Biochemistry Past Paper PDF (Up to Beginning of Enzymes) 2024/2025

MD 1: Biochemistry & Cell Biology (2024/2025) Abhishek Sah Frendo MDS 1038 – BIOCHEMISTRY 1 MD 1: Biochemistry & Cell Biology (2024/2025) Abhishek Sah Frendo 1. Acid-Base Concepts o Deﬁne an acid o Deﬁne a base o Describe proton hopping o Deﬁne a conjugate acid-base pair o Describe the Henderson-Hasselbach equation o Describe a titration curve for a conjugate acid-base pair o Give a deﬁnition of pK o Describe the products of metabolism in terms of pH o Describe the bu6ers found in the blood o Describe bicarbonate bu6er system (see blood bu*ers) o Explain how pH can be controlled by the lungs o Explain how pH can be controlled by the kidneys 2. Amino acids o Draw a titration curve for an amino acid o Explain why some amino acids are charged at a physiological pH o Explain which amino acids are charged at a physiological pH o Describe the characteristics of the histidine side-chain o Explain the concept of chirality using amino acids o Know which amino acids are essential MD 1: Biochemistry & Cell Biology (2024/2025) Abhishek Sah Frendo o Classify amino acids according to chemical properties o Know which amino acids are incorporated proteins o Understand that not all amino acids are incorporated into proteins 3. Protein Structure 1 o Describe the ﬂow of information from gene to protein o Understand the mitochondrial proteome o Draw a dipeptide & peptide bond o Explain the stereochemical limits of peptide bond o Describe phi, Φ & psi, Ψ (dihedral) angles 4. Protein Structure 2 o Describe 10, 2nd, 3rd, & 40 structure o Describe the di6erence between polypeptide chains & subunits o Explain why side chains are not involved in forming 2 structures o Describe alpha helix, beta sheet & forces stabilising them o Describe the types of non-covalent forces o Describe di6erence between apoprotein & holoprotein o Understand that protein families exist & their genetic relationships MD 1: Biochemistry & Cell Biology (2024/2025) Abhishek Sah Frendo 5. Protein Structure 3: Modiﬁcation & Targeting o Describe common post-translational modiﬁcations to proteins & the amino acids modiﬁed including: - glycosylation, phosphorylation, acetylation & carboxylation. o Describe how selenoproteins are made o Understand the intracellular sorting signals for proteins & how they are targeted to: - Mitochondria, ER, lysosomes, peroxisomes, & nucleus o Explain the synthetic processing & secretory pathway of insulin & its structures at each stage. o Use creatine kinase to explain the term tissue-speciﬁc isoforms o Describe how protein denaturation occurs 6. Globular Proteins 1 – Folding & Misfolding o Describe the folding mechanism for a globular protein o Describe how molecular chaperones aid proteins o Describe the ubiquitin-proteasome system in brief o Explain what amyloidosis is o Describe how prion proteins are transmissible MD 1: Biochemistry & Cell Biology (2024/2025) Abhishek Sah Frendo 7. Globular Proteins 2 – The Globins o Understand the di6erences between myoglobin & haemoglobin o Describe oxygen binding curves of myoglobin & haemoglobin o Know the meaning of distal & proximal histidine o Describe the various tetrameric forms of haemoglobin in development o Explain cooperativity & allostery o Describe how haemoglobin carries carbon dioxide o Explain how bisphosphoglycerate binds to haemoglobin o Describe how ﬁbres are formed in sickle red-blood cells o Understand di6erent forms of modiﬁed haemoglobins including carbaminoHb, carboxyHb, & Methaemoglobin 8. Globular Proteins 3 – Immunoglobulins & Membrane Proteins o Describe the structure of immunoglobulins o Understand Fab & Fac fragments o Name the di6erences between Ig isotypes o Describe the di6erences between mono-clonal & poly- clonal antibodies o Understand ‘clonal selection’ & antibody response o Explain epitopes & idiotypes MD 1: Biochemistry & Cell Biology (2024/2025) Abhishek Sah Frendo o Know the di6erence between channels & transporters o Describe the structure of aquaporins & ion channels o Understand the concepts & structural features of selectivity & gating o Describe how potassium channels are selective for potassium & not sodium o Describe the alternative access model for transporters. 9. Enzymes 1 - The Enzyme Reaction o Deﬁne the di6erences between substrate-binding & catalytic sites. o Describe induced ﬁt o Explain how enzymes speed up reactions o Explain how to generate a Michaelis-Menten plot o Recognise the Michaelis-Menten equation o Know the meaning of Vmax & Km o Describe the role of Mg2+ in reactions using diphosphates o Explain catalysis using carbonic anhydrase as an example o Describe the reaction of glucokinase o Understand the di*erences between hexokinase II & glucokinase 10. Fibrous Proteins o Describe the structure of immunoglobulins MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo o Understand Fab & Fac fragments o Name the di6erences between Ig isotypes o Describe the di6erences between mono - & poly-clonal antibodies ACID BASE CONCEPTS Define an Acid Arrhenius acid: substance that increases [H30+] in solution Lewis acid: accepts an electron pair (electron acceptor) Bronsted & Lowry acid: donates a proton (proton donor) Define a Base Arrhenius base: substance that increases [OH-] in solution Lewis base: donates an electron pair (electron donor) Bronsted & Lowry base: accepts a proton (proton acceptor) 1 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo What is a Proton? - A hydrogen nucleus: H+ - An elementary (subatomic) particle - Does not exist on its own; picked up by water molecules to become hydronium ions, H30+ (often omitted for clarity in equations) 2 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo A mechanism whereby H30+ ions are created from other H30+ ions Although they do not move physically, electrons are shuffled from H20 to H30+ ions in a chain of reaction; A proton can be found almost anywhere almost immediately o Protons are not static within the hydronium ion o They “hop” between water molecules through the H-bonds o This is accelerated by proton tunnelling 3 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo Define a Conjugate-Acid Base Pair Strong electrolytes (HCl & NaCl) dissociate completely Weak electrolytes (CH3COOH) do not dissociate completely (metabolic products dissociate weakly to release protons) Instead, they establish an equilibrium between the undissociated & dissociated forms These correspond to conjugated acid (protonated form HA) & conjugated base (A-) 4 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo Examples of weak biological electrolytes include: organic acids, phosphoric acid, & carbonic acid For every acid there is a conjugate base Remember: H – hydrogen, HA – anything that can give up H+ Describe the Henderson-Hasselbach equation 5 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo Henderson-Hasselbach equation shows the dissociation of a constant weak acid/base in a logarithmic equation, Illustrates relations between pH, pK & ration of acid and base o pH: -log10[H+] – a quantitative measurement of acidity or alkalinity of a solution o pKa: -log10 of the dissociation constant of the acid o [A-]: concentration of the conjugate base (unprotonated) o [HA]: concentration of the acid (protonated) 6 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo Describe a titration curve for a conjugate acid-base pair Curve Shape § A titration curve for a conjugate acid-base pair is typically sigmoidal. § It reﬂects pH change as a titrant (acid or base) is added to a solution. N.B. The above titration curve illustrates diEerent compounds, not conjugate pairs. 1.0 means same amount as titrant (e.g., lactic acid, NH3, acetic acid). Key Points on the Curve § For each compound (acid / base) there are conjugate acids & bases produced at end of the titration. § Bu*er Region: around pKa of the conjugate acid-base pair, the curve ﬂattens, showing resistance to pH changes. BUFFERING REGION! This means that 50% of the acid is deprotonated (ionised; dissociated). § Equivalence Point: where equivalents of acid equal the equivalents of base. (NOT IMP for exam but good to be aware of its existence). 7 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo Role of pKa § The pKa value corresponds to the midpoint of the buEering region. § For a conjugate acid-base pair, the pKa determines the strength of the acid or base. § pK values show why one is an acid & other is a base (note: previous titration curve illustrates diEerent compounds, not conjugate pairs). End Points o Curve starts & ends with the predominant forms of the compound: § At low pH: conjugate acid form predominates. § At high pH: conjugate base form predominates. Degree of Protonation At a ↓ pH (↑H+) < pKa, compound is protonated (unionised) At a ↑ pH (↓H+) > pKa, compound is deprotonated (ionised) When pH = pKa § 50% of compound is deprotonated & 50% is protonated (equal amounts of ionised & unionised) § Represents point where acid & conjugate base are in equilibrium § Around the pKa, system resists changes in pH when small amounts of acid / base are added. This is the BUFFERING EFFECT. 8 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo Titration Curve of Acetic acid A solution containing acetic acid (HA= CH3COOH) & acetate (A- = CH3COO-) with a pKa of 4.8 can resist a change in pH from pH 3.8-5.8 with maximum buffering capacity at pH 4.8. At pH < pKa the protonated form CH3COOH is main species. At pH > pKa the deprotonated base is predominant species. 9 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo Provide a definition of pK Negative logarithm of the dissociation constant (Ka) of an acid The pH at which a compound is 50% protonated & 50% unprotonated (ratio 1:1) Measure of an acid’ strength in aqueous solution o ↓ the pKa; stronger the acid > its proton donor ability o ↑ the pKa; weaker the acid < its proton donor ability The pK of an acid is the pH at which concentrations of the acid & its conjugate base are equal. When pH = pK, the system is at its buffering capacity; resisting changes in pH upon small additions of an acid/base. What are Buffers? A buffer consists of a weak conjugate acid/base pair. Buffers allow a solution to resist drastic changes in pH when H+ ions or OH- ions are added by absorbing or giving up protons. In other words, buffers are compounds that are at or near their pKa under physiological conditions. What is the role of Biological Buffers? A biological buffer is a biological compound capable of maintaining a constant pH. Examples include: bicarbonate buffer system, haemoglobin, plasma protein, hydrogen, ammonium + phosphate ions. The role of biological buffers is to maintain homeostatic conditions within the body & provide a means of removing acids which is vital. 10 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo Describe the products of metabolism in terms of pH Acidic Products of Metabolism Metabolism generates acids, which can ↓ pH if not buffered. § Carbon Dioxide (CO₂) – body’s main metabolic acid Produced during aerobic respiration CO₂ reacts with water to form carbonic acid (H₂CO₃), which dissociates into H⁺ and bicarbonate (HCO₃⁻): This contributes to acidification of blood if not removed by lungs. § Lactic Acid Generated during anaerobic glycolysis (e.g., in muscles during intense exercise). Lactic acid dissociates into lactate & H⁺, which can lower pH locally (causing muscle fatigue) or systemically. 11 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo § Ketone Bodies Produced during fat metabolism (e.g., in fasting or diabetes) when the body uses fat (ketones) instead of glucose for energy. Includes acetoacetic acid and beta-hydroxybutyric acid, which are acidic and can lead to ketoacidosis if produced in excess. § Phosphoric Acid Released from breakdown of phospoproteins or nucleotides. § Sulfuric Acid Produced from the metabolism of sulfur-containing amino acids (e.g., cysteine & methionine). Cys & Met release sulfur; S is oxidised to sulfate (SO42-) in the liver. Basic Products of Metabolism Some processes produce substances that can ↑ pH (reduce acidity). § Ammonia (NH₃) Generated during protein metabolism (amino acid deamination). Ammonia is basic and combines with H⁺ to form ammonium (NH₄⁺), which is less toxic and excreted by the kidneys. Excess ammonia production can increase local alkalinity. § Bicarbonate (HCO₃⁻) Produced in metabolic processes or ingested It neutralises acids to maintain a stable blood pH (in fact, gastric HCl is neutralised by pancreatic bicarbonate) Similary HCO3- can neutralise hydroxide ions (OH-) if necessary 12 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo Describe the buffers found in the blood The blood contains several buffering systems that work together to maintain its pH within a narrow range of 7.35–7.45, which is essential for proper physiological function. Acid-Base Disturbance Acidemia: a reduction in arterial pH < 7.35 Alkalemia: an increase in arterial pH >7.45 Acidosis: abnormal pathological processes that decrease pH Alkalosis: abnormal pathological processes that raise pH Respiratory acidosis / alkalosis: affect pCO2. Involves gain of H+ or loss of HCO3- (e.g. impaired breathing) Metabolic acidosis / alkalosis: directly cause a change in [HCO3-]. Involves gain of HCO3- or loss of H+. 13 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo Bicarbonate Buffer System § Primary buffer in blood plasma. § This system involves an equilibrium between carbonic acid (H₂CO₃) and bicarbonate (HCO₃⁻). § This reaction is accelerated by enzyme carbonic anhydrase present in RBCs. § Key Components CO₂: produced by cellular respiration; dissolved in blood. H₂CO₃: carbonic acid; formed when CO₂ reacts with water. HCO₃⁻: bicarbonate ion; buffers excess H⁺ ions. § Mechanism When blood becomes acidic (low pH), bicarbonate binds to H⁺ to form H₂CO₃, which is converted to CO₂ & exhaled by lungs. When blood becomes basic (high pH), H₂CO₃ dissociates to release H⁺, restoring pH balance. 14 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo § Continuous replenishment of carbonic acid QUESTION: The pKa of H₂CO₃ is 3.8, so at a physiological pH of 7.4, it is completely dissociated. If it is completely dissociated, how can it function as a buffer? Although H₂CO₃ is largely dissociated at a physiological pH of 7.4, it is constantly replenished by hydration of CO2 in the body. THE BICARBONATE BUFFER SYSTEM IS AN OPEN SYSTEM! This means that even though CO2 might itself be transient, the system maintains a dynamic equilibrium. § Effective pKa of the System While the pKa of H₂CO₃ is about 3.8, the effective buffering capacity of the bicarbonate system is due to the combined equilibria involving CO2 & HCO3-. At a physiological pH, the ratio of HCO3- to H₂CO₃ is ~ 20:1; which provides strong resistance to pH changes. § CO2 as a regulator The dissolved CO2, which in equilibrium with gaseous CO2 in the lungs acts as a ‘reservoir’ for the buffer system. The concentration of CO2 can be finely adjusted by changes in respiratory rate (hyperventilation & hypoventilation). § Negligibility of Bicarbonate Dissociation to Carbonate The pKa for HCO3- further dissociation to CO32- is 9.8, which makes the reaction negligible at the physiological pH. This reaction does not occur in bodily fluids; ensuring that the bicarbonate buffering system operates within optimal range. 15 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo Haemoglobin Buffer System § Located in the red blood cells (erythrocytes) § Haemoglobin (Hb), the oxygen-carrying protein in RBCs, plays a critical role in buffering pH by binding or releasing H⁺. § Mechanism: When Hb releases O2 to tissues, it binds H⁺ ions, helping prevent blood from becoming too acidic. Hb also binds CO₂ to form carboxyhaemoglobin, which helps transport CO₂ to the lungs for exhalation. Cells also transport H+ out in exchange of Na+ or they will transport HCO3- out of the RBC & into the plasma, in exchange for Cl- (CHLORIDE SHIFT); maintains electrostatic cell balance. 16 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo Phosphate Buffer System § Effective in intracellular fluid & urine buffering but also contributes to blood buffering. § The pKa of inorganic phosphate is 7.2 § Therefore, it is ionised at intracellular pH & can buffer H+ ions. § There are higher concentrations in the cells than in the ECF. § Involves a balance between dihydrogen phosphate (H₂PO₄⁻) and monohydrogen phosphate (HPO₄²⁻): H₂PO₄⁻ ⇌ H⁺ + HPO₄²⁻ § Mechanism: When blood becomes acidic, HPO₄²⁻ binds H⁺ to form H₂PO₄⁻. When blood becomes basic, H₂PO₄⁻ releases H⁺. Plasma Protein Buffer System § Proteins, such as albumin (most abdundant plasma protein) , contribute to buffering blood pH by their ability to bind or release H⁺ ions. § Mechanism: Proteins have amino acid side chains (e.g., histidine) that can accept or donate H⁺ depending on pH changes (amino acids have side chains with functional groups capable of buffering). At an acidic pH, proteins accept H⁺, reducing acidity. At a basic pH, proteins release H⁺, reducing alkalinity. 17 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo How can pH be controlled by the lungs Carbon Dioxide and pH Regulation § CO₂ produced during cellular respiration is transported in the blood. It reacts with water to form carbonic acid (H₂CO₃): CO₂ + H₂O ⇌ H₂CO₃ ⇌ H⁺ + HCO₃⁻ An ↑ in CO₂ levels ↑ conc. of H⁺ ions, ↓ blood pH (more acidic). A ↓ in CO₂ levels ↓ conc. of H⁺, ↑ blood pH (more basic). The lungs remove volatile acid – CO2 Ventilation and pH Control § The lungs adjust the blood's CO₂ level through the rate and depth of ventilation (breathing): Hyperventilation (↑ Breathing Rate – fast breathing): o More CO₂ is exhaled, reducing its concentration in the blood. o This ↓ H⁺ concentration, ↑pH. Hypoventilation (↓ Breathing Rate – shallow breathing): o Less CO₂ is exhaled, increasing its concentration in blood. o This ↑ H⁺ concentration, ↓ pH. Chemoreceptor Control Specialized chemoreceptors in the brainstem (medulla) and blood vessels (e.g., carotid bodies) detect changes in CO₂, pH, & O2 levels. If pH ↓ (acidosis), brain signals lungs to ↑ breathing; expel more CO₂ If pH ↑ (alkalosis), brain signals lungs to ↓ breathing to retain CO₂. 18 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo How can pH be controlled by the kidneys Reabsorption of Bicarbonate § Bicarbonate is ﬁltered by the kidneys in the nephron (functional unit of the kidney) at the glomerulus. § In the proximal tube, ﬁltered bicarbonate is almost entirely reabsorbed back into the bloodstream (preventing its loss). H⁺ + HCO₃⁻ ⇌ H₂CO₃ ⇌ CO₂ + H₂O § The CO₂ dipuses into the kidney cells, where it reacts with water to regenerate bicarbonate. The kidneys remove non-volatile acid – HCO3- Excretion of Hydrogen ions § The kidneys actively excrete H+ into the urine, which helps lower the blood’s acidity. § Mechanism of H+ excretion Titratable acids: H⁺ ions are secreted into the tubular ﬂuid, where they combine with phosphate ions (HPO42-) to form dihydrogen phosphate (H₂PO₄⁻), which is excreted in urine. Ammonium ion formation: NH3 in the tubular ﬂuid binds H+ to form ammonium (NH4+) which is also secreted. § Essentially, the above illustrates how H+ in kidney are bu6ered by phosphate & ammonia prior to being excreted as urine. § This process enables the kidneys to produce urine with a pH as low as 4.5 (usually between 5.5-7.0). 19 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo Clinical Correlation of pK: Aspirin Absorption Aspirin must be in the uncharged protonated form in order to diEuse through the cell membrane of the stomach mucosal lining. The stomach ph of approximately 2.0 is much lower than the pKa of the carboxyl group of aspirin (4). This shifts the equilibrium to the protonated form. The stomach mucosal intracellular pH is 6.8-7.1. This is above the pKa of aspirin and favours the unprotonated form of the drug. In the unprotonated form, aspirin is unable to diSuse across the cell membrane back the stomach lumen. The ionised (unprotonated) form of aspirin may become trapped in these cells, crystallise & cause mucosal cell rupture + gastric bleeding. Aspirin is acetylsalicylate which is the inactive form of the drug. The acetyl group is enzymatically removed in the stomach forming salicylate which is the active form. Most drugs are either weak acids or weak bases. Acidic drugs release a H+, this causes a charged anion A- to form. A drug passes through membranes more readily if it is uncharged. Therefore, for a weak acid the uncharged HA can permeate through membranes while the charged A- cannot. The eEective concentration of the permeable form of the drug at its absorption site is determined by the relative concentrations of the charged & uncharged forms. The ratio between these two forms is in turn determined by the pH at the site of absorption & by strength of the weak acid; which is represented by pKa. 20 MD 1 (2024-2025): Biochemistry; Topic 1: Acid-Base Concepts Abhishek Sah Frendo Remember the lower the pKa, the stronger the acid. In the acidic environment of the stomach aspirin is predominately in the un-ionised uncharged form and is thus preferentially absorbed. Alternatively in a basic environment such as the intestines, aspirin is likely to remain charged. However, even though this suggests that acidic drugs like aspirin are preferentially absorbed at low pH, there is still relatively little absorption in the acidic environment of the stomach, an organ unsuited for absorption. The stomach is a depot for drugs rather than an organ for drug absorption. Therefore, the rate of gastric emptying into the intestines greatly impacts the overall rate of absorption. This is due to the large surface area of the intestinal wall as a result of the villi & microvilli. Additionally, the duodenum primarily absorb drugs that are weak acids because of the acidic pH of stomach secretions. Effects of changes in pH § A ↓ in protein function § A ↓ in cardiac output § A ↓ in blood pressure § A constriction in small arterioles § May lead to arrhythmias § Neural excitation A decrease in pH reduces excitability; depresses CNS & may result in a loss of consciousness. An increase in pH can cause an increase in excitability: tingling sensation, nervousness, & muscle twitches. 21 MD 1 (2024-2025): Biochemistry; Topic 2: Amino Acids Abhishek Sah Frendo AMINO ACIDS Draw a titration curve for an amino acid 1. Titration Regions: The graph shows pH change as a base (OH⁻) is added to glycine in solution. The curve includes two buffering regions where the pH changes more gradually, centered around pK1 and pK2. 2. Two Ionizable Groups: Carboxylic acid group (COOH) with pK1=2.34. COOH loses a proton first to become COO- as the pH increases. Amino group (NH₃) with pK2=9.60. NH3+ loses its proton to become NH2 at a higher pH. 22 MD 1 (2024-2025): Biochemistry; Topic 2: Amino Acids Abhishek Sah Frendo 3. Isoelectric Point (pI): At pI=5.97, glycine exists primarily as a zwitterion. This is its neutral form with a positively charged amino group (NH₃⁺) and a negatively charged carboxylate group (COO⁻). At this point, the net charge of the molecule is zero. 4. pH Ranges: Low pH (< 2.34): At a very low pH, the solution is highly acidic, & there is an abundance of H+ ions. Glycine is fully protonated with COOH & NH₃⁺. It exists as a positively charged molecule (+1). pH 2.34–5.97: As the pH rises, COOH group deprotonates, forming COO⁻ since its pKa (pK1 = 2.34) is surpassed. The NH₃⁺ remains protonated. The resulting form is a zwitterion. pH 2.34-5.97-9.60: Glycine exists as a zwitterion. Net charge = 0. High pH (> 9.60): At a high pH, the solution is basic, & there are few H+ ions. The NH₃⁺ deprotonates, forming NH₂. Glycine becomes fully deprotonated with COO- & NH2. It is a negatively charged molecule (-1). 23 MD 1 (2024-2025): Biochemistry; Topic 2: Amino Acids Abhishek Sah Frendo Amino Acids at a physiological pH Physiological pH Definition The normal pH range in most bodily fluids, typically around 7.4, with slight variations depending on the specific tissue or organ system (e.g. arterial blood pH ranges between 7.35-7.45). At this slightly basic (alkaline) pH many biochemical processes function optimally. Amino acids at physiological pH Amino acids are amphoteric, meaning they can act as acids (proton donors) or bases (proton acceptors) Carboxyl group o pKa ≈ 2 o At pH < 2, carboxylic group remains protonated (COOH) o At pH 7.4 or above: COOH → Dissociates to COO⁻ + H+ Amino group o pKa ≈ 9.5 o At pH > 9.5, amino group is deprotonated (NH2) o At pH 7.4 or below: NH2 → Protonated to NH3⁺ 24 MD 1 (2024-2025): Biochemistry; Topic 2: Amino Acids Abhishek Sah Frendo Explain WHY some amino acids are charged at a physiological pH 1. Amino Group (NH₂/NH₃⁺) – always charged: NH2, can gain a H+, becoming positively charged (NH₃⁺). At physiological pH (~7.4), the amino group usually exists in its protonated form (NH₃⁺) since its pKa (~ 9-10) is higher than 7.4. 2. Carboxyl Group (COOH / COO⁻) – always charged: COOH can lose a H+, becoming negatively charged (COO⁻). At physiological pH (~7.4), the COOH group is typically deprotonated (COO⁻) because its pKa (~ 2) is lower than 7.4. 3. Side Chains of Some Amino Acids: They can be classified as acidic or basic based on their chemical compositions & pKa values. o Acidic Side Chains: aspartate and glutamate have carboxyl groups in their side chains, which are deprotonated & negatively charged at pH of 7.4 (pKa≈3−4). o Basic Side Chains: lysine (pKa≈10), arginine (pKa≈12), & histidine (pKa≈6) can have positively charged side chains at physiological pH, depending on their protonation state. 4. pH relative to pKa: pH < pKa: The group tends to be protonated. pH > pKa: The group tends to be deprotonated. 5. At physiological pH, amino acids exist as zwitterions with both a +ve & -ve charge, but overall charge depends on their side chains. 25 MD 1 (2024-2025): Biochemistry; Topic 2: Amino Acids Abhishek Sah Frendo Explain WHICH amino acids are charged at a physiological pH Positively charged (Basic side chains) o Histidine (his, H), o Arginine (arg, R), o Lysine (lys, K) o Proton acceptors o Hydrogen bond donors Negatively charged (Acidic side chains) o Aspartate/Aspartic acid (Asp, D) o Glutamate / Glutamic acid (Glu, E) o Proton donors o Hydrogen bond acceptors 26 MD 1 (2024-2025): Biochemistry; Topic 2: Amino Acids Abhishek Sah Frendo Explain the characteristics of the histidine side chain 1. pKa & Versatility in pH The imidazole group of histidine has a pKa ~ 6 which is close to the physiological pH (~ 7.4). This proximity allows histidine to exist in two states: protonated (positively charge) or deprotonated (neutral). For example: in haemoglobin, a specific histidine residue plays a critical role in O2 binding. The protonation state (neutral or +ve charged) of histidine varies between venous & arterial blood, influencing haemoglobin’s ability to bind & release O2. 2. Role as a Proton Donor & Acceptor It acts as an acid (proton donor) or a base (proton acceptor). The ability to switch between these states allows histidine to participate in proton transfer reactions. 3. Delocalised Double Bonds & Sensitivity to Environment The imidazole ring contains delocalized electrons; meaning that arrangement of double bonds can shift. This property makes histidine’s pKa & charge state sensitive to its local environment when it is part of a protein. 27 MD 1 (2024-2025): Biochemistry; Topic 2: Amino Acids Abhishek Sah Frendo Explain the concept of chirality using amino acids Chirality refers to a property of a molecule where it cannot be superimposed on its mirror image (similar to how your left hand cannot perfectly overlap your right hand). Molecules with this property are called chiral molecules. Structure of Amino Acids An amino acid consists of: A central carbon atom (alpha carbon). An amino group (NH₂). A carboxyl group (COOH). A hydrogen atom. A variable side chain (R group) that differs between amino acids. 28 MD 1 (2024-2025): Biochemistry; Topic 2: Amino Acids Abhishek Sah Frendo Chirality in Amino Acids The alpha carbon in most amino acids is chiral (or optically active) because it is attached to four different groups. Due to this chirality, amino acids can exist as two enantiomers (non-superimposable mirror images): 1. L-form (Levorotatory): found in naturally occurring proteins. 2. D-form (Dextrorotatory): rare in nature but present in some bacterial cell walls and antibiotics. Glycine as an Exception Glycine (simplest & smallest) is the only amino acid that is not chiral (achiral), as its R group is a hydrogen atom, making two of the groups on the alpha carbon identical. Hence, the compound is said to be optically inactive. Biological Roles of D-Amino Acids o Present in dietary sources o Metabolized by the liver & kidneys o Converted into L-amino acids or used in energy production pathways after metabolic processing. 29 MD 1 (2024-2025): Biochemistry; Topic 2: Amino Acids Abhishek Sah Frendo Know which amino acids are essential The following is the list of essential amino acids; which cannot be synthesized by the human body & must be supplied via one’s diet. 1. Histidine - His (H) (conditionally essential in children / rapid growth) 2. Isoleucine - Ile (I) 3. Leucine - Leu (L) 4. Lysine - Lys (K) 5. Methionine - Met (M) 6. Phenylalanine - Phe (F) 7. Threonine - Thr (T) 8. Tryptophan - Trp (W) 9. Valine - Val (V) 10. Arginine – Arg (R) (conditionally essential in children / rapid growth) These amino acids are crucial for protein synthesis. 30 MD 1 (2024-2025): Biochemistry; Topic 2: Amino Acids Abhishek Sah Frendo Classify amino acids according to chemical properties 31 MD 1 (2024-2025): Biochemistry; Topic 2: Amino Acids Abhishek Sah Frendo Know which amino acids are incorporated into proteins Proteinogenic Amino Acids 20 standard amino acids are directly encoded via genetic code. These amino acids are assembled into proteins during the process of translation on the ribosome. Chirality Most amino acids (except glycine) are chiral, meaning they have two enantiomers: L-form and D-form. The L-form is the active configuration used in protein synthesis in mammals. (Remember: Life loves left!) This stereospecificity ensures that proteins fold correctly into their functional 3D structures; which is essential for their biological roles. 32 MD 1 (2024-2025): Biochemistry; Topic 2: Amino Acids Abhishek Sah Frendo Understand that not all amino acids are incorporated into proteins Categories of Non-Proteinogenic Amino Acids: Metabolic Intermediates: o Ornithine & Citrulline: involved in the urea cycle o Citrate: regulates glycolysis & gluconeogenesis (inhibits PFK) o 3-Phosphoglycerate (3PG): precursor for serine Modified Amino Acids in Non-Ribosomal Peptides: o D-Alanine and D-Glutamate: found in bacterial cell walls. o Beta-Alanine: a precursor in synthesis of vitamin B5 o Gramicidin & vancomycin: antibiotics containing D-amino acids; helping them to interfere with bacterial cell wall synthesis. Neurotransmitters: o Gamma-Amino Butyric Acid (GABA): functions as an inhibitory neurotransmitter in the brain (derived from glutamate) o Acetylcholine: functions as an excitatory neurotransmitter in the brain (derived from choline & acetyl-CoA) o Nitric Oxide: functions as a gasotransmitter in the brain (derived from L-arginine by nitric oxide synthase - NOS). It is a retrograde neurotransmitter (released by postsynaptic neurons to influence presynaptic neurons). Selenocysteine: o Sometimes called the 21st amino acid, it is incorporated into specific proteins but is not directly encoded by the genetic code. 33 MD 1 (2024-2025): Biochemistry; Topic 2: Amino Acids Abhishek Sah Frendo Other Functions of Amino Acids: In the human body, amino acids have other biological roles in addition to protein synthesis. Tyrosine o Precursor for the following neurotransmitters: dopamine, adrenaline, & noradrenaline. Glutamate o Functions as an excitatory neurotransmitter in the nervous system. Tryptophan o Precursor for serotonin and melatonin, which regulate mood and sleep, respectively. Arginine o Synthesizes creatine phosphate, a critical energy store in muscle, brain, & blood. o Produces nitric oxide, a vasodilator; helps regulate blood flow. Cysteine and Glycine o Involved in the production of glutathione, a powerful antioxidant crucial for mitochondrial protection. Gamma-Aminobutyric Acid (GABA) It is both a neurotransmitter & an amino acid. It cannot be used to build proteins (non-proteinogenic) since it is the wrong type of amino acid. It is a gamma amino acid. 34 MD 1 (2024-2025): Biochemistry; Topic 2: Amino Acids Abhishek Sah Frendo Clinical Correlation: cysteine, cystinuria & kidney stones Free cysteine in the blood is involved in protein synthesis & other metabolic functions. However, it is prone to spontaneous non- enzymatic oxidative reactions forming cystine. Normally, amino acids are filtered & completed reabsorbed by the kidneys (glomerulus filters, proximal tubules reabsorbs most back). Individuals with cystinuria (autosomal recessive genetic disorder) have a defect in the amino acid transporter that reabsorbs cysteine. This results in cysteine accumulating in the urine. Cysteine is poorly soluble in water, especially at acidic pH of urine. In individuals with cystinuria, elevated cystine levels in urine lead to its precipitation & crystallization, forming cystine kidney stones. Long-term complication: CDK – chronic kidney disease. 35 MD 1 (2024-2025): Biochemistry; Topic 2: Amino Acids Abhishek Sah Frendo Clinical Correlation: Protein Malnutrition Protein malnutrition effects health due to the lack of essential amino acids; inhibits the synthesis of proteins required by the body. Hospital patients, post-operative, or recovering from major trauma or infections are often in a hypercatabolic state. This means their bodies must rapidly breakdown nutrients for protein synthesis & repair. These patients frequently require IV administration of nutrients. Inadequate protein intake during hospital recovery is associated with death from 2nd infections. Kwashiorkor: (severe form of malnutrition) protein deprivation in children who are weaned of milk too early in their development. The following are signs & symptoms associated with this disease: o Stunted growth: delayed development & growth in children o Fatty liver: due to impaired lipoprotein synthesis o Skin changes: rough blotchy skin with patches of depigmentation o Hair changes: thin, brittle hair, that may become depigmented o Oedema: swelling due to fluid retention (decreased albumin synthesis); especially evident in abdomen (enlarged belly) & legs. 36 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo PROTEIN STRUCTURE I Describe the flow of information from a gene to a protein The central dogma of molecular biology describes the flow of genetic information from DNA to RNA to protein. It has since been modified to account for complexities such as post-transcriptional modification and alternative splicing, which significantly influence gene expression and protein diversity. 37 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Central Dogma of Molecular Biology Transcription Starts in the nucleus where a gene’s DNA sequence serves as a template for synthesizing a complementary RNA molecule. The enzyme RNA polymerase binds to DNA at the promotor region & synthesizes a primary transcript (pre-mRNA), in eukaryotes. The primary transcript includes exons (coding regions) and introns (non-coding regions). Post-Transcriptional Modifications Introns are removed, and exons are joined together during splicing, creating the mature mRNA. The length of mature mRNA determines the final length of the protein because intronic sequences, are excluded from translation. This step also allows for alternative splicing, where different combinations of exons can be joined; enabling one gene to produce multiple protein variants (increasing protein diversity). 5’ Capping & 3’ Polyadenylation which facilitate nuclear export. Translation The mature mRNA is transported to the cytoplasm, where it is translated into a protein by ribosomes. Ribosomes read mRNA codons (groups of three nucleotides that encode specific amino acids) with the help of transfer RNA (tRNA). Each tRNA recognizes a specific codon through its anticodon & delivers the corresponding amino acid; building the 10 structure. 38 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo 39 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Understand the Mitochondrial Proteome The mitochondrial proteome refers to complete set of proteins found within mitochondria, which are critical organelles responsible for energy production and various cellular processes. Mitochondria has Dual Genetic Origin: mitochondrial & nuclear DNA Mitochondrial DNA (mtDNA): o Mitochondria have their own genome, separate from the nucleus. o In humans, mtDNA encodes 13 proteins, which are integral to the electron transport chain (ETC) and oxidative phosphorylation, key processes for ATP generation. Nuclear DNA: o Most mitochondrial proteins are encoded by nuclear DNA. o These proteins are synthesized in the cytoplasm and then imported into mitochondria using specialized protein-import machinery, such as the TOM (Translocase of the Outer Membrane) and TIM (Translocase of the Inner Membrane) complexes. 40 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Functional Categories of Mitochondrial Proteins Energy Production: proteins in ETC & ATP synthase. Metabolism: enzymes for TCA cycle, β-oxidation, & amino acid metabolism. ROS and Antioxidant Defence: proteins managing reactive oxygen species (e.g., superoxide dismutase, glutathione system). Apoptosis Regulation: cytochrome c and other proteins involved in programmed cell death. Mitochondrial Dynamics: proteins controlling fission, fusion, and mitophagy (selective degradation of damaged mitochondria ensuring proteome quality). Role in Cellular Signalling Mitochondrial proteins are involved in: o Calcium homeostasis: regulating intracellular Ca²⁺ levels. o Apoptotic signalling: cytochrome c release & activation of caspases (cysteine-dependent aspartate-specific proteases). o Metabolic signalling: sensing cellular energy status. Disorders related to Mitochondrial Proteome Dysfunction Mitochondrial diseases: caused by mutations in mtDNA or nuclear genes encoding mitochondrial proteins (e.g. Leigh syndrome). Neurogenerative diseases: dysfunctional mitochondria contribute to Parkinson’s’, Alzheimer’s’, & Huntington’s disease. Aging: accumulation of mitochondrial mutations & proteome dysregulation is linked to aging & aging-related diseases. 41 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Draw a dipeptide & peptide bond Reactants: Two amino acids participate in the reaction. Each amino acid has an amino group (NH₂) and a carboxyl group (COOH). Condensation Reaction: The COOH of one amino acid reacts with the NH₂ group of another. A molecule of water (H₂O) is released in the process. Peptide Bond Formation: A covalent bond, known as a peptide bond, forms between carbon of the COOH group and nitrogen of the amino group. Hence: the C-N bond. Product: The result is a dipeptide, a molecule consisting of two amino acids linked by a peptide bond. (Remember: This occurs in the ribosomes!) 42 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Explain the stereochemical limits of the peptide bond Resonance and Planarity: The peptide bond exhibits partial double-bond character due to resonance between carbonyl group (C=O) & amide nitrogen (N-H). This is known as a resonance hybrid; it exists between two resonance structures: o In one form, there is a single bond between the carbonyl carbon (C=O) & nitrogen (N). o In another, a lone pair of electrons on the nitrogen shift towards the carbon, giving the partial double bond character to C-N bond. This resonance delocalizes electrons across the bond, making the bond planar and rigid (this prevents free rotation about the bond). Consequently, the six atoms involved in the peptide bond (C, O, N, H, and the two adjacent alpha carbons) lie within the same plane. 43 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Restricted Rotation The partial double-bond character restricts free rotation around the peptide bond, locking it into a cis or trans configuration. o Trans configuration: favoured in most peptide bonds since it reduces steric hinderance between R groups of nearby amino acids. o Cis configuration: rare, except for peptide bonds involving proline, where steric hindrance is similar in both configurations. Polarity and Dipole Moment The peptide bond is polar due to the electronegativity differences between oxygen, carbon, and nitrogen: o The oxygen atom has a partial negative charge (δ⁻) o The nitrogen atom has a partial positive charge (δ⁺) This creates a dipole moment, which is critical for hydrogen bonding in secondary structures; alpha helices and beta sheets. Hydrogen Bonding Potential The polarity of the peptide bond enables the C=O group to act as a hydrogen bond acceptor & the N–H group to act as a hydrogen bond donor. These interactions stabilize the protein’s 20 & 30 structures. 44 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Describe phi, Φ & psi, Ψ (dihedral) angles A torsional / dihedral angle, measures the rotation about a specific bond between two atoms in a molecule. Phi (Φ): o The Phi angle refers to rotation around the N-Cα bond (the bond between the nitrogen of the amide group & the alpha carbon). o This angle determines the spatial orientation of the peptide bond on the N-terminal side of the alpha carbon. Psi (Ψ): o The Psi angle refers to rotation around the Cα-C bond (the bond between the alpha carbon and the carbonyl carbon). o This angle determines the spatial orientation of the peptide bond on the C-terminal side of the alpha carbon. Constraints on Φ and Ψ Angles Unlike the peptide bond, which is rigid due to its partial double-bond character, the Φ and Ψ angles can theoretically rotate freely. However: o Steric Hindrance: caused by large side chains (R-groups) or clashing atoms, restrict certain combinations of Φ and Ψ angles. Significance of Φ and Ψ Angles Secondary Structure o The pattern of Φ and Ψ determine if a region of the polypeptide will form an alpha helix, beta sheet, or other structure. Tertiary Structure o These dihedral angles also play a major role in defining the overall 3D structure & stability of the protein. 45 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Limits to Protein Structure Sequence diversity o A protein with n amino acids can theoretically have 20n possible sequences (20 standard amino acids). o However, only a small fraction of these sequences form stable, functional proteins. Restricted side chain conformations o Amino acid side chains are constrained due to steric hinderance & rotational limits of bonds within the side chain. Peptide Bond Rigidity o The peptide bond is rigid & planar due to its partial double-bond character; limiting its flexibility. Limited backbone flexibility o Backbone flexibility depends on the Φ (phi) and Ψ (psi) torsional angles, which describe rotation around the N–Cα & Cα–C bonds. o Steric hinderance further restricts allowable values Φ and Ψ as mapped in Ramachandran plots. Proline constraints o Proline has a unique cyclical structure that restricts its backbone conformation; making it more rigid than other amino acids. o It exhibits cis-trans isomerism; affects protein folding & stability. Disulfide bonds o Disulfide bonds between cysteine residues creates strong covalent cross-links that stabilise & restrict the protein’s 3D shape, reducing flexibility. 46 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo PROTEIN STRUCTURE II Describe 1°, 2°, 3°, & 4° structure Primary Structure The linear sequence of amino acids in a polypeptide chain, linked by peptide bonds. It is encoded directly by gene’s nucleotide sequence in DNA It determines the protein's overall structure and function. Secondary Structure Localized folding of the polypeptide chain into regular structures such as: o α-helix: a coiled structure stabilized by hydrogen bonds between the backbone atoms. o β-sheet: a sheet-like arrangement formed by hydrogen bonding between parallel or antiparallel strands. These structures are stabilized by hydrogen bonds between the carbonyl and amide groups of the peptide backbone. 47 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Tertiary Structure The overall 3-dimensional shape of a single polypeptide chain. Stabilized by interactions between side chains (R groups), including: o Hydrogen bonds (polar R groups with other polar R groups) o Ionic bonds (oppositely charged R groups bond together) o Hydrophobic interactions (amino acids orient outwards; H20) o Disulﬁde bridges (covalent bonds between cysteine residues) Determines protein's functional shape (enzymes' active sites). Quaternary Structure The arrangement of two or more polypeptide chains (subunits) into a multi-subunit complex. Stabilized by similar interactions as the tertiary structure. Examples include: haemoglobin (a tetramer with four subunits), insulin, and antibodies. 48 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Not all proteins have quaternary structures. Quaternary structure is seen in proteins made up of more than one polypeptide chain, and these subunits can be either: Homomeric: when the protein is composed of multiple identical subunits. Each subunit is the same in terms of its amino acid sequence and structure. § Example: haemoglobin in humans is a homomeric protein with four identical subunits (two alpha chains and two beta chains in the case of adult hemoglobin; 2 alpha chains & two gamma chains in children). Heteromeric: when the protein consists of diperent subunits, meaning each subunit may have a distinct amino acid sequence & structure. § Example: RNA polymerase in eukaryotes is a heteromeric protein composed of diEerent subunits, each contributing to its function in transcription. 49 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Describe the difference between polypeptide chains & subunits Polypeptide Chains: linear chains of amino acids connected by peptide bonds. A single polypeptide chain may fold to form a functional protein or a domain within a larger protein. Subunits: individual polypeptide chains within a protein that has quaternary structure. Subunits are assembled to form a functional multi-polypeptide protein complex, such as adult haemoglobin, which has four subunits (two α and two β chains). 50 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Explain why side chains are not involved in forming 2° Side chains (R groups) are not directly involved in forming secondary structures because these structures are stabilized by hydrogen bonds between backbone atoms of the polypeptide chain, specifically: The carbonyl oxygen (C=O) of one amino acid & the amide hydrogen (N-H) of another. 51 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Describe the alpha helix, beta sheet & forces stabilising them Alpha Helix (α-helix) Structure: o A right-handed spiral; meaning it twists in a clockwise direction; as you move along the axis of the helix. o This is the most common form of α-helix found in proteins. o The backbone forms the helix, with side chains (R groups) extending outward from the helix; enabling it to interact with the surrounding environment or other parts of proteins. o Length of helix: a complete helix consists of 3.6 amino acids. Stabilization: o Stabilized by hydrogen bonds between the carbonyl oxygen (C=O) of one residue and the amide hydrogen (N-H) four residues earlier. o These H-bonds are parallel to the axis of the helix. o Additionally stabilised by hydrophobic interactions between non-polar side chains (especially in interior of the protein). Location o It is present in the following proteins: § keratin – fibrous protein; found in hair & skin § ferritin – globular protein; found in liver, spleen, bone marrow § myoglobin – globular protein; found in skeletal & cardiac muscle 52 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo 53 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Beta Pleated Sheet (β-sheet) Structure: o A zigzag, pleated structure, with atoms arranged in a plane. o Formed by segments of polypeptide chain lying side by side. o The beta sheets may be parallel (strands run in the same N- to C-terminal direction) or antiparallel (strands run in the opposite direction). o Side chains alternate above & below the plane of the sheet; enabling interactions with other molecules or protein regions. o Form large, flat or twisted surfaces providing rigidity for structural scaffolds in proteins or as binding interfaces for other molecules. Stabilization: o Stabilized by hydrogen bonds between the carbonyl oxygen (C=O) of one strand & amide hydrogen (N-H) of an adjacent strand (rather than within the same strand as in alpha). o These H-bonds are perpendicular to the polypeptide chains. o Additionally stabilised by hydrophobic interactions between non-polar side chains (particularly when the beta-sheet is buried in the protein core). Location o It is present in the following proteins: § Silk Fibroin – fibrous protein; found in spider silk. § Amyloid fibrils – fibrous protein; found in Alzheimer’s disease. § Immunoglobulins (antibodies) – globular protein; core of fold. 54 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo 55 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Explain the variation that exists in 1° structures The primary structure (1°) of a protein refers to the specific sequence of amino acids that are linked together in a polypeptide chain. This sequence is determined by the genetic code and varies significantly between different proteins. The variation in primary structures is what gives proteins their unique functional properties. Amino Acid Sequence Variation Every protein has a unique sequence of amino acids. Even a single change in the sequence can have a significant effect on the protein's structure and function. The sequence is determined by the nucleotide sequence of the gene encoding the protein. This sequence includes 20 standard amino acids, each with distinct side chains. Influence on Protein Function The variation in the 1° structure leads to different shapes, folding patterns, & properties; critical for the protein's biological activity. Examples: o haemoglobin's 10 structure allows it to fold into a 40 structure; capable of binding & transporting O2 efficiently in the blood. o collagen's 10 structure forms a triple helix; providing structural support to connective tissues like skin, bone & collagen. o lysozyme’s 10 structure determines its active site; it allows it to break down bacterial cell walls by cleaving peptidoglycan. 56 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Mutations A mutation in a protein’s 10 structure can disrupt folding & function, potentially resulting in a disease. For instance, sickle cell anaemia is caused by a single amino acid change in haemoglobin’s 10 structure. o A single point mutation in the HBB gene, which encodes the beta-globin subunit of haemoglobin. o This mutation causes glutamic acid (Glu / E) to be replaced by valine (Val / V) at position 6 of the beta-globin chain (Glu6Val). o This substitution causes haemoglobin molecules to aggregate into rigid fibres under low O2 conditions. o These fibres deform red blood cells (RBCs) into a sickle shape, impairing their flexibility. o Consequently, these sickle-shaped RBCs result in: blockage in blood vessels, leading to pain (vaso-occlusive crises), organ damage & anaemia due to rapid RBC breakdown. 57 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Explain the difference between polymorphisms & mutations Polymorphisms and mutations both refer to changes in the sequence of amino acids in proteins, but they differ in their frequency, impact, and implications for protein function. POLYMORPHISMS These are variations in the protein sequence that naturally occur in a population at a frequency of 1% or greater. They are generally not harmful & represent natural genetic diversity. Many have no clinical impact but serve as genetic markers. Conservative substitutions are common examples of polymorphisms, where one amino acid is replaced by another with similar properties. For instance: o Lysine (positively charged) could be substituted for Arginine (also positively charged). o Leucine (branched chain hydrophobic) could be replaced by Isoleucine (also hydrophobic and branched). o Threonine (hydroxyl group) could be substituted for Serine (also hydroxylated). These changes do not necessarily impact the protein’s function significantly since properties of amino acids involved are similar. The frequency of polymorphisms is higher than that of mutations. Polymorphisms often vary between populations due to evolutionary pressures, such as migration, adaptation or genetic drift. 58 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Examples of Polymorphisms Human Leukocyte Antigen (HLA) proteins in the immune system. o There are over 20,000 variations of these proteins across the human population. These variations are responsible for differences in immune responses, such as tissue rejection in organ transplants, since HLA proteins help recognize self from non-self cells. Sickle Cell Trait arises from a single nucleotide mutation (Glu6Val) in the HBB gene, where individuals inherit one normal hemoglobin gene (HbA) & one mutated gene (HbS), making them carriers (heterozygous). o This trait is common in populations from malaria-endemic regions, as it provides a survival advantage by conferring resistance to malaria. Although the trait generally causes no symptoms, it is maintained in these populations through natural selection, making it a widespread genetic variation within certain groups. Eye colour arises from genetic variations in the OCA2 and HERC2 genes, with multiple alleles leading to different eye colors, such as brown, blue, green, and hazel. o These variations occur in a significant portion of the population, and their distribution is influenced by genetic inheritance and evolutionary factors. The variation in eye color is largely determined by amount & type of melanin in the iris, & while not affecting health, it contributes to genetic diversity. 59 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo MUTATIONS These are variations in the protein sequence that rarely occur in a population at a frequency of 1% or lower. They often lead to altered biological function (loss, no, or gain) & so tend to be harmful; potentially resulting in diseases or conditions. Many have significant clinical impacts – protein mutations cause dysfunction in an inherited disease/increase susceptibility to a disease. Nonconservative substitutions (where the substituted amino acid has very different properties from the original) often lead to mutations that affect protein function. These changes usually impact the protein’s function, catalysis, cellular targeting, or degradation significantly since properties of amino acids involved differ substantially. The frequency of mutations is lower than that of polymorphisms since mutations are typically harmful or deletions. Mutations like polymorphisms, are also influenced by evolutionary pressures & their frequences can vary between populations. 60 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Examples of Harmful Mutations Sickle Cell Anaemia o Substitution Mutation: a single point mutation in HBB gene where adenine is replaced by thymine; resulting in glutamic acid being replaced by valine at position 6 of the beta-globulin chain (Glu6Val). o Effect: causes haemoglobin S (HbS) to form stiff fibres under low O2 conditions leading to sickle-shaped RBCs. This can block blood flow, causing painful episodes (vaso-occlusive crises), organ damage, & chronic haemolytic anaemia (due to premature RBC destruction). Cystic Fibrosis o Frameshift Mutation: a deletion of 1 amino acid in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene. CTFR gene encodes a protein that regulates the transport of Cl- & Na+ ions across the cell membranes in epithelial cells of the lungs & of the pancreas. o Effect: disrupts the CFTR protein, impairing chloride ion transport across cell membranes; leading to thick, sticky mucus that causes severe lung, & digestive system complications. Duchenne Muscular Dystrophy (DMD) o Frameshift Mutation: a deletion of 1 amino acid in the DMD gene. DMD gene encodes dystrophin; a protein essential for muscle function. o Effect: leads to the absence of functional dystrophin, causing progressive muscle weakness & wasting, & eventually, over time, the loss of motor function. 61 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Polymorphisms vs Mutations Definition & Impact on Disease: Polymorphisms are natural genetic variations found within a population that do not typically lead to disease. They often have minimal or no functional impact on proteins and can occur in both coding and non-coding regions of the genome. Mutations are changes in the DNA or protein sequence that usually result in disease. They often disrupt protein structure or function. Frequency & Commonality: Polymorphisms are more common in the population, often occurring in more than 1% of individuals; have usually benign or neutral effects. Mutations are typically rarer and may only affect a small portion of the population. Mutations often have a significant biological impact. Types of Genetic Variation Polymorphisms can include single nucleotide polmorphisms (SNPs), insertions or deletions (indels) or even copy number variations (CNVs) that do not substantially alter gene function. Mutations generally refer to any genetic change, including point mutations, frameshifts, insertions, deletions, or duplications that alter gene or protein function. Evolutionary Significance Polymorphisms may arise due to evolutionary pressures & might provide an adaptive advantage in various environments. Mutations are usually random & less likely to provide an adaptive benefit; usually causing deleterious effects. 62 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Describe the difference between an apoprotein & a holoprotein The difference between apoproteins and holoproteins lies in the presence or absence of non-protein groups (prosthetic groups) that are essential for the protein's full functionality. APOPROTEIN Definition: polypeptide chain of a protein without its associated non-protein groups (prosthetic groups). It is the inactive form of the protein (an incomplete protein). An apoprotein requires the binding of a prosthetic group or cofactors to gain functional activity. Examples: o Haemoglobin apoprotein consists of just the polypeptide chains (α and β subunits). It requires a haem group (prosthetic group) to be functional, allowing it to bind & release O2 effectively. o Transferrin apoprotein does not bind to iron. It requires the binding of Fe3+ ions to become a functional holotransferrin. This Fe-bound form is critical for transport of Fe to various tissues. o Apolipoprotein B (apoB) is a key apoprotein that serves as the protein component of lipoproteins such as low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL). In its apoprotein form, apoB is inactive & requires the association with lipids, such as cholesterol & phospholipids, to form a functional lipoprotein complex. The apoB-containing lipoproteins play a crucial role in lipid metabolism & cholesterol transport to tissues & organs. Importance: apoproteins are typically inactive in their biological role until they are combined with their required prosthetic groups. 63 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo HOLOPROTEIN Definition: a complete, functional protein that includes both its polypeptide chain & its prosthetic groups. These non-protein groups are essential for the protein to perform its biological activity. When an apoprotein binds to its prosthetic group, it forms a holoprotein; it is now capable of carrying out its intended function. Importance: holoproteins are the active forms of proteins in biological systems; they are fully functional & capable of their specific tasks. 64 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Understand that protein families exist & their genetic relationships In the context of protein families, the proteins related to a common ancestor gene are called paralogs. These proteins evolve due to gene duplication events followed by mutations, which may result in structural & functional divergence while still retaining similarities. Myoglobin and Haemoglobin Myoglobin and the different chains of haemoglobin are examples of paralogs. The common evolutionary origin of myoglobin & haemoglobin lies in their shared ancestry from a single ancestral globin protein. This ancestral globin likely arose over 500 million years ago and underwent gene duplication followed by divergent evolution, leading to the development of the distinct but related functions seen in myoglobin & haemoglobin today. 65 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo MYOGLOBIN Structure: a monomer: single polypeptide chain with one haem group. Function: binds & stores O2 in muscle cells; providing a reserve for mitochondrial energy production. Location: found primarily in muscle (cardiac & skeletal) cells. Oxygen Binding: 1 O2-binding site per molecule. Oxygen Affinity: high affinity for O2, designed for storage. Oxygen Dissociation Curve: a hyperbolic curve due to its simple, non-cooperative binding of O2. Lifespan & Turnover: has a relatively stable presence within muscle cells without frequent turnover. HAEMOGLOBIN Structure: a tetramer made up of 4 globin chains (2 alpha & 2 beta), each with a haem group (a total of 4 haem groups). Function: transports O2 in RBCs from lung to tissues & assists in CO2 transport back to lungs. Location: found in red blood cells. Oxygen Binding: 4 O2-binding site per molecule. Oxygen Affinity: moderate affinity for O2, designed for delivery to tissues based upon demand. Oxygen Dissociation Curve: a sigmoidal curve due to cooperative binding, enabling it to release O2 effectively in tissues where O2 is low. Lifespan & Turnover: has a lifespan tied to RBCs (~120 days). 66 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Relationship Between Myoglobin and Haemoglobin Gene Duplication o The alpha globin gene evolved from the beta globin gene via a gene duplication event. o Myoglobin results from an ancestral duplication of the alpha globin gene. o Consequently, alpha globin gene & beta globin gene are paralogs, as are myoglobin and globin chains of haemoglobin. Genetic Variants o Additional examples of the globin family include: § Zeta globin: expressed in embryonic haemoglobin during early development. Pairs with epsilon globin. § Epsilon globin: expressed in embryonic haemoglobin during early development. Pairs with zeta globin. § Gamma globin: found in foetal haemoglobin (HbF), which has a higher oxygen affinity compared to adult haemoglobin. 67 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo PROTEIN STRUCTURE III Describe the common post-translational modifications to proteins & the amino acids modified Post-translational modiﬁcations (PTMs) are chemical alterations made to proteins after their synthesis, which inﬂuence their structure, function, stability, localization, and interactions. Common PTMs: include phosphorylation, glycosylation, acetylation, ubiquitination, and carboxylation. 68 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Glycosylation Description: The addition of sugar molecules (glycans) to proteins, commonly on extracellular or secreted proteins. Glycosylation is critical for proper folding, stability, & cell-cell communication. Types: o N-linked glycosylation: attachment to nitrogen atom in the side chain of asparagine (Asn) residues. o O-linked glycosylation: attachment to oxygen atom in the side chains of serine (Ser) or threonine (Thr). Amino Acids Modiﬁed: Asparagine, Serine, and Threonine. Phosphorylation Description: addition of a phosphate group to proteins, often regulating enzymatic activity, signal transduction, and protein- protein interactions; can change the way proteins react to epectors. Enzymes involved: o Kinases: add phosphate groups o Phosphatases: remove phosphate groups Amino Acids Modiﬁed: serine, threonine, & tyrosine hydroxyl groups 69 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Acetylation Description: addition of an acetyl group (COCH3), typically to the amino group of lysine residues. Acetylation can regulate protein stability, localisation & interactions. Enzymes involved: o Histone acetyltransferases (HATs): acetylate lysines on histones, enhancing transcription o Histone deacetylases (HACs): remove acetyl groups; often repressing transcription. Amino Acids Modiﬁed: lysine Carboxylation Description: addition of a carboxyl group (COOH), enhancing calcium-binding properties, critical for blood coagulation proteins & bone matrix formation. Enzymes involved: o Vitamin K-dependent gamma-glutamyl carboxylase Amino Acids Modiﬁed: glutamate à gamma-carboxyglutamate 70 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Describe how selenoproteins are made Selenoproteins are described as the 21st amino acid. They are synthesized through a unique mechanism that incorporates amino acid selenocysteine (Sec, U) into the protein sequence. It resembles sulphur in terms of its properties. Unique Codon Usage Selenocysteine is specified by the UGA codon, which typically signals a stop in standard genetic code – termination of protein synthesis during translation (alongside UAA or UAG). However, in selenoprotein genes, a specialized selenocysteine insertion sequence (SECIS) in the mRNA allows UGA to be recoded for selenocysteine incorporation. Biosynthesis of Selenocysteine Selenocysteine does not exist as a free amino acid in the cell. It is synthesized while attached to a specialized tRNA, tRNASec, which has an anticodon recognizing the UGA codon. Initially, serine is enzymatically attached to tRNASec & is then converted to selenocysteine by the action of selenocysteine synthase, using a selenium donor. Incorporation During Translation Selenocysteine is incorporated into the growing polypeptide chain on the ribosome, guided by the SECIS element and associated translation factors, just like any other amino acid. Example: Glutathione peroxidase is a well-known enzyme that relies on selenocysteine for its antioxidant activity. This enzyme protects cell from oxidative damage by reducing hydrogen peroxide & lipid peroxides. 71 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo 72 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Understand the intracellular signals for proteins & how they are targeted to Intracellular sorting signals direct proteins to speciﬁc organelles or cellular compartments, ensuring proper localization and function. These signals are typically short amino acid sequences within the protein or added during processing. Mitochondria Signal: proteins destined for the mitochondria have an N-terminal mitochondrial targeting sequence (MTS), which is rich in positively charged (Arg, Lys) and hydrophobic residues, allowing recognition and transport into the mitochondria. Mechanism: o Proteins are synthesized in the cytosol as precursors. o MTS is recognized by translocase of the outer membrane (TOM). o The precursor protein is translocated through TOM and then through the translocase of inner membrane (TIM). o MTS is cleaved by Mitochondrial Processing Peptidase (MPP) to yield the mature protein. Example: cytochrome c oxidase subunits. It is involved in the ETC. 73 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Endoplasmic Reticulum (ER) Signal: proteins destined for ER have an N-terminal ER signal sequence (6-12 hydrophobic residues followed by a cleavage site). Mechanism: o Signal is recognized by the signal recognition particle (SRP) during translation. o SRP directs the ribosome to SRP receptor on the ER membrane. o The protein is translocated into the ER lumen or membrane via a translocation channel. o The signal sequence is cleaved by signal peptidase after the protein is translocated. Example: secretory proteins like insulin. 74 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Lysosomes Signal: mannose-6-phosphate (M6P) tag (a glycosylation modification) is added to soluble lysosomal enzymes for targeting. Mechanism: o In the Golgi, proteins destined for lysosomes are tagged with M6P. o M6P receptors in the trans-Golgi network direct proteins into clathrin-coated vesicles. o Vesicles fuse with late endosomes, which deliver proteins to lysosomes. Example: acid hydrolases (enzymes involved in breaking down biomolecules in the lysozyme) are delivered to lysozymes via this pathway. 75 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Peroxisomes Signal: peroxisomal targeting signal (PTS) o PTS1: C-terminal tripeptide, commonly SKL (Ser-Lys,Leu) o PTS2: N-terminal nonapeptide. Mechanism: o The PTS is recognized by cytosolic receptors called PEX proteins (e.g. PEX5 for PTS1 & PEX7 for PTS2). o The receptor-bound protein is transported to the peroxisome membrane, where it interacts with the docking complex on the peroxisomal membrane. o The docking complex facilitates the transfer of the cargo protein through a specialised translocon into the peroxisome matrix. Example: catalase – an enzyme responsible for breaking down hydrogen peroxide into H20 & O2. 76 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Nucleus Signal o Nuclear Localisation Signals (NLS): a sequence rich in basic residues, directing proteins to the nucleus. o Nuclear Export Signals (NES): a signal, often leucine-rich, directing proteins from nucleus to cytoplasm. Mechanism: o NLS is recognized by importins in the cytoplasm. o The importin-protein complex is transported through nuclear pore complexes (NPCs) via an energy-dependent process utilising the Ran-GTP cycle to drive directionality. o Once inside the nucleus, if the proteins needs to be exported, it will have an NES, which is recognised by exportins, faciliting transport back to the cytoplasm. Internal Signals: o NLS & NES are essential internal signals that determine the protein’s directionality between nucleus & cytoplasm. Example: Transcription factors, like NF-κB, which contains NLS for nuclear import to bind DNA & NES for export after regulating gene expression. 77 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Explain the synthetic processing & secretory pathway of insulin & structures at each stage The synthetic processing and secretory pathway of insulin is a highly regulated process that involves multiple steps to ensure proper folding, processing, and secretion of this essential hormone. 78 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Transcription and Translation (Preproinsulin) Preproinsulin (110 amino acids): the initial product of the INS gene (1 single gene) is synthesized as preproinsulin on ribosomes associated with rough endoplasmic reticulum (rER). Signal Sequence: N-terminal signal sequence directs the ribosome to dock on the ER membrane via interaction with the signal recognition particle (SRP). Signal Cleavage: once inside the ER, the signal sequence is cleaved by signal peptidase, producing proinsulin. Folding and Disulfide Bond Formation (Proinsulin) Proinsulin (86 amino acids): A single polypeptide that includes the A chain, B chain, and C peptide. Proper folding occurs in the ER, assisted by molecular chaperones, forming three disulfide bonds: o Two interchain bonds between the A and B chains. o One intrachain bond within the A chain. The oxidizing environment of the ER is essential for disulfide bond formation (it cannot occur in reducing environment of the cytosol). Golgi Processing and Cleavage (Mature Insulin) Proinsulin is transported from ER to Golgi apparatus in vesicles. In the Golgi, proinsulin is packaged into secretory granules, where proteolytic enzymes cleave the C-peptide. This cleavage produces mature insulin, which consists of: o Two polypeptide chains: A chain (21 AA) & B chain (30 AA). o Three disulfide bonds. 79 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Storage and Secretion Storage Form: in secretory vesicles, insulin is stored as a hexamer, stabilized by Zn2+ coordinated with histidine residues from 3 insulin monomers. Each hexamer contains 2 Zn2+ and is formed from 2 trimers. Secretion: in response to glucose or other stimuli (incretin, metformin) insulin is secreted into the blood via exocytosis. C-peptide: is packaged in Golgi secretory granules in beta- pancreatic cells & simultaneously secreted with insulin in equimolar amounts. Significance of C-Peptide Used as a diagnostic marker of endogenous insulin production (from pancreas) since it is not present in exogenous insulin therapies (injections). C-peptide has biological activity in stimulating some cells via membrane receptors; especially in nerve & kidney cells. It has therapeutic potential in improving kidney function and vascular health in patients with Type 1 diabetes (where patients do not produce C-peptide; since there is no insulin production). 80 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Use creatine kinase to explain the term tissue-specific isoforms Tissue specific isoforms are proteins that differ somewhat in their primary structure but retain similar functions. They are generated via alternative splicing (process during RNA processing). An example is Creatine kinase (CK); enzyme involved in energy metabolism, catalysing the reversible conversion of creatine and ATP to phosphocreatine and ADP. It has 3 distinct isoforms: CK-MM (Skeletal Muscle Isoform) Found predominantly in skeletal and cardiac muscle. Provides a rapid energy supply during muscle contraction. MM represents the two identical subunits: homodimer CK-BB (Brain Isoform) Localized primarily in the brain and other tissues with high energy demands (such as the lung tissues). Supports neuronal energy metabolism. BB represents the two identical subunits: homodimer CK-MB (Cardiac Isoform) Found predominantly in cardiac muscle; As a hybrid of CK-MM and CK-BB subunits (heterodimer). Its specific expression in the heart makes it clinically significant as a myocardial infarction (heart attack) marker. 81 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Significance of Tissue-Specific Isoforms Functional Specialization: each isoform adapts to the energy requirements of its tissue, ensuring efficient energy transfer & utilization. Clinical Relevance: the presence of specific isoforms in blood can be used as diagnostic tools: o Elevated CK-MB indicates cardiac damage. § Thus, increases after a myocardial infarction o Elevated CK-MM can suggest skeletal muscle injury or disease. 82 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Isoforms vs Isoenzymes Isoforms Definition: isoforms are different forms of a protein that arise from a single gene through processes like alternative splicing or post-translational modifications. Function: isoforms may perform similar or related functions but may have slight differences in structure, localization, or activity. Example: different isoforms of the troponin protein in cardiac & skeletal muscle. Troponin is a complex of 3 regulatory proteins (troponin C,I, & T) found in muscle fibres (skeletal & cardiac muscles). Cardiac troponin (cTn) is also used as a biomarker for heart injury (e.g. during myocardial infarction) since it is released into the blood. Isoenzymes (Isozymes) Definition: isoenzymes are different forms of an enzyme that catalyze the same reaction but differ in their amino acid sequence or structure. They can have distinct biochemical properties (varying kinetic properties or regulatory mechanisms). Function: isoenzymes perform the same function but may be adapted for specific tissues, developmental stages, or environmental conditions. They often exhibit different substrate affinities, pH optima, or regulation. Example: Lactate dehydrogenase (LDH) catalyses the reversible conversion of pyruvate to lactate during anaerboic glycolysis. LDH has several isoenzymes (LDH-1 to LDH-5), each found in different tissues. 83 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Describe how protein denaturation occurs Protein denaturation refers to the process by which a protein loses its native structure, leading to the unfolding and disruption of its secondary, tertiary, & sometimes quaternary structures. Causes of Protein Denaturation Heat (Let ‘em cook): o Heat increases the kinetic energy of molecules, disrupting non- covalent interactions such as hydrogen bonds, van der Waals forces, and hydrophobic interactions. o Covalent bonds, such as peptide bonds & disulﬁde bonds, usually remain unaEected. o Proteins have an optimal temperature for biological activity, beyond which they denature. o Example: cooking an egg causes albumin proteins to denature and coagulate irreversibly. 84 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo pH: o Extreme pH levels disrupt ionic interactions & hydrogen bonds: § At low pH: negatively charged carboxyl groups (COO⁻) become protonated (COOH), altering ionic interactions. § At high pH: positively charged amino groups (NH₃⁺) are deprotonated (NH₂), disrupting ionic bonds. o Proteins have an optimal pH for activity, and deviations cause denaturation. o Example: acidic conditions in ceviche denature ﬁsh proteins without using heat. UV Radiation: o Ultraviolet light can break certain bonds and generate free radicals that damage the protein's structure. Denaturing Agents: o Chemical agents such as organic solvents (e.g., alcohol), detergents, and heavy metal ions (e.g., lead, mercury) disrupt hydrophobic interactions and protein folding. Ionic Strength: o High salt concentrations disrupt ionic interactions and protein solubility, often leading to precipitation. 85 MD 1 (2024-2025): Biochemistry; Topic 3: Proteins Abhishek Sah Frendo Melting Temperature (Tm) Tm is the temperature at which half of the protein in a solution is denatured. It serves as a measure of a protein's thermal stability. Factors such as ionic strength & pH inﬂuence Tm. Reversibility of Denaturation: Under ideal conditions, some proteins can refold into their native conformation after the removal of the denaturing agent, regaining their function. Example: small, simple proteins like ribonuclease (Rnase) Irreversible Denaturation: o Most proteins become permanently disordered and insoluble once denatured, forming aggregates or precipitates. o Example: cooking an egg — denaturation of ovalbumin proteins in egg whites is irreversible. Misfolded proteins can also contribute to pathological irreversible conditions such as Alzheimer’s disease. 86 MD 1 (2024-2025): Biochemistry; Topic 4: Globular Proteins Abhishek Sah Frendo GLOBULAR PROTEINS I Describe the folding mechanism of a globular protein Protein folding is a highly ordered process by which a linear po

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