Notes on Biochemistry PDF

1 1/14 Introduction to biochemistry Briefing Document: I. Core Themes and Concepts in Biochemistry Definition of Biochemistry: The study of the chemistry of living matter, focusing on how collections of inanimate molecules interact to maintain and perpetuate life. Characteristics of Life: ​...

1 1/14 Introduction to biochemistry Briefing Document: I. Core Themes and Concepts in Biochemistry Definition of Biochemistry: The study of the chemistry of living matter, focusing on how collections of inanimate molecules interact to maintain and perpetuate life. Characteristics of Life: ​ Complexity and Organization: Life exhibits a high degree of structural complexity, with defined functions at various levels. ​ Extraction, Transformation, and Systematic Use of Energy: Organisms require energy to maintain their dynamic state, extracting it from sources such as sunlight or fuels. ​ Sensing and Responding to Changes: Living systems can perceive and react to their environment, adapting to changing conditions. ​ Self-Replication with Potential for Evolution: Life can reproduce itself, passing on genetic information with enough variability to drive evolution. Cellular Basis of Life: ​ The cell is the fundamental unit of life. ​ There are three domains of life: Bacteria, Archaea, and Eukarya, distinguished by their cellular and molecular characteristics. ​ Bacterial cells are simpler, lacking membrane-bound organelles. ​ Eukaryotic cells are more complex, possessing a nucleus and other organelles. ​ Plant cells are also eukaryotic cells ​ Cellular components can be separated and studied to understand their chemistry Energy and Carbon Sources: All organisms need energy and carbon for life. They can be classified based on the source of both. Importance of Specific Elements: Biomolecules are mainly carbon-based and include elements such as H, O, N, P, and S. Metal ions (K+, Na+, Ca2+, Mg2+, Zn2+, Fe2+) are essential for metabolism. The Versatility of Carbon: Carbon's unique bonding properties allow it to form a wide range of molecules with diverse structures (linear, branched, cyclic) and functional groups. This versatility enables the formation of complex biomolecules with distinct functions. Functional Groups and Molecular Structure: Biomolecules are often polyfunctional, possessing multiple functional groups. The three-dimensional structure of a molecule is crucial for its function. Stereoisomers and Specificity: Stereoisomers, molecules with the same chemical bonds but different spatial arrangements, have distinct biological properties. Their interactions with other molecules are often stereospecific. Molecular Conformation: Some conformations of molecules are more energetically favorable than others. II. Key Takeaways ​ Biochemistry is a fundamental scientific discipline that provides the foundation for understanding life at the molecular level. ​ Living systems are complex and highly organized, relying on intricate chemical processes to extract energy, grow, and reproduce. ​ The structure and function of biomolecules are inextricably linked, and even subtle differences in molecular configurations can have profound biological consequences. ​ Biochemical research has vast potential to address societal challenges but also comes with ethical obligations and a responsibility to avoid potential harms. ​ The cases of Moderna (a rapid and ethical success), and Theranos (an ethical and scientific failure), provide critical lessons on how scientific endeavor should proceed. Glossary Biochemistry: The study of the chemical processes and substances that occur within living organisms. Biomolecules: Molecules produced by living organisms, including carbohydrates, proteins, lipids, and nucleic acids. Cell: The basic structural and functional unit of all known living organisms. Chiral Molecule: A molecule that is not superimposable on its mirror image.  2 Cis Isomer: A stereoisomer in which substituent groups are on the same side of a double bond or ring. Conformation: The specific three-dimensional shape of a molecule achieved through rotation of single bonds. Eukaryotic Cell: A cell characterized by the presence of a nucleus and other membrane-bound organelles. Functional Group: Specific groups of atoms within molecules that have distinctive chemical properties. Metabolism: The set of chemical processes that occur within an organism to maintain life. Organic Compound: A molecule that contains carbon, often combined with hydrogen and other elements. Prokaryotic Cell: A cell lacking a nucleus and other membrane-bound organelles. Stereoisomer: Molecules with the same chemical formula and bonds but different three-dimensional arrangements of atoms. Stereospecificity: The ability of a biomolecule to interact or bind with another molecule only if the other molecule has a specific stereochemical configuration. Trans Isomer: A stereoisomer in which substituent groups are on opposite sides of a double bond or ring. Biochemistry Study Guide 1. What are the four key characteristics that define life according to the study of biochemistry? Life is characterized by complexity and organization; the extraction, transformation, and use of energy; the ability to sense and respond to changes; and self-replication with the capacity for evolution. 2. Explain the relationship between biochemistry and the laws of physics and chemistry. Biochemistry studies how inanimate molecules in living things interact to maintain and perpetuate life. The molecules and processes within living things must still conform to all physical and chemical laws. 3. Describe the main differences between prokaryotic and eukaryotic cells. Prokaryotic cells are generally simpler and lack a nucleus and membrane-enclosed organelles, while eukaryotic cells are more complex and contain a nucleus and various organelles. 4. What is the role of the cell membrane in a bacterial cell? The cell membrane in a bacterial cell acts as a permeability barrier, controlling what enters and exits the cell. It is made of lipids and proteins. 5. Where do plant cells get their energy, and how does this differ from animal cells? Plant cells obtain energy from sunlight through photosynthesis, while most animal cells obtain energy by consuming fuels. 6. What are the four most common elements found in biomolecules? The four most common elements in biomolecules are hydrogen (H), oxygen (O), nitrogen (N), and carbon (C). 7. Why is carbon such a versatile element for building biomolecules? Carbon is versatile because it can form single, double, and triple bonds with other elements. This allows it to create a wide variety of molecules with different shapes, sizes, and compositions. 8. What are stereoisomers and how do they differ? Stereoisomers are molecules with the same chemical bonds but different spatial arrangements of atoms. This difference in configuration can affect their properties. 9. How does a molecule’s three-dimensional structure influence its function? A molecule’s three-dimensional structure determines its shape, which is essential for interacting with other molecules and performing its specific biological function. 10. Briefly explain why the interactions between biomolecules are specific. Interactions between biomolecules are specific due to factors like shape and spatial arrangement of atoms, which allow them to fit and bind with other molecules.  3 Essay Questions 1. Discuss the interconnectedness between the four defining characteristics of life as studied through biochemistry. How does each characteristic contribute to the overall maintenance and continuation of life, and how do they influence each other? 2. Contrast and compare the structural and functional differences between prokaryotic and eukaryotic cells. Consider the implications of these differences in terms of evolutionary history and the diverse roles they play in living organisms. 3. Describe the central role of energy in biological systems. Discuss the various sources of energy for different organisms and explain how energy is extracted, transformed, and used to maintain complexity and drive life processes. 4. Explore the chemical properties of carbon that make it essential for building biological molecules. How do carbon’s bonding capabilities and structural versatility contribute to the diversity and function of biomolecules? 5. Analyze the importance of three-dimensional structure and stereochemistry in the function of biomolecules. Explain how variations in molecular shape influence biological interactions and why stereospecificity is necessary for many biological processes.  4 1/16 Water, Aqueous Solutions, and Non-Covalent Interactions Briefing Document I. The Structure of Water & Hydrogen Bonding Water's Unique Properties: Water makes up 70% of most organisms. Molecular Structure: The oxygen atom in water has four electron pairs arranged in a distorted tetrahedron around it. Two are bonding with hydrogen atoms, and two are nonbonding lone pairs. This geometry and the high electronegativity of oxygen creates a net dipole moment, with partial negative charge on oxygen and partial positive charges on the hydrogens. Hydrogen Bonding: The dipole moment of water enables it to act as both a hydrogen bond donor and acceptor, resulting in up to four H-bonds per molecule. Consequences of H-bonding: These interactions are responsible for water's anomalously high boiling and melting points, and its high surface tension. While hydrogen bonds are weak individually (20 kJ/mol) compared to covalent bonds (420 kJ/mol), they are critical in bulk water. Hydrogen Bonding in Ice: Ice forms a regular hexagonal lattice, with each water molecule forming four hydrogen bonds. Hexagonal ice has lower density than liquid water; ice floats. This regular structure gives ice low entropy. Other Hydrogen Bonds: Hydrogen bonds can occur between other molecules (or parts of molecules), particularly between electronegative atoms like nitrogen and oxygen. Ideal hydrogen bonds are linear to maximize electrostatic interaction. II. Water as a Solvent & The Hydrophobic Effect Solvent Properties: Water is a polar solvent, effectively dissolving charged and polar substances that can form hydrogen bonds with water, i.e. "substances that can form H-bonds to water are more soluble in water than substances that do not." Hydrophilic vs. Hydrophobic: Hydrophilic substances are "water-loving" and dissolve easily in water. Hydrophobic substances are "water-fearing" and are nonpolar molecules. Nonpolar substances, like gases, aromatic moieties, and aliphatic chains, are poorly soluble in water. Why Oil and Water Don't Mix: Bulk water has a high entropy (disordered state). Water near a hydrophobic solute becomes highly ordered, which has lower entropy and is thermodynamically unfavorable. The Hydrophobic Effect: This is not a direct attractive force, but rather an association of nonpolar molecules in water due to the system’s drive to increase its entropy by minimizing ordered water molecules around nonpolar substances. "Non-polar portions of the amphipathic molecule aggregate so that fewer water molecules are ordered. The released water molecules will be more random and the entropy increases." Importance: The hydrophobic effect drives protein folding, protein-protein association, formation of lipid micelles, and binding of steroid hormones to their receptors. It also explains the binding of many drugs to hydrophobic sites in proteins. "Many drugs are designed to take advantage of the hydrophobic effect." III. Van der Waals Interactions Description: Van der Waals interactions are weak, non-covalent interactions occurring between all atoms, regardless of polarity. Components: They consist of an attractive force (London dispersion) and a repulsive force (steric repulsion). ​ Attractive Force: Arises from temporary dipoles caused by random electron fluctuations. ​ Repulsive Force: Prevents atoms from getting too close. Distance Dependence: Attraction dominates at longer distances (0.4-0.7 nm); repulsion dominates at short distances. The optimal distance is the van der Waals contact distance. Significance: These interactions determine steric complementarity, stabilize biological macromolecules (like DNA base stacking), and facilitate the binding of polarizable ligands. Weakness and Reversibility: Individually, they are weak and easily broken, allowing for reversible interactions. IV. Summary of Non-Covalent Interactions The main non-covalent interactions are: ​ Hydrogen bonds  5 ​ Ionic (Coulombic) interactions: electrostatic interactions between charged species or between ions and permanent dipoles ​ The Hydrophobic effect ​ Van der Waals Interactions These interactions are weak individually, but their cumulative effect is significant in biological processes such as protein structure and ligand binding. V. Ionization of Water Process: Water dissociates into H+ (proton) and OH- (hydroxide) ions in a rapid, reversible process, described as H2O ⇌ H+ + OH-. The equilibrium strongly favors undissociated water. Proton Hopping: Protons move rapidly through water via "hops" between hydrogen-bonded water molecules, which facilitates rapid acid-base reactions in aqueous solutions, much faster than diffusion of other ions. "Short “hops” of protons between a series of hydrogen-bonded water molecules result in an extremely rapid net movement of a proton over a long distance". Quantitative Treatment: The equilibrium constant (Keq) for water ionization is 1.8 x 10^-16 M. The ionic product of water (Kw) is [H+][OH-] = 1.0 x 10^-14 M^2 at 25°C, and this value remains constant. ​ In pure water [H+] = [OH-] = 10^-7 M. VI. pH Scale Definition: pH is the negative logarithm of the hydrogen ion concentration (pH = -log[H+]). Relationship to pOH: pH and pOH always add up to 14 (pH + pOH = 14) Neutral Solution: In a neutral solution, [H+] = [OH-], and the pH is 7. Logarithmic Scale: pH is a logarithmic, not arithmetic, scale; a change of 1 pH unit represents a 10-fold change in [H+]. Biological Relevance: pH significantly affects enzyme activity, such as pepsin's preference for acidic conditions and trypsin's preference for neutral pH, and a range of other biological processes. VIII. Weak Acids and Bases Ionization: Weak acids and bases do not fully ionize in water. Conjugate Pairs: A conjugate acid-base pair consists of a proton donor and its corresponding proton acceptor. For example, acetic acid (CH3COOH) is the proton donor, and its conjugate base, acetate (CH3COO-), is the proton acceptor. Acid Dissociation Constant (Ka): Ka quantifies a molecule’s tendency to lose a proton. "Ka = [H+][conjugate base] [conjugate acid]" pKa: pKa = -log Ka. A lower pKa indicates a stronger acid and is a measure of a molecule’s propensity to lose H+. "pKa quantifies a molecule’s propensity to lose a H+" Titration Curves: Titration curves are used to determine the pKa of a weak acid. At the midpoint of the curve, pH = pKa. IX. Biological Buffer Systems Importance: Maintenance of intracellular pH is important for enzyme activity, solubility of polar molecules, and equilibrium of reactions. Buffers: Buffers resist changes in pH when small amounts of acid or base are added, consisting of a mixture of a weak acid and its conjugate base. Buffering Capacity: Buffering capacity is greatest at pH = pKa where there is a 50:50 mixture of the acid and its anion form, and is lost when pH differs from pKa by more than 1 pH unit. Biological Buffers: Important biological buffers include phosphate (pKa = 6.86), bicarbonate, and histidine. "Buffer systems in vivo are mainly based on – phosphate, concentration in millimolar range, pKa = 6.86 – bicarbonate, important for blood plasma – histidine, efficient buffer at neutral pH" In Vitro Buffers: In vitro buffers are often based on sulfonic acids of cyclic amines like HEPES, PIPES, and CHES. Henderson-Hasselbalch Equation: The equation relates pH, pKa, and buffer concentration and is useful for understanding buffer behavior: pH = pKa + log ([A-]/[HA]). Glossary  6 Hydrogen Bond: A relatively weak electrostatic attraction between a hydrogen atom covalently linked to an electronegative atom (such as oxygen or nitrogen) and another electronegative atom. Dipole Moment: A measure of the separation of positive and negative charges in a molecule. Electronegativity: The ability of an atom to attract electrons in a chemical bond. Hydrophobic: "Water-fearing"; refers to nonpolar molecules that do not readily dissolve in water. Hydrophilic: "Water-loving"; refers to polar or charged molecules that readily dissolve in water. Amphipathic: A molecule that contains both polar (or charged) and nonpolar regions. Hydrophobic Effect: The phenomenon where nonpolar molecules aggregate in water to minimize disruption of water's structure and increase entropy of the water molecules. Van der Waals Interactions: Weak, short-range attractive or repulsive forces between atoms, including London dispersion forces and steric repulsion. London Dispersion Forces: Temporary, induced dipole-dipole forces caused by random fluctuations in electron distribution around an atom. Steric Repulsion: A repulsive force that arises when atoms approach each other too closely, due to electron cloud overlap. Ion Product of Water (Kw): The product of the concentrations of hydrogen ions (H+) and hydroxide ions (OH-) in water at equilibrium, Kw = [H+][OH-]. pH: A measure of the acidity or alkalinity of a solution, defined as the negative logarithm of the hydrogen ion concentration (pH = -log[H+]). pKa: A measure of the acidity of a weak acid, defined as the negative logarithm of the acid dissociation constant (Ka), where a lower pKa indicates a stronger acid. Acid Dissociation Constant (Ka): An equilibrium constant that indicates the strength of a weak acid. Buffer: A solution that resists changes in pH upon the addition of small amounts of acid or base, consisting of a mixture of a weak acid and its conjugate base. Conjugate Acid-Base Pair: A pair of molecules or ions that are related by the gain or loss of a proton (H+). Henderson-Hasselbalch Equation: An equation relating the pH, pKa, and the ratio of concentrations of the conjugate base and acid of a buffer solution, pH = pKa + log([A-]/[HA]). Proton Hopping: The rapid movement of protons through a network of hydrogen-bonded water molecules, facilitating fast proton transfer in water. Study Guide 1. Describe the structure of a water molecule and explain how its geometry and electronegativity lead to its unique properties. Water molecules have a distorted tetrahedral shape with four electron pairs around the oxygen atom. The high electronegativity of oxygen creates a dipole moment, with partial negative charges on the oxygen and partial positive charges on the hydrogens, making the molecule polar. 2. Explain how hydrogen bonds contribute to water’s high boiling point and surface tension. Hydrogen bonds, which form between the partial positive charge on a hydrogen atom of one water molecule and the partial negative charge on the oxygen atom of another, are relatively strong intermolecular attractions. These interactions must be overcome for water to boil or display surface tension. 3. Why is ice less dense than liquid water? In ice, water molecules form a regular hexagonal lattice structure with hydrogen bonds. This structure is more open than the arrangement of water molecules in liquid water, leading to a lower density. 4. What is the hydrophobic effect and how does it influence the behavior of amphipathic molecules in water? The hydrophobic effect is the tendency of nonpolar molecules to aggregate in water. Amphipathic molecules, which have both polar and nonpolar regions, arrange themselves so that the nonpolar portions cluster together to minimize disruption of water's structure. 5. Briefly describe Van der Waals interactions and their two components.  7 Van der Waals interactions are weak, short-range forces that occur between all atoms. They include an attractive force (London dispersion) caused by temporary dipoles, and a repulsive force (steric repulsion) based on atomic size. 6. Explain the concept of proton hopping in water. Proton hopping is the rapid movement of protons through a network of hydrogen-bonded water molecules, rather than individual protons traveling long distances. It facilitates the rapid transfer of protons through solution. 7. What is the ion product of water (Kw), and what does it indicate about the concentrations of H+ and OH- ions in pure water? The ion product of water (Kw) is the product of the concentrations of H+ and OH- ions in water at equilibrium, equal to 1.0 x 10^-14 M^2 at 25°C. In pure water, [H+] = [OH-] = 10^-7 M. 8. What is the pH scale and how does it relate to the concentration of hydrogen ions? The pH scale is a logarithmic scale used to measure the acidity or alkalinity of a solution. It is defined as the negative logarithm of the hydrogen ion concentration, and each pH unit represents a tenfold difference in [H+]. 9. Describe the concept of pKa and its relationship to the strength of a weak acid pKa is the negative logarithm of the acid dissociation constant (Ka). A lower pKa indicates a stronger acid, meaning it more readily loses its proton. 10. Explain how a buffer system works, and identify the optimal buffering capacity of such a system. A buffer system is a mixture of a weak acid and its conjugate base. It resists changes in pH by absorbing added H+ or OH-. The optimal buffering capacity occurs when the pH of the solution is equal to the pKa of the buffer. Essay Questions 1. Discuss the unique properties of water that make it essential for life, focusing on how its molecular structure and hydrogen bonding contribute to these properties. 2. Explain the hydrophobic effect and its significance in biological systems. How does this effect influence the structure and function of proteins, cell membranes, and other biological molecules? 3. Compare and contrast the different types of non-covalent interactions, and discuss their individual and collective roles in biological systems. 4. Describe how the concept of pH is essential to understanding biological systems, and provide an explanation of the concept of buffering and how biological systems are able to maintain a stable pH. 5. Explain the relationship between pKa, buffer capacity, and the Henderson-Hasselbalch equation and discuss how this equation can be used to solve problems related to buffer solutions.  8 1/21 Amino Acids, Peptides, and Proteins Briefing Document: 1. Key Concepts and Themes: Carbon's Versatility: The unique role of carbon in forming diverse biological molecules. Its capacity for single, double, and triple bonds, along with its ability to bond with various elements allows for the creation of molecules with different shapes, sizes, and compositions. No other element forms molecules of such different shapes, sizes, and compositions Chirality: A carbon atom with four different substituents creates two stereoisomers with identical chemical but different physical and biological properties. Structural nuance has a significant biological impact. Amino Acids as Protein Building Blocks: Amino acids are defined as the basic building blocks of proteins, possessing properties well-suited for biological roles: ​ Capacity to polymerize (form chains) ​ Useful acid-base properties. ​ Varied physical and chemical properties. Amino Acid Structure and Classification: The common structure of amino acids, with an alpha-carbon, an amino group, a carboxyl group, and a unique R-group. Amino acids are further classified into five groups based on their R-group properties: ​ Nonpolar, aliphatic ​ Aromatic ​ Polar, uncharged ​ Positively charged ​ Negatively charged Spectroscopic Detection: The aromatic amino acids (tryptophan and tyrosine in particular) absorb UV light, which is a method used to quantify and characterize these molecules. "Tryptophan and tyrosine are the strongest chromophores". Ionization and Buffering: Amino acids and peptides can act as buffers due to the presence of ionizable groups (amino and carboxyl). The pKa values of these groups dictate the molecule's net charge at different pH levels. Isoelectric Point (pI): where the net charge is zero. The point at which an amino acid has minimal solubility and will not migrate in an electric field. The calculation of pI is explained for amino acids with and without ionizable side chains. Peptide Formation and Structure: Peptides are formed via condensation reactions linking amino acids with peptide (amide) bonds. The numbering of a peptide starts at the amino terminus (N-term). Different functions can be associated with peptides, from hormones and pheromones to antibiotics and toxins. Proteins and Their Complexity: Proteins are composed of polypeptide chains, and they can further include cofactors, coenzymes, prosthetic groups, and other post-translational modifications. There are levels of structure in proteins, and the sequence defines the structure and thus function of the protein. Protein Purification and Analysis: Techniques for separating and analyzing proteins including various types of chromatography (size-exclusion, ion-exchange, and affinity), and gel electrophoresis, specifically SDS-PAGE (used to determine molecular weight). Protein Sequencing: The process of determining the amino acid sequence is discussed as a way to understand protein function and evolutionary relationships. Methods for protein sequencing, like Edman degradation, are touched on. The idea that protein sequences define protein structures and function, and that similar proteins from different species have similar sequences is emphasized: "Different functions = different sequences." Evolutionary Relationships through Protein Sequences: Using protein sequence comparisons to determine how closely related organisms are. "If two organisms are closely related, the sequences of their proteins should be similar" 2. Key Ideas: Beers Law: A = ε·c·l. Used for calculating concentration of a substance. Amino Acid R-Group Diversity: The variation in amino acid R-groups contributes to the varied chemical and physical properties and functional capabilities of proteins. Post-translational Modifications: Modifications after translation like phosphorylation are critical in protein regulation and signaling, and these modifications can be reversible.  9 Peptide Bonds: Form via a condensation reaction (removal of water) resulting in a substituted amide linkage. Nomenclature: Peptide sequences are written from the N-terminus to the C-terminus. Standard abbreviations, both three-letter and one-letter codes, are used for amino acids. Protein Size: Proteins can vary greatly in size and complexity; "PKZILLA-1 & 2" are mentioned as very large examples. The average amino acid in a protein is approximately 110 Daltons. SDS-PAGE: SDS gives proteins a uniformly negative charge, allowing separation based solely on size. Specific Activity: Increases during purification as non-target proteins are removed. Mutations and Disease: Mutations in proteins can alter their sequence and lead to disease, with many diseases caused by a single amino acid change. Glossary Amino Acid: An organic molecule containing both an amino group (-NH2) and a carboxyl group (-COOH), the building blocks of proteins. Alpha (α) Carbon: The central carbon atom in an amino acid to which an amino group, carboxyl group, a hydrogen atom, and a distinctive R-group are attached. R-group: Also known as the side chain, this is the variable group that makes each of the 20 common amino acids unique and dictates their chemical properties. Chiral Molecule: A molecule that is not superimposable on its mirror image, often because it has an asymmetric carbon atom with four different substituents attached. Stereoisomer: Molecules with the same chemical formula and bonds, but with different three-dimensional arrangements of atoms, resulting in different physical and biological properties. Beer's Law: A law stating that the absorbance of a solution is directly proportional to the concentration of the substance and the path length of the light through the solution. (A= εcl) Molar Extinction Coefficient (ε): A constant that is a measure of how strongly a chemical species absorbs light at a given wavelength. Chromophore: A part of a molecule that absorbs light, typically causing the substance to have color. Isoelectric Point (pI): The pH at which a molecule has no net electric charge. Peptide Bond: The amide bond formed between two amino acids during protein synthesis. Condensation Reaction: A reaction in which two molecules are joined together with the elimination of a small molecule such as water. Residue: An amino acid unit within a peptide or protein chain. N-terminus: The end of a polypeptide chain that has a free amino group (-NH2). C-terminus: The end of a polypeptide chain that has a free carboxyl group (-COOH). Cofactor: A general term for functional non-amino acid component such as metal ions or organic molecules. Coenzyme: An organic cofactor used to designate an organic molecule, e.g. NAD+ in lactate dehydrogenase. Prosthetic Group: A cofactor that is covalently attached to a protein, e.g. Heme in myoglobin. Chromatography: A technique used to separate mixtures of substances, often proteins, based on differences in their physical and chemical properties. Electrophoresis: A technique used to separate molecules, such as proteins, by their rate of movement in an electric field. SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis; a common method to separate proteins based on size, by binding them to a charged detergent. Edman Degradation: A method for sequencing amino acids in a peptide, removing one amino acid at a time from the N-terminus. Phylogenetic Tree: A diagram that represents the evolutionary relationships among species. Study Guide 1. What are the four key properties of amino acids that make them suitable for a variety of biological functions? Amino acids have the capacity to polymerize, have useful acid-base properties, have varied physical properties, and possess varied chemical functionality. These properties allow them to be used in diverse biological roles within proteins and peptides.  10 2. How do biochemists name amino acids, and how does this differ from standard organic nomenclature? Biochemists start naming amino acids from the α-carbon and move down the R-group, while organic nomenclature starts from one end of the molecule. This biochemical designation method is useful for understanding the structure and properties of amino acids in the context of proteins and peptides. 3. Describe the five basic classifications of amino acids based on their R-group properties and give an example of an amino acid in each class. The five classifications are: nonpolar, aliphatic (e.g., alanine); aromatic (e.g., phenylalanine); polar, uncharged (e.g., serine); positively charged (e.g., lysine); and negatively charged (e.g., aspartate). Each classification is based on the chemical properties of the R-group, which dictate the interactions the amino acids can make in biological systems. 4. Explain how Beer's Law can be used to quantify the concentration of a substance, and list the variables in the equation A = εcl. Beer's Law (A = εcl) is used to quantify the concentration of a substance by measuring its light absorbance (A). The molar extinction coefficient (ε), concentration (c), and the path length of the sample (l) must be known to calculate the concentration of a compound in a solution. 5. How do aromatic amino acids differ in their light absorbance in the UV region, and which are the strongest chromophores? Aromatic amino acids absorb light in the UV region, with tryptophan and tyrosine being the strongest chromophores due to their aromatic rings. This property is used to detect and quantify these amino acids within proteins or peptides. 6. Explain how the isoelectric point (pI) is determined for amino acids with and without ionizable side chains, and why it is significant. The isoelectric point (pI) is the pH at which a molecule has no net electric charge. For amino acids without ionizable side chains, pI is the average of the pKa values of the α-carboxyl and α-amino groups; for amino acids with ionizable side chains, it's the average of the two pKa's that include molecules with a net charge of zero. The pI is important because at this pH the molecule is least soluble and does not migrate in an electric field. 7. Describe the process of peptide bond formation, including the type of reaction and the byproduct of the reaction. A peptide bond is formed through a condensation reaction (removal of water) between the carboxyl group of one amino acid and the amino group of another. This substituted amide linkage joins the amino acids together forming a peptide. 8. List three different functions that peptides can perform within biological systems. Peptides can act as hormones and pheromones (e.g., insulin), neuropeptides (e.g., substance P), and antibiotics (e.g., polymyxin B). Their diverse functionalities highlight their importance in various biological processes, from signaling to defense. 9. Explain how SDS-PAGE electrophoresis separates proteins and describe the purpose of using a detergent like SDS in the process. SDS-PAGE separates proteins based on their size, in part because SDS binds to and unfolds all proteins and gives them a uniformly negative charge. After this unfolding, the rate of movement depends on size, allowing smaller proteins to move faster. 10. What is the significance of protein sequence and how can it be used to understand evolutionary relationships between species? Protein sequence is crucial because it defines the protein’s structure, which ultimately determines its function. Comparing sequences across species reveals evolutionary relationships, with similar sequences indicating a closer relationship. Essay Questions  11 1. Discuss the importance of amino acid R-group properties in determining the overall structure and function of proteins. Consider both individual amino acid characteristics and the cumulative effects of different types of amino acids within a polypeptide. 2. Describe how the concept of isoelectric point (pI) can be used to separate and analyze amino acids and proteins. Provide examples of how the pI can be experimentally determined and how it can influence protein behavior. 3. Explain how different methods of protein separation can be used in conjunction to purify a protein from a complex cellular extract. Discuss the chemical and physical principles that underpin each of these methods, and propose a logical order to execute a protein purification protocol. 4. How do post-translational modifications of proteins contribute to the diversity and function of a proteome? Provide specific examples of such modifications and explain their biological roles. 5. Describe how protein sequence can be used as a molecular clock to understand evolutionary relationships and provide a detailed analysis of how protein sequence differences can lead to changes in protein function and potentially human disease.  12 1/23 Protein Structure, Function, and Folding (Part 1) Briefing Document: Structure Dictates Function: A protein's function is directly dependent on its specific three-dimensional structure. Proteins adopt a unique 3D conformation in an aqueous solution. ​ The "native fold" is the specific, stable structure a protein assumes to perform its biological function. ​ "Most proteins exist in one or a small number of stable structural forms." Non-Covalent Interactions Stabilize Protein Structure: Protein folding is driven by favorable interactions within the protein that overcome the conformational entropy cost of forming a specific native fold. ​ Key non-covalent interactions that stabilize protein structure: ○​ Hydrophobic Effect: "Release of water molecules from the structured solvation layer around the molecule as protein folds increases the net entropy" and "hydrophobic amino acids often buried in protein interior." This effect is a major driving force for protein folding. ○​ Hydrogen Bonds: Interactions between N-H and C=O groups of the peptide bond lead to regular structures like alpha-helices and beta-sheets. ○​ Van der Waals Interactions: "Medium-range weak attraction between all atoms contributes significantly to the stability in the interior of the protein." ○​ Electrostatic Interactions: "Long-range strong interactions between permanently charged groups," especially "salt-bridges…buried in the hydrophobic environment strongly stabilize the protein." Structural Hierarchy: Protein structure is organized hierarchically, starting from the primary sequence of amino acids to more complex folding patterns. ​ Secondary Structure: Local spatial arrangements of the polypeptide chain, with the alpha-helix and beta-sheet being the two primary forms. ​ Alpha-Helix: A helical backbone held together by hydrogen bonds within the chain. Described as "Right-handed helix with 3.6 residues (5.4 Å) per turn." and with a diameter of "10 – 12 Å" including the side chains. Certain amino acids are strong helix formers such as "Ala and Leu" while "Pro acts as a helix breaker and Gly acts as a helix breaker" ​ Beta-Sheet: A more extended, sheet-like arrangement held together by hydrogen bonds between strands. Strands can be "parallel or antiparallel" and "Amino acids forming neighboring strands can be located far away in protein amino acid sequence." ​ Beta-Turns: The 180 degree turn is made over four amino acids, often stabilized by hydrogen bonds. "Proline in position 2 or glycine in position 3 are common in b-turns". The Peptide Bond: ​ The peptide bond is rigid and planar, with partial double bond character. ​ Rotation is possible around bonds connecting the alpha-carbon to the nitrogen (phi, denoted as "f") and to the carbonyl carbon (psi, denoted as "y"). ​ dihedral angles (torsion angles): ○​ f (phi): angle around the X-carbon—amide nitrogen bond ○​ y (psi): angle around the X-carbon—carbonyl carbon bond ​ The Ramachandran plot illustrates the allowed and disallowed combinations of phi and psi angles, dictating the possible conformations of the polypeptide backbone. "Ramachandran plot shows the distribution of f and y dihedral angles that are found in a protein" and "shows the common secondary structure elements". Categorizes Proteins into tertiary structures: ​ Fibrous proteins are typically insoluble; made from a single secondary structure ​ Globular proteins are water-soluble globular proteins ​ Other types include lipid-soluble membranous proteins and Disordered proteins / domains Key Facts: Amino Acid Sequence is Determinative: The amino acid sequence of a protein dictates its final 3D structure. Protein Structures are not Static: While proteins adopt specific stable conformations, it is mentioned that these structures are not static, which implies that they can undergo conformational changes.  13 The Ramachandran Plot: This plot provides a crucial tool for understanding protein structure, revealing the favorable and unfavorable backbone conformations. Helix Stability: Not all polypeptide sequences readily form alpha-helices. Hydrophobic residues such as Ala and Leu are strong helix formers, while Pro and Gly act as helix breakers due to conformational restrictions. Glossary Amino Acid Sequence: The linear order of amino acids in a polypeptide chain, which determines the protein's primary structure and ultimately, its 3D structure. Dihedral Angles (φ and ψ): Torsion angles that define the conformation of the polypeptide backbone. Phi (φ) measures rotation around the alpha-carbon—amide nitrogen bond, and psi (ψ) measures rotation around the alpha-carbon—carbonyl carbon bond. Electrostatic Interactions: Long-range strong interactions between permanently charged groups, often forming salt bridges. Favorable Interactions: Noncovalent interactions, including hydrogen bonds, Van der Waals interactions, hydrophobic effects, and electrostatic interactions, that contribute to protein stability. Fibrous Proteins: Proteins characterized by their elongated shape and insolubility, typically composed of a single type of secondary structure. Globular Proteins: Water-soluble proteins that adopt a compact, roughly spherical shape. Hydrophobic Effect: The tendency of nonpolar molecules to cluster together in an aqueous environment, a key factor in protein folding. Hydrogen Bonds: Weak interactions between hydrogen atoms and electronegative atoms such as oxygen or nitrogen. Native Fold: The specific, stable 3D conformation of a protein in its functional state. Peptide Bond: A covalent bond formed between the carboxyl group of one amino acid and the amino group of another amino acid. Ramachandran Plot: A graph that shows the distribution of phi and psi dihedral angles that are found in a protein. Secondary Structure: Local spatial arrangement of a polypeptide chain, commonly either alpha helices or beta sheets. Tertiary Structure: The overall 3D arrangement of atoms in a polypeptide chain. Van der Waals Interactions: Weak, short-range attractions between all atoms, which contribute significantly to the stability in the interior of the protein. Study Guide Quiz 1. What is the primary determinant of a protein's 3D structure? The primary determinant of a protein's 3D structure is its amino acid sequence. This sequence dictates how the polypeptide chain will fold and interact with itself and its environment, leading to a unique three-dimensional shape. 2. Describe the role of the hydrophobic effect in protein folding. The hydrophobic effect is a significant driver of protein folding. As a protein folds, hydrophobic amino acids move to the interior, away from water. This release of water molecules from around these amino acids increases the net entropy of the system. 3. What are the two key dihedral angles in a peptide bond, and what do they measure? The two key dihedral angles in a peptide bond are phi (φ) and psi (ψ). Phi measures the rotation around the alpha-carbon—amide nitrogen bond, while psi measures the rotation around the alpha-carbon—carbonyl carbon bond. 4. What is a Ramachandran plot, and what information does it provide?  14 A Ramachandran plot is a graph that shows the distribution of phi and psi dihedral angles found in a protein structure. It reveals which combinations of angles are favored and allowed, highlighting common secondary structure elements and unusual backbone structures. 5. What are the two common types of secondary structures in proteins? The two common types of secondary structures in proteins are the alpha helix and the beta sheet. Both are stabilized by hydrogen bonding patterns between the backbone atoms of the polypeptide chain. 6. Describe the key features of an alpha helix, including the type of bonding. An alpha helix is a spiral structure held together by hydrogen bonds between the backbone amides and carbonyl oxygen groups four amino acids away. It typically is a right-handed helix with 3.6 residues per turn, with sidechains projecting outward. 7. How does the structure of a beta sheet differ from an alpha helix? A beta sheet is a more extended, sheet-like structure in comparison to an alpha helix. It is formed by hydrogen bonds between backbone amides and carbonyl oxygens of adjacent strands of the polypeptide chain. 8. What are beta turns, and what is their function? Beta turns are short, 180-degree turns that frequently occur in proteins. They are often found connecting strands in beta sheets and are typically stabilized by hydrogen bonds with a proline or glycine. 9. What are the major classes of protein tertiary structures? The major classes of protein tertiary structures are fibrous proteins, globular proteins, lipid-soluble membrane proteins, and disordered proteins/domains. Fibrous proteins are typically insoluble, globular proteins are water-soluble, and membrane proteins are lipid-soluble. 10. Briefly explain how noncovalent interactions contribute to the stability of proteins. Noncovalent interactions such as hydrogen bonds, Van der Waals interactions, hydrophobic effects, and electrostatic interactions all work in concert to stabilize protein structure. Individually they are weak, but when they act collectively, they provide a strong stabilizing force. Essay Questions 1. Discuss the relationship between a protein's amino acid sequence, its 3D structure, and its biological function. Explain how changes in the amino acid sequence can affect both structure and function. 2. Describe the different types of noncovalent interactions that stabilize protein structure. Explain how each type of interaction contributes to the overall stability and proper folding of a protein. 3. Compare and contrast the structures of alpha helices and beta sheets. Discuss the types of amino acids that favor each secondary structure and how these structures are organized within the three-dimensional architecture of a protein. 4. Discuss the importance of the Ramachandran plot in understanding protein structure. Explain how this tool helps to identify favorable and unfavorable combinations of dihedral angles, and its usefulness in the analysis of newly discovered protein structures. 5. Explore the various classes of protein tertiary structure (fibrous, globular, membrane, and disordered). Explain the properties of each class, along with examples of proteins that belong to each class and their biological functions.