiotics.

(d)

Explanation: Two-dimensional gel electrophoresis separate proteins in two steps, according to two independent properties: the first-dimension is isoelectric focusing (IEF), which separates proteins according to their isoelectric points (pI); the second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins according to their molecular weights (MW).

(b)

Explanation: SDS-PAGE (sodium dodecyl sulfate- polyacrylamide gel electrophoresis), is a technique which is combined with IEF for the two-dimensional electrophoresis of proteins. Here proteins complexes are separated by their mass.

(a)

Explanation:

SDS-PAGE (sodium dodecyl sulfate- polyacrylamide gel electrophoresis) is a technique, which is combined with IEF for the two-dimensional electrophoresis of proteins. Here proteins complexes are separated by their mass.

(b)

Explanation. Sodium dodecyl sulphate (SDS) is an anionic detergent, which denatures secondary and tertiary structures.

(a)

Explanation: Depending on their partition coefficient, the peptides will separate on the basis of their hydrophilicity and hydrophobicity. The hydrophobic peptides will move faster than hydrophilic ones.

(a)

Explanation: Downstream processing is recovery and purification of biosynthetic products particularly pharmaceuticals from natural sources such as animal or plant tissue.

(b)

Explanation: Electrospray Ionization (ESI) can analyze polar molecules ranging from less than 100 Da to more than 1,000,000 Da in molecular mass.

(a)

Explanation: Insulin is a protein produced in the mammalian pancreas, which plays a role in catabolism of glucose, failing of which develops diabetes.

(c)

Explanation: Oxytocin (protein) is a reproductive hormone, can be produced by direct chemical synthesis.

(b)

Explanation: It can be possible to create transgenic animals by direct gene transfer (i.e. microinjection) into stem cell or egg cells and produce antibodies, vaccines, proteins, and other therapeutically valuable products on a commercial scale. This technique is known as Molecular Pharming.

(d)

Explanation: A mass spectrometer is an analytical device that measures the molecular weight of chemical compounds by converting them into ions. It separates molecular ions according to their mass-to-charge ratio (m/z).

(c)

Explanation: Matrix Assisted Laser Desorption Ionization (MALDI) is a soft ionization technique, which is used for the analysis of proteins or other biochemical. It deals well with the compounds especially those of high molecular mass with high sensitivity.

(d)

Explanation:Papain is used in biochemical research involving the analysis of proteins, in preparations of various remedies for indigestion, in tenderizing meat, and in enzyme-action cleansing agents for soft contact lenses.

83. The major enzyme responsible for clot breakdown is known as

a. thrombin.

b. plasmin.

c. erythropoietin.

d. platelets.

Ans. (b)

Explanation. Plasmin is an important enzyme present in blood that degrades many blood plasma proteins, most notable, fibrin clots. As an enzyme, t-PA catalyzes the conversion of plasminogen to plasmin.

(d)

Explanation: Blood clotting factor VIII for treatment of haemophilia A, factor IX for treatment of haemophilia B, hepatitis B vaccine for prevention of hepatitis are produced by recombinant DNA technology.

(c)

Explanation. OKT3 is a highly effective therapeutic agent in preventing graft tissue rejection and it is able to reverse even acute rejection of transplanted kidney.

(d)

Explanation. OKT3 is a highly effective therapeutic agent in preventing graft tissue rejection and it is able to reverse even acute rejection of transplanted kidney.

(a)

Explanation. Hemophilia A is the most common type of hemophilia. It is also known as factor VIII deficiency or classic hemophilia.

(d)

Explanation. Hemophilia A is the most common type of hemophilia. It is also known as factor VIII deficiency or classic hemophilia.

(a)

Explanation. The gene for factor VIII is located in the X chromosome. This gene was isolated and introduced into Chinese Hamster Ovary (CHO) cell line.

(c)

Explanation. As an enzyme, it catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for clot breakdown. Because it works on the clotting system, tPA is used in clinical medicine to treat stroke.

(a)

Explanation: An anticoagulant is a substance that prevents coagulation (i.e. it stops blood from clotting). Heparin and sodium citrate are examples of anticoagulants.

(a)

Explanation. The first biotechnology product human insulin (Humulin) was produced through genetically engineered bacteria (E. coli).

(d)

Explanation. The first genetically engineered human insulin (humulin) was produced in 1980 by using genetically engineered E. coli. Human insulin gene was transferred into E. coli cells, which produce insulin.

(c)

Explanation. Alpha interferon (INF- a ) produced by blood leukocyte. It is used to cure hepatitis C.

(a)

Explanation. Gamma interferon (INF- g) produced by blood lymphocytes. It is used for chronic granulomatous disease and osteopetrosis.

(b)

Explanation. Hexokinase is used in quantitative estimation of glucose levels in serum. A biosensor has been prepared using glucose oxidase, which oxidizes glucose within 20 seconds.

(c)

Explanation. Bromelain (from pineapple) is used to tenderize meat by hydrolyzing peptide bonds.

(b)

Explanation. Subtilisin (27.5 kDa) is a bacterial protease (protein digesting protein), which is secreted, in large amount from many Bacillus species. It is a serine endopeptidase obtained from Bacillus subtilis initially.

(c)

Explanation. Subtilisin is a serine endopeptidase obtained from Bacillus subtilis initially. To improve the power of laundry detergents, detergent manufacturers supplement subtilisin in their products.

(d)

Explanation. The only recombinant vaccine currently in use in humans is the Hepatitis B Virus (HBV) vaccine, which is a recombinant subunit vaccine.

(d) Electrophoresis is the process of separating charged molecules through a gel matrix by the application of an electric field. The gel matrix has a sieving effect that allows molecules to be separated on the basis of size, while the electric field separates molecules on the basis of charge. In simple electrophoresis, the mobility of proteins is due to their charge, which is pH dependent.

(d) Edward Buchner was born in Munich in 1860. He was awarded the Nobel Prize in 1907 as a result of biochemical investigations and discovery of non-cellular fermentation. He also discovered that yeast enzymes and not yeast cells cause alcoholic fermentation of sugars and hence discovered zymase.

(c) Insulin is a hormone that's secreted by your pancreas to help regulate blood sugar level and promotes glycogen storage. Individuals with diabetes mellitus supplement insulin to make up for their body's inability to produce sufficient amounts.

(b) GRAS stands for Generally Recognized As Safe - The classification used by the FDA to denote a food substance that is considered to be safe for human consumption. GRAS listed microbes are non-pathogenic, non-toxic and generally should not produce antibiotics.

(b) Subtilisin (serine endopeptidase) is a protease (a protein-digesting enzyme) initially obtained from Bacillus subtilis. Subtilisins belong to the group of serine proteases, which initiate the nucleophilic attack on the peptide (ester) bond through a serine residue at the active site. The active site features a charge-relay network involving Asp-32, His-64, and active site Ser-221 arranged in a catalytic triad.

(c) a- chymotrypsin is the active form of hydrolytic enzyme chymotrypsin, synthesized in the pancreas that has an unusually reactive serine residue in the active site. The inactive precursor enzyme is termed chymotrypsinogen and the fully active enzyme is called alpha- chymotrypsin.

(a) Serine and Glutamine are polar amino acids. Amino acids are broadly divided into three groups-hydrophobic, polar and charged and therefore are capable of interacting within the protein in a variety of non-covalent interactions which drive proteins to fold in a particular way.

(d) Horseradish peroxidase is ideal in diagnostic kit because it is smaller, more stable and less expensive than other popular alternatives. The enzyme horseradish peroxidase (HRP) is used extensively in molecular biology applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule. HRP is often used in conjugates (molecules that have been joined genetically or chemically) to determine the presence of a molecular target. For example, an antibody conjugated to HRP may be used to detect a small amount of a specific protein in a western blot. Here, the antibody provides the specificity to locate the protein of interest and the HRP enzyme, in the presence of a substrate, produces a detectable signal. Horseradish peroxidase is also commonly used in techniques such as ELISA and Immunohistochemistry.

(c) Hydrophobic interactions are the interactions between molecules in which nonpolar portions of the molecule tend to avoid interaction with polar water molecules. The molecule will align itself so that the hydrophobic portions have the least chance of coming into contact with the other polar molecules. These forces (usually dispersion forces) exist between nonpolar molecules or nonpolar groups within a molecule or interactions that rely on the tendency of non-polar groups to aggregate to avoid contact with a polar solvent.

(a) V.A. Ingram first confirmed the difference between normal haemoglobin and sickle cell haemoglobin in 1957 by a technique called protein finger printing or peptide mapping in the famous laboratory of Molecular Biology (LMB), Cambridge, UK.

(a) Chymotrypsinogen is a precursor of the digestive enzyme chymotrypsin (zymogen). This molecule is inactive and must be cleaved by trypsin, and then by other chymotrypsin molecules before it can reach its full activity. Its activity is the conversion of proteins to amino acids.

(c) High grade proteins like polyclonal antibody have been used for therapeutic purposes. These proteins produced by recombinant DNA technology have found therapeutic use. High-grade proteins are also used as therapeutic hormones and growth factors.

(d) Subtilisin (serine endopeptidase) is a protease (a protein-digesting enzyme) initially obtained from Bacillus subtilis. The stability and activity of subtilisin can be increased by genetic engineering (site-directed mutagenesis).

(b) Proteins are large molecules required for the structure, function, and regulation of the body's cells, tissues, and organs. Each protein has unique functions. Proteins are essential components of muscles, skin, bones and the body as a whole. Protein is also one of the three types of nutrients used as energy sources by the body.

(a) Beta Thalassaemia syndromes are a group of hereditary disorders characterized by a genetic deficiency in the synthesis of beta-globin chains. In the homozygous state, beta Thalassaemia (i.e., Thalassaemia major) causes severe transfusion-dependent anemia. In the heterozygous state, the Beta Thalassaemia trait (i.e., Thalassaemia minor) causes mild-to-moderate microcytic anemia. Mutations in globin genes cause Thalassaemias. Alpha Thalassaemia affects the alpha-globin gene(s). Beta Thalassaemia affects one or both of the beta-globin genes. These mutations result in the impaired synthesis of the beta globin protein portion, a component of Hb, thus causing anemia.

(c) The term "Probiotic" has been given many meanings, but it now generally recognized as a fermented dairy product containing live lactic acid bacteria that have been specially selected to provide specific health benefits. Yogurt will be manufactured using two different varieties of probiotic cultures. Lactobacillus acidophilus, Lactobacillus subsp. casei, and Bifido-bacteria may be added to yogurt as probiotic cultures. Probiotic cultures benefit human health by improving lactose digestion, gastrointestinal function, and stimulating the immune system.

Streptococcus thermophilus is one of the most important bacteria used in dairy industry (in the manufacture of yogurt, together Lactobacillus bulgaricus and some cheese products). These species are included in the GRAS group (Generally Recognized As Safe). Streptococcus thermophilus are generally used for the fermentation of milk but as well as for its role as probiotic, alleviating symptoms of lactose intolerance or other gastrointestinal disorders and for its antimicrobial effects.

(b) Proteins are the workhorse of a biological system, performing all of the biochemical functions necessary to maintain cellular metabolism, architecture and growth. Different tissues are made up of certain cell types that each have a unique population of proteins that make up the cellular environment. Because of this, different cell types have a unique protein profile, or fingerprint. Protein fingerprinting became a useful technique to compare same proteins from different sources.

(d) Two-dimensional gel electrophoresis is a gel electrophoresis method in which a protein sample is separated by isoelectric point in one dimension and by size in a second, perpendicular dimension. This technique separate proteins in two steps, according to two independent properties: the first-dimension is isoelectric focusing (IEF), which separates proteins according to their isoelectric points (pI); the second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins according to their molecular weights (MW).

(b) Molecular pharming is a new application of genetic engineering that takes genes, primarily of human and animal origin, and introduces them into plants or farm animals to inexpensively produce medically important substances. The premise is using plants as efficient chemical factories for producing antibodies, vaccines, blood proteins, and other therapeutically valuable proteins.

(c) Modern household laundry detergents must effectively clean and remove the most difficult stains at low washing temperatures and short wash times. Addition of detergent enzymes to laundry detergents and direct-application stain removers improves detergent cleaning performance, renews whiteness, color and appearance of garments. Enzymes designed for use in cleaning applications are compatible with all commonly used detergent components such as nonionic and most anionic surfactants, builders, anti-redeposit ion agents, optical brighteners and oxygen-releasing bleaching agents. Use of enzymes in household laundry detergents is environment friendly, since all enzyme products are non-toxic completely, biodegradable and helps reduce clothes-washing energy consumption.

(a) A prion is thought to be an infectious agent that, according to current scientific consensus, is comprised entirely of a propagated, mis-folded protein. The mis-folded form of the prion protein has been implicated in a number of diseases in a variety of mammals, including bovine spongiform encephalopathy (BSE, also known as "mad cow disease") in cattle and Creutzfeldt-Jakob disease (CJD) in humans. All hypothesized prion diseases affect the structure of the brain or other neural tissue, and all are currently untreatable and are always fatal.

(b) The serine proteases were so named as they have a highly reactive serine residue, Ser-195, which attacks the carboxyl group of the substrate. This results in an acylenzyme intermediate consisting of the substrate covalently bound to the enzyme at this serine. However, the reactivity is dependent upon the arrangement of the serine side chain with two other polar side chains, approximately in a straight line, which is characteristic of all serine proteases. The Ser-195 is positioned at one end of this line; while at the other end is Asp-102, with His-57 in the middle. This is called the catalytic triad. These three residues are far apart in the sequence- the tertiary structure of the polypeptide chain brings them together in the required arrangement.

(b) The enzyme active sites consist of a specificity sub-site and a reaction sub-site, and in these protein groups are positioned around different parts of the substrate. In the specificity sub-site the enzyme uses polar and nonpolar groups to make weak interactions with the substrate; in the reaction sub-site other groups on the enzyme carry out the reaction. In some cases the same amino-acid residue may participate in both specific substrate binding and catalysis.

(c) Proteins are large chains of amino acids combined by dehydration synthesis, where by molecules join by removing water. Hydrolysis breaks down proteins into amino acids. Pinocytosis is a method of ingesting large insoluble molecules, and is a form of active transport.

(d) The sickle-cell disease occurs when the sixth amino acid, glutamic acid is replaced by valine to change its structure and function.

(c) The data generated from peptide mapping become so large that computers became necessary to do homology searches leading to the birth of Bioinformatics.

(b) Isolation and separation of proteins from a biological tissue or a microbial culture involves various separation techniques, which are known as downstream processing. Downstream processing is recovery and purification of biosynthetic products particularly pharmaceuticals from natural sources such as animal or plant tissue.

(a) Mass spectrometry is an analytical technique that identifies the chemical composition of a compound or sample based on the mass-to-charge ratio of charged particles. A sample undergoes chemical fragmentation forming charged particles (ions). The ratio of charge to mass of the particles is calculated by passing them through electric and magnetic fields in a mass spectrometer.

(d) Alcalase is a protease used to remove protein-based stains in both laundry and automatic dish wash detergents. Alcalase has very good washing performance at relatively low pH (7-9) and is also gentler on wool and silk than other proteases. Alcalase is suitable for use in soaking preparations as well as liquid/powder detergents.

(a) The milk serum protein of milk can be converted to a sellable product such as Whey Protein Concentrate (WPC), and Whey Protein Isolate (WPI) used as nutraceutical protein. The first step in making cheese is to curdle the milk. The whey is the watery liquid that separates from the curds. Whey is often considered a waste product of cheese making. Whey protein is the name for a collection of globular proteins that can be isolated from the whey. It is a mixture of beta-lactoglobulin (~65%), alpha-lactalbumin (~25%), and serum albumin (~8%), which are soluble in their native forms. When dried and powdered, it is considered a good source of protein. Whey Protein Isolate (WPI) is when the whey protein is filtered to remove any lactose, fat, and carbohydrates from the original protein.

(c) Using site-directed mutagenesis of the subtilisin gene in E.coli, the methionine was substituted by a variety of other amino acids, and the enzyme activity measured in the presence of bleach because native subtilisin is easily inactivated by bleach. The substitution of Met²²² with alanine was the best in terms of activity and stability.

(c) Whey protein is the ultimate source of protein - it is made from milk and occurs during the process of turning milk into cheese at which point whey protein is separated out. Protein itself is found in many foods such as fish, beef, and chicken & dairy products as well as eggs, cottage cheese, soy and vegetable protein also contain good amounts of protein. None of these can be compared in quality or ease of use with whey protein. Whey protein has the highest value in providing branched-chain amino acids, which result in building and retaining muscle tissue. Whey protein provides the body with the necessary building blocks to produce amino acids that are used for building muscle tissue. All serious bodybuilders, athletes & sports professionals understand the importance of protein supplementation. Studies that compare whey protein to other sources found that whey protein contains the perfect combination of overall amino acid profile and in the correct concentrations for optimal performance in the body.

(b) Protein after synthesis could undergo posttranslational modifications. It is estimated that protein could undergo as many as 200 different types of these modifications. Due to the reason the relationship between number of genes and number of proteins is non-linear.

(d) It has been discovered that certain ‘rouge proteins’ which are incorrectly shaped can cause diseases such as the mad cow disease. These proteins called prions can cause normal proteins to turn diseased which again seems to involve an alteration in protein structure.

(c) An epitope is the part of a macromolecule that is recognized by the immune system, specifically by antibodies, B cells, or T cells. Although epitopes are derived from nonself proteins, sequences derived from the host that can be recognized are also classified as epitopes.

(d) Site-specific mutagenesis is a technique to change one or more specific nucleotides within a cloned gene in order to create an altered form of a protein with one or more specific amino acid changes. a.k.a. oligonucleotide-directed mutagenesis; oligonucleotide-directed site-specific mutagenesis.

(a) Papain is extracted from the papaya, effectively breaking down proteins, papain serves as an ideal meat tenderizer.

(b) The proteome is the entire complement of proteins expressed by a genome, cell, tissue or organism. More specifically, it is the expressed proteins at a given time point under defined conditions. The term is a blend of proteins and genome.

(d) Although the mutational effects were rather small, this strategy proved to be successful since half of the mutants showed an increased stability. This stability may originate from the suppression of unfavorable interactions between protein molecules. For example, introduction of a charge at the surface of the protein may provide a new coulombic interaction on the protein surface.

(c) The biological value of a protein refers to the how much of the nitrogen content of food is retained by the body. The biological value of proteins ranges from 50 to 100 percent and is a measure of how much dietary protein source can support growth. Animal proteins have biological values of 70 percent or higher, and plant proteins have biological values of 50 to 70.

(a) Sanger determined the complete amino acid sequence of insulin and in doing so proved that proteins have specific structures. He also helped develop the chain termination method of DNA sequencing. As a result of these discoveries Sanger was awarded Nobel Prizes for both, once in 1958 and again in 1980.