Exam 1 Cell Biology Study Guide

Cellular reactions with positive DG˚' can occur because

2. Input of energy; Coupled reactions • Endergonic reactions (+DG) are coupled to exergonic reactions (-DG) • In coupled reactions, product of 1st reaction is often a reactant or substrate in the 2nd reaction

Dissociation constants of binding reactions reflect affinity of interacting molecules

Dissociation constant (Kd): The reciprocal of the equilibrium constant (ie. Kd=[reactants]/[products]).

Four Broad Structural Categories of Proteins

*Globular proteins - generally water-soluble, compactly folded structure *Fiborous proteins - large, elongated, often stiff molecules *Integral membrane proteins - embedded within the phospholipid bilayer of membranes *Intrinsically disordered proteins - No well-ordered structure in their native conformation. Rather, polypeptide chains are flexible with no fixed conformation. • Few to no hydrophobic residues • Interact with multiple partner proteins and only fold into defined conformations through such interactions.

Phosphorylation and dephosphorylation covalently regulate protein activity

*Protein Kinases: • Transfer terminal phosphate group from ATP to specific residues of target proteins. • Conformational change induced by negative charge of phosphate • May activate or inactivate protein function • Most kinases have multiple target proteins. • Protein Phosphatases: • Hydrolyze phosphate group off protein • Similarly, may be activating or inactivating

Organelles of the Endomembrane System

- Endoplasmic reticulum (ER) system of interconnected flattened sacs, cisternae - continuous with outer nuclear membrane. - Synthesize: Lipids, fatty acids, membrane bound proteins and proteins that will be exported from cell. - Smooth versus Rough ER - absence vs presence of ribosomes -Rough ER - produces proteins found in the endomembrane system Smooth ER - produces lipids that comprise the cell membrane system. And serves as a detoxification station for the cell. -Golgi apparatus - Cisternal stacks that are discontinuous from ER. - Receives proteins and lipids from ER. - Processes, modifies, packages and transports these molecules to various intracellular destinations. -Endosomes and Secretory Vesicles - Small vesicles that transport between organelles - Drive endocytosis for ingesting fluid and particles, and exocytosis for release of material from cell. - Endocytosed vesicles fuse with sorting stations called endosomes. - From endosomes these molecules may be recycled back to plasma membrane or delivered to lysosomes for degradation. -Lysosomes - Animal cell specific organelles - Contain diverse acid hydrolases: Enzymes that degrade a variety of biological molecules - these hydrolases function only at low pH, maintained by proton pumps that import H+ into lumen of lysosomes. - Perform autophagy: Digestion of aged, poorly functioning organelles - Recycles nutrient building blocks back into cytosol -Peroxisomes: Dense crystalline core of oxidase enzymes H2O2 then converted to water via the action of the enzyme catalyase - this critical reaction rids cells of the dangerous H2O2, and is used to degrade other toxins like alcohol and formaldehyde

Major Cellular Macromolecules

- The polymers and their monomers: 1. Protein - Amino acids - Give cells structure and perform most cellular tasks. Molecular polymer: a chain of repeated identical or highly similar subunits joined by covalent bonds. 2) Nucleic acids (RNA and DNA) - Nucleotides - Carry coded information for making proteins at the right time and place 3) Carbohydrates - simple sugars - Provide structural support, energy storage and the source of many small molecules.

Adenosine Triphosphate (ATP)

- a universally conserved energy rich molecule is produced by conserved mechanisms and is hydrolyzed to release energy that drives many cellular activities.

Features of Eukaryotic cells

- has a complex and highly dynamic cytoskeleton -• Mechanical strength • Controls cell shape • Organizes cytoplasm • Drives and guides movement, both of the cell, as well as molecules and organelles internally. -Microtubule based cilia and flagella extend from plasma membrane and provide motility and generate forces that can circulate extracellular fluids. DNA organized into chromatin: long linear polymers associated with histone proteins (shared by Archaea). • Nuclear envelope: Composed of two lipid membranes (outer and inner) that are continuous with one another. • Membranes fused at nuclear pore complexes, sites where molecular transport into and out of nucleus is regulated. • Rigid shape of nucleus maintained by internal network of intermediate filaments called lamins, generating the nuclear lamina.

deoxyribonuleic acid

-(DNA) - All life carries it's genetic info in DNA -within DNA, discrete units called genes encode the proteins that perform or direct most cellular activities

pH and temperature dependence of enzyme activity

-Acid-base catalysis is pH dependent: • Catalysis requires a particular ionization state of one or more amino acid side chains in the catalytic site. • For example, the pKa of histidine is 6.8, at or below this histidine is protonated and cannot activate serine in the active site of chymotrypsin. • Other proteases, like lysosomal hydrolases, have evolved sequences that maintain catalytic activity at low pH (like that in lysosomes).

Types of Prokaryotic cells

1. Domain Archaea: Methanogens, Halophiles, Acidophiles, Thermophiles 2. Domain Bacteria • Includes the smallest known cell - the mycoplasma bacteria. • As well as cyanobacteria - photosynthetic bacteria.

Enzymes as Biological Catalysts Overcoming the Activation Energy Barrier

-Activation energy (ΔG‡) is required for any chemical reaction. EA is a barrier that inhibits formation of a thermodynamically unstable intermediate, the transition state. -Enzymes decrease ΔG‡ by binding more tightly to the transition state than to the substrate or product, thereby stabilizing this intermediate.

Dissociation constants of binding reactions reflect affinity of interacting molecules

-Macromolecules can have distinct binding sites for multiple ligands.

Chemistry of Life

-Molecular complementarity: enables molecules with complementary shapes and chemical properties to form biomolecular interactions. -Polymerization: Small molecule building blocks form larger cellular structures and polymers such as DNA through either covalent or noncovalent associations. - Chemical equilibrium Chemical reactions are reversible. Ratio of products to reactants depends on the rate constants (k) of the forward and reverse reactions. Energy driving many cellular activities is derived from hydrolysis of high-energy phosphoanhydride bonds in Adenosine Triphosphate (ATP).

Primary culture and cell strains have a finite lifespan

-Rare cells in culture will escape senescence by acquiring oncogenic mutations that maintain telomere length. Such cells are said to be transformed and have played powerful roles in study of cell function. -Cultured cells with indefinite life spans are called cell lines. -The first human cell line, in use since 1952, is named HeLa derived from the cervical cancer of a Baltimore woman Henrietta Lacks - the history and ethics of this line is a powerful tale of abuse of authority and civil rights violations.

chemical reactions in cells are at a steady state

-Steady state: Prevents buildup of toxic intermediates, generated in normal pathways. Because these intermediates are immediately consumed in other reactions, their concentrations are maintained at relatively low levels, which cause no deleterious effects. -Importantly, establishing and maintaining steady state is the principle means of driving chemical reactions in cells - it establishes concentrations of reactants and products that promote only a certain direction of reaction.

Mechanisms of Enzyme Catalysis

-Substrate Orientation: Multiple substrates brought together in correct orientation to catalyze reaction. -Changing Substrate Reactivity: substrate influenced by amino acid side chains at active site that alter chemical properties (e.g., charge) of substrate. -Inducing Strain in the Substrate: enzyme changes conformation of substrate to bring closer to conformation of transition state.

Non-Polymer macromolecules

1) Lipids - structural (cell membranes) or energy source (fatty acids) • Include phospholipids: the conserved building blocks of all cellular membranes. • Serve as building materials for other molecules. • Play important roles in cell signaling • The non-phospholipid cholesterol alters membrane dynamics and is the source of all steroid hormones

The Cell Theory

1. All organisms are composed of one or more cell. 2. The cell is the structural unit of life. 3. Cells arise from pre-existing cells by division. -articulated in the mid 1800's

four major types of noncovalent bonds

1. Ionic bonds - attractions between fully charged atoms: • Weakened in the presence of water (hydration shell) • As well as the concentration of other ions in solution • As we'll see, regulating concentration of cytosolic ions modifies many cellular activities • Ionic interactions result from attraction between fully charged ions cations (+) and anions (-). • Much weaker than covalent bonds, but contribute greatly to shape of most biological molecules 2. Hydrogen bonds - occur when covalently bound hydrogen has a partial positive charge and attracts electrons of a second atom (usually oxygen) that has a partial negative charge. • H-bonds determine the structure and properties of water. • H-bonds in biological molecules promote conformation: • protein structure • annealing strands of DNA • Interaction between two molecules 3. Van der Waals Interactions - Attractions between nonpolar molecules, due to transient dipole formation. - Electron density may temporarily shift to one side of the nucleus. - This generates a transient charge to which a nearby atom can be either attracted or repelled - Attractive bond energy: 0.1 to 0.3 kcal/mol 4. Hydrophobic Interaction - occurs when nonpolar molecules associate, minimizing their exposure to polar molecules. Plays an important role in determining the final shape (conformation) of proteins.

model organisms

A non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the model will provide general insight into the workings of other organisms.

5 different bases used to build nucleic acids

Bases are purines or pyrimidines. • Purines: Adenine and guanine, have a double ring. Found in both DNA and RNA. • Pyrimidines: Single ring. Cytosine and thymine in DNA. Uracil rather than thymine in RNA

Five Nucleotides used to Build Nucleic Acids

Basic structure of a nucleotide: Each consists of three parts: - A five-carbon sugar - A nitrogenous base on the 1' carbon - A phosphate group on the 5' carbon - May be mono-, di- or tri-phosphate - They are acidic and at physiological pH - the phosphate group is deprotonated 1. Nucleosides: A sugar (ribose or deoxyribose) covalently attached to a nitrogenous base. Important roles as building blocks of other organic molecules. 2. Nucleotides: A sugar (ribose or deoxyribose) covalently attached to a nitrogenous base and one to three phosphate groups. Polymerize to form DNA and RNA and play important roles in energy transduction.

Carbohydrates

Carbohydrates include simple sugars and sugar polymers. Principle roles: Energy storage and structural molecules. • Ketones: carbonyl on an internal carbon (ketose) • Aldehydes: Carbonyl on a terminal carbon (aldose). • Glycosidic bonds are -C—O—C- links between sugar -OH groups. • Disaccharides a source of readily available energy (like lactose and sucrose) • Oligosaccharides (3-10 sugars): bound to cells surface proteins and lipids, and used for cell recognition, alter protein solubility and ability to interact with other proteins • Polysaccharides ( > 10 sugars): Energy storage and structural molecules. • Isomers: Molecules with same chemical formula, but different structure • Stereoisomers with a single different stereogenic center are called epimers • Additional stereoisomers generated from orientation of -OH and -H around the linkage Polysaccharides: identical sugar monomers but dramatically different properties Storage Polysaccharides: Polymers of sugars joined by glycosidic a(1-4) linkage with branches formed by a(1-6) linkages. • Glycogen: Animal product made of branched glucose polymers. • Starch: Plant product made of both branched and unbranched glucose polymers. - These glycosidic bonds can be readily hydrolyzed by animal enzymes, releasing sugar monomers as an energy source. Structural Polysaccharides: Polymers joined by b(1-4) glycosidic linkage • Cellulose: Principle plant structural polysaccharide • Chitin: found in the exoskeleton of invertebrates • Most animals lack digestive enzymes that can hydrolyze b glycosidic bonds. Therefore, such carbohydrates cannot be digested.

Chaperonin-mediated protein folding

Chaperonins: folding chambers into which all or part of an unfolded protein can be bound in an appropriate environment, giving it time to fold properly (aka Hsp60). • Multi-subunit chamber • ATP hydrolysis closes the lids providing an environment free of interacting molecules • Release of ATP opens chamber and allows folded protein to exit

Enzyme Inhibitors

Enzyme inhibitors slow the rates for enzymatic reactions. • Irreversible inhibitors bind tightly to the enzyme. • Reversible inhibitors bind loosely to the enzyme. - Competitive inhibitors bind enzyme active sites and therefore inhibit the normal substrate from being acted upon. Competitive inhibitors • Resemble substrate in structure (ie, are molecular analogs). • Bind the enzyme active site. • Maximum velocity is not affected. • KM is increased, decreased affinity for substrate. • Can be overcome with high substrate/inhibitor ratio. - Non-competitive inhibitors bind enzyme at location other than active site, altering enzyme conformation and ability to bind substrate Noncompetitive inhibitors • Bind to position other than active sites. • Vmax cannot be reached. It's like reducing enzyme concentration • KM is unchanged • Cannot be overcome with high substrate/inhibitor ratios.

Enzymes as Biological Catalysts

Enzymes: Protein catalysts that increase the rate of chemical reactions (a small number of RNA enzymes called ribozymes). • Each has a unique 3-dimensional conformation that allows it to interact only with specific substrate molecule(s). • Substrate: The substance(s) which an enzyme binds and catalyzes a chemical reaction(s). • Work at cell-specific temperature and pH • Are highly specific for their substrates. • Catalyze reactions in an orderly manner to prevent production of by-products that could negatively impact cell. Like all catalysts, enzymes: • Are required only in small amounts • Are not consumed or altered irreversibly by the reaction • Have no effect on the thermodynamics of the reaction Importantly, enzymes can be regulated to meet the needs of a cell.

Culturing and visualizing cells

Historical challenges to studying cells in vivo spawned techniques to isolate and culture clones of cells - a genetically homogenous population derived from a single founder cell.

Intrinsically Disordered Proteins

Induced fit - the interaction between two molecules results in conformational changes that allow the molecules to interact with greater affinity for one another.

The Michaelis constant: Km

KM for most reactions typically ranges between 10-1 and 10-7 M and is inversely proportional to substrate affinity: • Smaller KM greater affinity • Higher KM weaker affinity KM, the Michaelis constant - specific empirical value for each enzyme. Independent of either enzyme or substrate concentration. Key concepts: •KM is equal to the substrate concentration at exactly ½ of Vmax •A measurement of the relative affinity of enzyme for its substrate. •Enzymes with a low KM have a high affinity for their substrate, and •Enzymes with a high KM have a poor affinity for their substrate

Dissociation constants of binding reactions reflect affinity of interacting molecules

Key concept: For protein-protein and protein-DNA interactions: • Strong affinity/tight binding Kd ~ 10-10-10-9 M • Moderate affinity ~ 10-6 M • Weak affinity 10-3 M

Mechanisms of Protein Regulation

Key concept: Protein activity or function can be modified by altering its 3-D conformation: • Covalent modifications - Covalently attached molecules can alter proteins' chemical composition and conformation. • Examples include - phosphorylation, glycosylation, ubiquitination, methylation, acetylation • Allosteric modification - Non-covalent, physical association with another molecule can produce modest to extreme changes in protein conformation. • Examples include - interaction with metal co-factors, coenzymes or nucleotides.

Biochemical Energetics

Kinetic energy is the energy of movement - motion of molecules Potential energy is stored energy: • Chemical potential energy - energy stored in bonds connecting atoms in molecules; e.g., conversion of glucose to ATP and NADH. Energy must be expended to form bonds and is released when bonds are broken. • Concentration gradients - potential energy created by storing molecules on one side of a membrane and allowing them to flow across the membrane barrier spontaneously; e.g., oxidative phosphorylation • Electric potential energy - potential energy produced by separating differently charge ions on opposite sides of the membrane; e.g., membrane potential

enzyme kinetics

Kinetics - the study of rates of enzymatic reactions under various experimental conditions. • Reaction velocity eventually slows and stops. Saturation: All enzymes are occupied by substrate • Vmax, Maximal velocity: is enzyme concentration dependent, but can be used to determine an important property of an enzyme, its turnover number (aka catalytic constant, kcat) - the number of reactions at saturation catalyzed by a single enzyme per unit time. typical Kcat values range from 1 - 103/s, but may be as high as 106

Important Concepts about Biochemical Building Blocks

Lipids: A diverse group of molecules that are poorly soluble in water, or completely insoluble. Contain large hydrophobic (hydrocarbon) regions. • Include: - Sterols: like cholesterol and its derivatives - fats: glycerol linked by ester bonds to three fatty acids. - Phospholipids: Like fats, but one FA chain is replaced by a charged phosphate group - Lipids like phospholipids, with a large hydrophobic region and a charged hydrophilic region, are amphipathic

Lipids

Lipids: Diverse group of molecules, characterized by poor to no solubility in water. - Fats: aka triacylglycerol: Composed of the alcohol glycerol linked by ester bonds to three fatty acids (FAs). • FAs are unbranched hydrocarbons with one carboxyl group; they are amphipathic. • Saturated FAs lack C=C double bonds and are solid at room temperature. • Unsaturated FAs have one or more C=C double bonds (polyunsaturated) and are liquid at room temperature. • Steroids are animal lipids derived from cholesterol Lipids • Hydrocarbon skeleton with 4 rings • Different groups (usually with little polarity) attached to each steroid • Phospholipids are amphipathic lipids and major components of cell membranes. • Amphipathic - Hydrophilic polar group - Hydrophobic fatty acid tails • Phosphoglycerides - Glycerol backbone (3 carbon sugar) - 2 fatty acids (attached by ester bonds to -OH of glycerol) - Phosphate group (attached to the third -OH) with small polar group attached

Enzymes as biological catalysts cont

Many enzymes are non-covalently conjugated to non-protein components that facilitate chemical catalysis. • Cofactors inorganic atoms or molecules, mostly metal ions. Such ions participate directly in catalysis through ionic interactions with the substrate - the molecule(s) being acted upon. • Coenzymes are organic enzyme conjugates, usually derived from vitamins, that generally function as intermediate carriers of electrons, specific atoms or functional groups that are transferred in the catalyzed reaction.

Culturing animal cells requires nutrient rich media and special solid surfaces

Media requirements: • Amino acids: 9 essential and 3 not produced by many cell types • Vitamins, salts, fatty acids and glucose • Blood serum: The fluid remaining after blood cells clot. • Serum contains: • insulin, which stimulates cells to absorb glucose • Transferrin, which supplies iron in a useable form • Diverse growth factors that stimulate proliferation SURFACE REQUIREMENTS: • Fibroblasts, cells of connective tissue, like dermis, are among the hardiest and easiest cells to culture and study.

Primary culture and cell strains have a finite lifespan

Most normal cells divide approximately 50 times before entering senescence (exiting the cell cycle) -Phase I: Seeded cells grow to confluence (fill the plate) -Phase II: Cells can be removed, diluted and re-plated and will continue to grow -Phase III: After approximately 50 generations, cells exit the cell cycle and become senescent - primarily due to shortening of telomeres following each round of DNA replication. Exception being embryonic stem cells, and some immortalized lines.

amino acids differ in their side chains

Polar charged - R groups act as strong organic acids or bases 1. Almost always fully charged (ionized) at pH 7 (aspartic acid, glutamic acid and the two basic amino acids lysine, arginine). 2. As a result, can form ionic bonds; For example histone proteins have many lysine and arginine residues that bind negatively charged phosphate groups of DNA. 3. Histidine - A basic amino acid is only partially charged at pH 7; important role in enzyme active sites through ability to gain or lose a proton in physiologic pH ranges. -Polar uncharged - R groups weakly acidic or basic; therefore not ionized at pH 7; • Can form Hydrogen bonds with atoms of other molecules that have partial charge (part of polar covalent bonds). Through the -OH of serine, threonine and tyrosine Or two H bonds via the carboxyamide of glutamine and asparagine *Nonpolar - R groups hydrophobic; generally lack O & N; cannot interact with water or for electrostatic bonds; vary primarily in size & shape; allows them to pack tightly into protein core 1. Associate with one another via hydrophobic & van der Waals interactions in protein interior. Unique amino acids: glycine, proline, cysteine and histidine 1. Glycine - R = H, makes backbone flexible, useful in protein hinges 2. Proline - R group forms a ring with the amino group (imino acid); Proline does not readily fit into secondary structure (a-helix) and creates a kink in the polypeptide. 3. Cysteine - R group has reactive sulfhydryl (—SH); forms disulfide bridge (—S—S—) with other cysteines reinforcing and maintaining protein conformation. -Unique amino acids: glycine, proline, cysteine and histidine 4. Histidine: The imidizole ring of histidine can shift from being positively charged in an acidic environment to unprotonated under slightly alkaline conditions. The activities of many proteins, especially enzymes, are modulated by localized shifts in environmental pH through protonation or deprotonation of histidine residues.

First Level of Protein Structure

Primary structure • Specific linear sequence of amino acids • Information for sequence encoded in DNA • Protein function - derived from 3D structure (conformation), which is determined by the amino acid sequence and intramolecular noncovalent interactions. Structure of a polypeptide: • Peptide bond - formed by dehydration reaction between one amino acid -COO- to another amino acid NH3 +. • Polypeptide - linear polymer has a free amino end (N-terminus) and a free carboxyl end (Cterminus).

Domains are modules of tertiary structure

Protein Domains: parts of tertiary structure that function and evolve independently of the rest of the protein. Three general kinds of protein domains: • Functional domains - exhibit specific activity, usually independent of other regions of the protein, even when isolated from the rest of the protein. • Structural domain - region of 40 or more residues arranged as a single, stable, distinct structure often comprised of one or more secondary structures. Over 1000 unique structural domains. • Topological domain - regions of proteins defined by their spatial relationship to the rest of the protein; e.g., membrane spanning proteins have extracellular, membrane embedded and cytoplasmic domains. Each may comprise multiple structural and/or functional domains.

Overview of protein structure and function

Protein hierarchical structure: • Primary structure - linear sequence of amino acids linked by peptide bonds. • Secondary structure - local a-helices or bsheets • Tertiary structure - peptide three -dimensional shape • Quaternary structure - association into multipeptide complexes • Supramolecular complexes - can be very large, consisting of tens to hundreds of subunits

The formation of noncovalent interactions results in a release of energy and a more stable structure (lower energy state).

Proteins fold into a 3-dimensional shape that requires the least amount of energy to maintain.

Bioenergetics Gibbs Free Energy

Reactions are favorable, spontaneous and exergonic if DG is negative (-DG) Conversely, reactions are unfavorable, endergonic and nonspontaneous if DG is positive (+DG) Key point: Endergonic reactions can be coupled to exergonic reactions in order to drive them forward.

Second Level of Protein Structure

Secondary structure: • Discrete regional conformation of amino acids into α-helix, β-sheet, hinges, turns, loops, or finger-like extensions. • These stable spatial arrangements of polypeptide regions are held together by hydrogen bonds between backbone amide and carbonyl groups The a helix, a common secondary structure: • Prolines - can't participate in hydrogen bonding and are excluded from a-helices.

Spontaneous vs non-spontaneous reactions

Spontaneous processes are favorable and exergonic. Can occur without external energy. Nonspontaneous processes: Require external energy, i.e., are endergonic and unfavorable. Many biological reactions are non-spontaneous and require an input of energy.

Establishing function through building unique conformation

Structural Motifs: Regular combinations of secondary structure sometimes, but not always, with an associated specific function. -Three common motifs (but there are several more): a) Coiled-coil motif: Two α helices wound around each other (stabilized by hydrophobic interactions). Used for protein-protein interactions. b) EF hand motif: One of several types of helix-loophelix motifs. The loop chelates calcium Ca2+ to stabilize the motif structure. Used for either protein-protein or protein-DNA interactions. c) Zinc-finger motif: One or more Zn2+ coordinate an a-helix and a small b-sheet. Present in many RNA and DNA-binding proteins.

Multicellularity

Such division of labor requires cells to be organized into different tissue types, the 4 principle types being: • Epithelia - sheets of tightly associated cells • Mesenchyme - Autonomous free moving cells • Neuronal - Autonomous and conductive • Muscle - contractile - And to be associated with an extracellular matrix (ECM)and with one another: • Various cell adhesion molecules (CAMs) join cells together into tissues. • Other membrane associated proteins anchor some cells to the ECM. And organized into organs with complementary and coordinated functions.

polar covalent bonds

Such polar bonds establish an electric dipole, a positive charge separated from a negative charge. • Quantitative measurement of the separation of the charges is called the dipole moment (µ) and reflects the combination of charge strength and distance of separation. • As many molecules will possess a number of polar bonds, the total strength of polarity depends on the total of all dipoles as well as the geometry of the molecule. • Take away: Covalent bonds are stable and contain lots of energy. -Functional groups—Covalently attached molecular units confer distinct chemical characteristics and properties to resulting molecule. Such functional groups establish the chemical properties of all organic molecules.

Tertiary Level of Protein Structure

Tertiary structure: • Conformation of entire polymer. • Stabilized by 1) hydrophobic and Van der Waals interactions between nonpolar side chains and 2) hydrogen bonds: polar side chains and backbone amino and carboxyl groups. • Because these are weak forces, the tertiary conformation is not rigidly stabilized and undergoes constant minor fluctuations. • Can also be easily altered by addition of several post-translational modifications. Tertiary structure: • Disulfide bonds (strong covalent bonds) between cysteine residues also play role in stabilizing tertiary structure (very important for secreted proteins).

Bioenergetics The First Law of Thermodynamics

The First Law of Thermodynamics - aka the law of conservation of energy: Transduction - conversion of energy from one form to another while being transferred. - Photosynthesis (solar energy to chemical bonds) - Muscle contraction (chemical bonds to physical force) - Chemical concentration gradients across a membrane to movement of other substances across that membrane. • Reactions causing the system to lose energy are exergonic. • Reactions causing the system to gain energy are endergonic.

Bioenergetics The Second Law of Thermodynamics

The Second Law of Thermodynamics states that in the universe, or any closed system, the degree of disorder can only increase. • Entropy (S): a measure of the disorder of a system. Higher entropy means lower order, in other words increased randomness. • Increasing entropy leads to loss of available energy in the system. • Free energy (G): The internal energy of a system that is available to perform work (total energy minus unusable energy).

Four ways to present protein structure

a) Cα backbone trace - depicts how the polypeptide is tightly packed into a small volume b) Ball-and-stick representation - reveals locations of all atoms c) Ribbon diagram - emphasizes secondary structures and their positions within the protein d) Water-accessible surface model - reveals protein surface topology with positive charge (blue) and negative charge regions (red) e) hybrid models combine two of the approaches

chemical building blocks of cells

Three of the four major macromolecules are polymers of monomer subunits: • Proteins - amino acids • Nucleic acids - nucleotides • Polysaccharides (carbohydrates)- monosaccharides • Covalent bonds that join monomers result from dehydration reactions: The net loss of a -H from one monomer and a hydroxyl (-OH) from the other. • Breaking such bonds is a hydrolysis reaction, involving the addition of a water molecule (an -H and -OH). • Peptide bond • Phosphodiester bond • Glycosidic bond

Bioenergetics Combining First and Second Laws: Gibbs Free Energy

To determine if two reactions can be coupled, it's necessary to determine spontaneous vs non-spontaneous and the amount of energy released/required by the reactions. -Entropy (S): A measure of randomness or disorder. (in units of energy/temperature). -Energy unavailable to do work = TΔS. • T is absolute temperature ˚C + 273 • Δ stands for "change in" -Free Energy (G): Energy available to do work

Molecular chaperone-mediated protein folding

Two general families of chaperones, distributed into all organelles and compartments of eukaryotic cells. • Molecular chaperones: Bind short segments of a nascent protein and stabilize unfolded or partly folded regions, preventing aggregation or degradation. • For example, HSP70 and HSP90 family proteins bind emerging proteins and prevent inappropriate interactions (HSP = heat shock protein). • Chaperonins: Form folding chambers into which all or part of a nascent protein can be sequestered without interference from other molecules. Process up to 15% of the cells' proteins.

Activity of trypsin serine proteases

Two key non-covalent interactions promote specificity. a) Side chain specificity binding pocket has correct size, depth and acidic asparagine reside to accept and hydrogen bond to arginine/lysine side chains

Dissociation constants of binding reactions reflect affinity of interacting molecules.

What's the use of Kd? It can tell us the relative affinity of molecules for one another. Kd = [P][D] /[PD] • The lower the Kd, the lower the concentration of protein needed to bind half of the DNA. • In other words, a low Kdmeans the protein has high affinity for the DNA

Metazoa

animals. Multicellularity and embryonic development.

Concentration of molecules in reaction influences DG

ll chemical reactions eventually arrive at equilibrium: Forward and reverse reaction rates are equal. • There is no net change in concentration of any solute. • There is no more exchange of energy with the surroundings. - In other words, at equilibrium DG=0, and the system can do no work Key point: Keq can be used to predict the favored direction of a chemical reaction. - Keq > 1: Forward reaction favored -Keq < 1: Reverse reaction favored

covalent bonds and electronegativity

• Electrons can be shared equally producing non-polar covalent bonds: ie, the electrons spend an equal amount of time around both nuclei in the bonds. • Alternatively, the electrons maybe pulled toward one of the nuclei and spend more time there, producing polar covalent bonds: generating weak positive (d+) and negative (d- ) charge with the two atoms. • Electronegativity: The extent of an atom's ability to attract an electron. The covalent bonding of atoms with very different electronegativities results in polar covalent bonds.

Flow Cytometry used to separate and sort cell types

• Fluorescence activated cell sorter (FACS): A flow cytometer that streams a column of single cells through a laser beam that excites the fluorophore.

Noncovalent binding of regulatory molecules alters protein activity

• Allosteric regulation (Greek for "other shape") is not just a form of enzymatic regulation. It is broadly a change in conformation, and therefore function, of a protein through physical association with another molecule • Allosteric effectors bind sites on target proteins that alter their conformation. • Allosteric regulation may be either positive or negative: - it could expose a binding site, for example, allowing a protein to interact with another - It could hide an active site, preventing an enzyme from binding its substrate - Such negative regulation is often seen at the end of an enzymatic pathway, where the final product binds and allosterically inhibits an enzyme earlier in the pathway - feedback inhibition. • Cooperativity: Unique allosteric regulation seen in proteins with multiple binding sites for same ligand. Cooperative binding can be positive or negative.

Biological fluids have characteristic pH values

• Amphoteric molecules are those that can act as either acids or bases, dependent on the pH of the solution.

Enzymes as Biological Catalysts The Active Site

• An enzyme interacts with its substrate to form an enzyme-substrate (ES) complex. • Substrate binds enzyme in region called the active site, which is composed of: • Substrate binding site • Catalytic site • The active site and the substrate have complementary shapes (conformations) that promote substrate specificity through electrostatic and hydrophobic interactions. • Highly specific, alteration of just a couple of atoms is sufficient to disrupt association.

Non-covalent binding of Ca2+ as an allosteric switch

• Calmodulin: widely distributed cytosolic protein that serves as a switch protein to activate other proteins

Reaction Rates

• Catalysts accelerate the rates of chemical reactions by lowering their activation energy. • Enzymes are biological catalysts - ie proteins that facilitate increased rates of spontaneous reactions.

Activation energy of uncatalyzed and catalyzed chemical reactions

• Catalysts only increases the rate (speed) of the reaction! • Catalysts do not influence the thermodynamics of a reaction!!!

Key features of enzyme catalysis

• Catalytic sites have evolved to promote transition state stability, which lowers activation energy and accelerates the reaction. • Uniquely positioned residues interact with the substrate, often through a multistep process. • Acid-base catalysis is often employed to break and form new bonds, therefore pH plays a huge role in enzyme activity

chemical reactions are reversible and direction is concentration dependent

• Chemical equilibrium: rates of forward and reverse reactions are equal. No net change in the concentrations of reactants and products. • Equilibrium constant (Keq): Ratio of product to reactant concentrations at equilibrium. • A catalyst can speed up rate of reaction, but it will not change the Keq

Reaction rate depends on activation energy necessary to energize the reactants

• Chemical reactions involve the breaking and reformation of covalent bonds • Both processes require atoms of reactant molecules to adopt highenergy, transient configurations: transition states • Reactants in that state are the transition state intermediate • Activation energy (ΔG‡) is the added energy needed to reach this transition state. Required even for spontaneous reactions.

Ligand Binding Regulates the Function of Many Proteins

• Collectively, these two regions of the heavy and light chains constitute the complementarity-determining region (CDR).

other features of eukaryotic cells

• Densely packed heterochromatin is inactive - not being transcribed. • More loosely packed euchromatin can be transcribed. • Nucleolus: Site of ribosomal gene transcription and assembly of ribosomes (RNA and protein). - Diverse specialized organelles perform discrete cellular activities

The GTPase switch

• GTPase superfamily (aka G proteins) are regulated by GTP vs GDP-bound state • When bound to GTP G proteins are active and can bind to and regulate activity of target proteins • Possess Intrinsic GTPase activity: hydrolyzes GTP to GDP • Bound to GDP such proteins adopt an inactive conformation Allosteric Regulators of G proteins: • GEFs (guanine nucleotide exchange factors): Promote exchange of GDP for GTP and therefore activate G proteins. • GAPs (GTPase-activating proteins): Enhance GTPase activity which therefore speeds up the inactivation of G proteins. • GDIs (Guanine nucleotide dissociation inhibitors): Prevent exchange of GDP for GTP, and therefore keep G proteins in an off state.

Michaelis-Menten Kinetics

• KM is equal to the concentration [S] at exactly ½ Vmax. • Not in your textbook: a comparison of Kd and Km: KM essentially equals Kd

Ligand Binding Regulates the Function of Many Proteins

• Ligand - Molecule to which a protein binds • Ligand binding often results in a conformation change (change in 3-D shape). • This conformational change is integral to protein function and is often important in regulating protein function. • Specificity - Ability of a protein to bind one molecule or a small group of molecules in preference to all other molecules • Affinity -The tightness or strength of binding between the protein and ligand Measured by Kd (the dissociation constant). Low Kd = high affinity; high Kd = lower affinity. • Molecular complementarity required for specificity and affinity - complementary shapes and numerous non-covalent interactions drive both • Ligand binding by vertebrate specific antibodies great example of specificity and affinity. • Circulating antibodies recognize specific antigens - usually macromolecules present on non-self structures, like the surface of a bacterium. • Each antibody recognizes a very specific molecular substructure - its epitope. A single antigen may have dozens/hundreds of regions that can be epitopes for different antibodies.

Important Concepts about Protein Binding and Enzyme Catalysis

• Ligand: The molecule to which a protein binds. Ligand binding often induces conformational change in the protein and alters its function. • Enzymes accelerate rates of cellular reactions by lowering activation energy and stabilizing transition-state intermediates. Enzymes act upon substrate molecules. • Enzymes often use acid-base catalysis mediated by one or more amino acid side chains. • Cofactors often associate with enzymes and aide in catalysis • Metabolic pathway enzymes may be associated as domains of a monomeric protein, subunits of a multimeric protein, or components of a protein complex assembled on a common scaffold.

Key features of eukaryotes

• Many are unicellular, but several independent lines where true multicellularity evolved, including animals, the Metazoa. • Membrane enclosed subcellular compartments, called organelles, separate cell processes. • Cytosol: Organelle-free solution of water, dissolved ions, small molecules and proteins. • Cytoplasm: Cytosol + organelles. • True nucleus containing several linear DNA chromosome

Oxidation-Reduction Reactions

• Many cellular reactions are oxidation-reduction or redox reactions in which electrons are transferred between atoms of reacting species. • The molecule being oxidized (the electron donor) donates the electron to the molecule being reduced (electron acceptor) • May be total transfer - resulting in ionization • May be partial transfer - New polar covalent bond formation with electron pairs pulled more closely to an atom in the new bond

Free energy change: Coupled reactions

• Many cellular reactions are unfavorable, products have more available energy than reactants (i.e., anabolic reactions that build sugars from CO2 and H20). • As we'll explore, such reactions can be propelled forward by being coupled to favorable reactions.

Proteolytic cleavage irreversibly activates or inactivates some proteins

• Many signaling molecules like insulin, are produced as long precursors called prohormones that must be cleaved into active hormones before release from the cell. • Zymogens are inactive enzyme precursors that have not yet been activated by proteolytic cleavage.

difference between Motif and Domain

• Motifs are primarily structural in nature and are usually not independently stable outside of a protein • Domains are defined by having specific functions and are composed or one or more secondary structures and motifs.

Defining features of prokaryotes

• No nucleus, but genome is extensively folded and condensed into a region called the nucleoid. • Additional genetic material in small, circular plasmids that can be exchanged between cells. • No significant processing of messenger RNA (mRNA) • Complex extracellular cell wall composed of peptidoglycan (protein + sugar) (bacteria only, not Archaea) • Gram negative: Thin cell wall with second exterior lipid membrane • Gram positive: Think cell cell with no second membrane

non covalent bonds

• Non-covalent bonds are much weaker than covalent bonds.

Nucleotide polymerization and Base Pairing

• Nucleotides polymerize through phosphodiester bonds producing single-stranded nucleic acid polymers. • Ester bonds result from dehydration reaction between a carboxylic acid (COOH) and an alcohol (-OH).

Metabolism Oxidation-Reduction: A Matter of Electrons

• Oxidation-reduction (redox) reactions involve a change in the electronic state of reactants. • The relative oxidation state of an organic molecule can be determined (roughly) by the number of hydrogen vs. oxygen and nitrogen atoms per carbon atom. ie, more hydrogen à more reduced, and more stored energy

Important Concepts about Protein Folding

• Peptide amino acid sequence is the primary determinant of 3D conformation and therefore function of the protein. • Most domains will fold spontaneously after translation, however some protein regions require help to prevent inappropriate associations • ATP-dependent molecular chaperones and chaperonins assist protein folding in vivo. • Misfolded/denatured proteins can form highly-ordered amyloid fibril aggregates that can cause diseases, including Alzheimer's disease and Parkinson's disease. • Conformation: The physical 3-dimensional shape of a molecule • Native conformation (native state): The normal conformation that a polypeptide will fold into under standard physiological conditions. • Nascent protein: A protein that is being translated or has not yet adopted it's fully folded native conformation. (Nascent: Something just coming into existence and beginning to display signs of future potential).

Ubiquitin- and proteasome-mediated proteolysis

• Proteasomes: Large molecular machines (>60 subunits) that degrade targeted proteins. Such proteins must be tagged before being recognized and degraded. • Cells tag proteins to be degraded by proteasomes by covalently attaching chains of the small protein ubiquitin to specific lysine residues (polyubiquitinylation). • E3 proteins, specifically, are the ubiquitin ligase that transfer single ubiquitin proteins from E2 to the substrate protein. • Health perspective: Several successful cancer chemotherapy drugs are proteasome inhibitors - especially effective against multiple myeloma, cancerous white blood cells that produce high levels of improperly folded antibodies. By inhibiting proteasomes, the antibodies cannot be cleared and rises to toxic levels - leading to death of the cancer cells.

Hypothetical protein-folding pathway

• Proteins assume their native conformation through a series of steps. • Chaperones are proteins that bind unfolded/denatured proteins and facilitate proper folding by preventing association with other molecules.

Quaternary Level of Protein Structure

• Rather they join with other polypeptides as subunits of larger protein complexes that have a collective and complex function - for example the transcription initiation complex. • Such complexing reflects quarternary structure, which is mostly achieve through non-covalent interactions - but some do require covalent bonds

Serine proteases example of active site

• Serine proteases: A large family of enzymes that catalyze cleavage of specific peptide bonds via hydrolysis reactions • Trypsin, for example, preferentially cleaves the peptide bond C-terminal to residues with positively charged side chains (arginine and lysine).

Covalent Bonds

• Shared pairs of electrons between atoms generate covalent bonds - strongest of the atomic interactions, and key to establishing the structure of organic molecules. • Molecules: Stable combinations of atoms held together by covalent bonds. • Non-covalent interactions: Ionic bonds, Hydrogen bonds, van Der Waal's interactions and the hydrophobic effect are weaker than covalent bonds, but likewise play instrumental roles in both the shape and chemistry of biological molecules. - Bonded to 4 different atoms (or groups) in a non-planar orientation a carbon atom (or any other, for that matter) is said to be asymmetric - the atoms bonded to carbon can be arranged in two different ways - mirror images of one another. -• Such molecules are called optical isomers, or stereoisomers, and can have very different biochemical properties. • Example, Novrad is a pain reliever while its stereoisomer Darvon is a cough suppressant.

Gene homology

• Such genes with shared ancestry and function are called homologs. • Similarly, the human homolog of this gene, Pax6, is required for eye development, and mutations in this gene result in the defect anaridia - in which the iris fails to form (left).

basic structure of an amino acid

• The R group provides unique chemical characteristics to each amino acid, referred to as a residue when incorporated into a polypeptide. • R groups may be polar charged; polar uncharged; nonpolar • Amino acids are linked together by peptide bonds - amide type covalent bonds between amino group of one monomer and the carboxylic acid group of another. • Their polymers are called peptides, or polypeptides • Only when they fold into a functional 3-dimensional shape (conformation) are they referred to as proteins.

acids, bases and buffers

• The cytoplasm of eukaryotic cells has a pH near neutral, pH= 7.2-7.4, • Many biological molecules have both acid and base groups • Amino acids for example have both carboxylic acid and an amino base group • At pH =7 both groups are charged: the carboxylic acid is deprotonated and the amino group protonated. • Such an ion is called a zwitterion. • At extreme pH only one of the groups is charged (amino at low pH, -COOH at high) • Low pKa, strong the acid ..... High pKa, strong base • It also explains the function of buffers, acid/conjugate base pairs that serve to keep the pH of a solution (or biological system) within a defined window by neutralizing any introduced acid or base. - Buffers in living systems resist changes in pH • The ability of a buffer to minimize change in pH is its buffering capacity and is dependent on the buffer concentration and the relationship of its pKa to the solution pH as described by the Henderson-Hasselbalch equation.

Oxidation and reduction reactions

• The readiness with which an atom or molecule gains an electron is its Reduction Potential (E) and the tendency to lose electrons is its oxidation potential.

the distinction between RNA and DNA nucleotides

• The sugar is a ribose in RNA • Deoxyribose in DNA

Biological fluids have characteristic pH values

• Water is the biological solvent • In aqueous solution acids release protons (H+) • Bases accept protons • Released protons associate with water molecules to form hydronium ions (H3O+) which represent the solutions proton concentration [H+] • Both H+ and OH- are highly reactive and can alter biological molecule's functions, by forming or breaking covalent bonds

Misfolded proteins can produce disease pathologies

• amyloid fibrils are resistant to degradation