Chlamydomonas Cell Structure & Features

Cycle 1: Major Structural Features of a Chlamydomonas Cell

• The cell wall is composed of glycoproteins and lacks cellulose.
• The plasma membrane encloses the cell and regulates transport.
• The chloroplast contains chlorophyll for photosynthesis and presents a cup-shaped structure.
• The eyespot (stigma) is a photoreceptive organelle that enables the cell to detect light.
• Two anterior flagella facilitate motility.
• The pyrenoid is associated with CO2 fixation within the chloroplast.
• Contractile vacuoles help regulate osmotic pressure.
• The nucleus houses genetic material, which includes a haploid genome.

Features of Chlamydomonas Grown in the Lab

• Organisms thrive in liquid or agar media under controlled light and temperature conditions.
• Autotrophic growth, using CO2, or mixotrophic growth is possible.
• They can be easily genetically manipulated.
• They have a short doubling time (approximately 10 hours at 25°C), making them ideal for experiments.

Advantages of Haploid (N) Cells for Research

• Mutations manifest directly without being masked by a second allele.
• Easier genetic screening for functional studies is enabled.
• Rapid genetic modification and identification of mutant phenotypes is permitted.

Growth Media: Macronutrients vs. Micronutrients

• Macronutrients are required in large amounts and include carbon, hydrogen, oxygen, nitrogen, phosphorus, potassium, magnesium, and sulfur (C, H, O, N, P, K, Mg, S).
• Micronutrients are essential, but in small amounts, including iron, copper, zinc, molybdenum, boron, and manganese (Fe, Cu, Zn, Mo, B, Mn).

Importance of SO4 (Sulfur) and Cu (Copper) in Chlamydomonas and Humans

• Sulfur (SO4) is essential for amino acids like cysteine and methionine, coenzymes, and vitamins.
• Copper (Cu) is needed for electron transport, specifically cytochrome c oxidase, photosynthesis, and respiration.

Growth and Doubling Time (10 Hours at 25°C)

• Doubling time refers to the period required for the population to double.
• Growth occurs logarithmically during the exponential phase of cell division.
• Influencing factors on growth include temperature, light intensity, and nutrient availability.

Microbial Growth Curve

• The lag phase involves cells adapting to the environment with minimal division.
• The exponential or log phase is characterized by rapid cell division under optimal conditions.
• The stationary phase occurs when nutrient depletion and waste accumulation slow growth.

Bacterial vs. Eukaryotic Flagella

• Bacterial flagella rotate and are composed of flagellin, powered by a proton gradient.
• Eukaryotic flagella exhibit whip-like motion and are composed of microtubules, powered by dynein.

Chlamydomonas Phylogeny and Relationship to Plants & Animals

• It diverged from land plants approximately 1 billion years ago.
• It shares genes with both plants (photosynthesis) and animals (flagellar structure).

Why Do Chlamydomonas and Humans Have Flagella, but Plants Do Not?

• A hypothesis for this: plants lost flagella during evolution as they adapted to a sessile lifestyle, while motile cells, such as sperm, retained them.

Basics of Cilia Structure

• Microtubules are based on a "9+2" arrangement.
• Key proteins: dynein (motor protein), nexin, and radial spokes.

Cilia and Human Disease (Ciliopathies)

• Defects in cilia or flagella lead to disorders like polycystic kidney disease, Bardet-Biedl syndrome, and respiratory dysfunctions.

Differences Between Motile and Non-Motile (Sensory) Flagella

• Motile flagella are used for movement, as seen in Chlamydomonas and sperm cells.
• Non-motile (sensory) flagella act as signaling structures, exemplified by primary cilia in humans.

Comparative Genomics of Chlamydomonas

• It has 7,476 identified proteins.
• Shared with humans: genes related to cilia/flagella.
• Shared with Arabidopsis (plants): genes related to photosynthesis and chloroplasts.
• Shared with both: genes for basic cellular functions like metabolism and gene regulation.

Organelle Definition

• An organelle is a specialized subunit within a cell with a specific function.
• Modern definition: Includes non-membrane-bound structures like the eyespot, ribosomes, and centrioles that perform essential cellular functions.
• Traditional definitions: Only membrane-bound structures like nuclei and mitochondria were considered organelles.

Basic Organization and Functional Features of the Eyespot

• The eyespot (stigma) is a light-sensitive organelle found in unicellular photosynthetic organisms like Chlamydomonas reinhardtii.
• Made of pigment granules arranged in layers, creating a directional light filter.
• Channelrhodopsin (ChR) is present in the plasma membrane.
• The eyespot helps detect light direction and intensity to guide phototaxis, which is movement toward or away from light.
• It filters and reflects light to optimize the activation of photoreceptors.
• It regulates flagellar movement to adjust swimming behavior.

Structure & Function of Channelrhodopsin and Voltage-Gated Na* Channels

• Channelrhodopsin (ChR) consists of seven transmembrane domains and a retinal pigment bound to opsin.
• Function: Upon light absorption, opens allowing cation (H+, Na+, Ca2+, K+) influx, leading to membrane depolarization.
• Voltage-Gated Na+ Channels consist of a transmembrane protein with four domains that contain voltage-sensing regions.
• Function: Opens in response to membrane depolarization, allowing Na+ influx, which propagates an action potential.

Basics of How an Action Potential is Generated

• Resting Membrane Potential: Maintained at -70mV by Na+/K+ ATPase and leaky K+ channels.
• Depolarization: Stimulus opens voltage-gated Na+ channels, allowing Na+ influx (membrane potential becomes more positive).
• Threshold: At -55mV, an action potential is triggered.
• Rising Phase: Voltage increases rapidly due to more Na+ channels opening.
• Peak: Na+ channels inactivate and voltage-gated K+ channels open at approximately +40mV.
• Repolarization: Restoring a negative membrane potential when K+ exits the cell.
• Hyperpolarization: Cell becomes more negative than resting potential due to excessive K+ efflux.
• Return to Resting Potential: Na+/K+ ATPase restores ion gradients.

Channelrhodopsin as a Photoreceptor

• Photoreceptor = Protein (opsin) + Pigment (retinal).
• Retinal is derived from Vitamin A and changes conformation upon light absorption.
• Opsin is a G-protein-coupled receptor (GPCR) that interacts with retinal to transduce light into a cellular signal.

Structural Characteristics of Pigment Molecules

• Conjugated Systems: Pigments like retinal and chlorophyll have alternating single and double bonds in a carbon chain.
• Importance: Delocalized π-electron system allows the molecule to absorb specific wavelengths of light.

Light Absorption & Fundamental Principles

• Light Absorption: A molecule absorbs a photon, exciting an electron to a higher energy state.
• *Chlorophyll (Chl) vs. Excited Chlorophyll (Chl)**:
  • Chl (ground state) absorbs light.
  • Chl* (excited state) can transfer energy or participate in photochemical reactions.

Differences in Light Absorption Among Blue, Red, and Green Light

• Blue light (450-495 nm) has the highest energy and excites electrons to higher states.
• Red light (620-750 nm) has lower energy and is absorbed efficiently by chlorophyll.
• Green light (495-570 nm) is the least absorbed; chlorophyll reflects green, making plants appear green.

Absorption Spectrum

• A graph showing the wavelengths of light absorbed by a molecule.
• Example: Chlorophyll absorbs blue and red light but reflects green.
• Retinal's absorption shifts when bound to opsin, allowing different opsins to detect different wavelengths.

Relative Energies of Blue, Green, and Red Photons

• Blue photons: Highest energy (~2.75 eV).
• Green photons: Intermediate energy (~2.3 eV).
• Red photons: Lowest energy (~1.8 eV).

Photoisomerization of Retinal & Conformational Change in Opsin

• Photoisomerization: Light absorption causes retinal to shift from 11-cis to all-trans form.
• This conformational change activates opsin, triggering a signaling cascade.

Photoreceptor Molecules in Rods and Cones

• Rod and cone cells are non-motile cilia in the retina.
• Rhodopsin (rods) and cone opsins (cones) are embedded in membrane disks.
• Function:
  • Rods: Night vision (high sensitivity, no color).
  • Cones: Color vision (lower sensitivity, three opsins for RGB detection).

Channelrhodopsin vs. Rhodopsin

• Channelrhodopsin (ChR) in Eyespot functions as an ion channel (light-gated) and absorbs blue-green light, directly allowing cation influx.
• Rhodopsin in the Human Eye functions as a GPCR (activates G-protein) and has different opsins for red, green, and blue light, indirectly modulating ion channels via GPCR.

Photochemistry: Eyes vs. Chloroplasts

• Eyes & Eyespots: Photoreception (light causes a conformational change in opsin, leading to signaling or ion flow).
• Chloroplasts: Photosynthesis (light excites chlorophyll, triggering electron transfer for ATP/NADPH production).

Optogenetics: Expressing Chlamy Opsin in Brain Cells

• Goal: Control neuronal activity with light.
• Process: Insert ChR genes into neurons, so light can activate/inhibit brain circuits.
• Applications:
  • Studying neural circuits.
  • Treating neurological disorders (e.g., Parkinson's, depression).

Optogenetic Experiment: Basic Steps

• Select opsin: Choose ChR or a modified variant.
• Introduce gene: Use viral vectors (e.g., AAV) to insert ChR DNA into target neurons.
• Control expression: Use cell-specific promoters.
• Deliver light: Insert an optical fiber to shine light on neurons.
• Record response: Use electrophysiology to measure activity.

Cycle 2:

Purpose of RNA Blot Analysis

• RNA blot analysis measures the presence and quantity of specific RNA molecules in a sample.
• Used to study gene expression by detecting specific mRNA transcripts.
• Helps understand how genes are regulated in different conditions.

Basic Steps of RNA Blot Analysis

1. RNA Extraction - Isolate total RNA from cells.
2. Gel Electrophoresis – Separate RNA molecules based on size.
3. Transfer to Membrane – RNA is blotted onto a membrane (nylon or nitrocellulose).
4. Hybridization – A labeled single-stranded DNA or RNA probe binds to complementary mRNA.
5. Detection – The labeled probe is visualized (e.g., radioactive, fluorescent, or chemiluminescent detection).

Importance of a Labeled, Single-Stranded Probe

• The probe must be single-stranded to hybridize with target mRNA through complementary base pairing.
• Labeling (radioactive/fluorescent) allows detection of the probe-bound mRNA.

Complementary Base Pairing in RNA Blot Analysis

• The probe and the mRNA sequence bind through hydrogen bonding between complementary bases:
  • A (adenine) pairs with U (uracil)
  • G (guanine) pairs with C (cytosine)
• This ensures specific binding to the target transcript.

Hydrogen Bonding in Nucleic Acids

• DNA and RNA strands are held together by hydrogen bonds:
  • 2 H-bonds between A & U (or A & T in DNA)
  • 3 H-bonds between G & C (stronger interaction)
• This bonding allows specific and stable pairing during hybridization.

Gene Expression and Information Flow:

Definition of a Gene

• A gene is a segment of DNA that contains the instructions for making a functional product (usually a protein or RNA molecule).

Three Major Classes of Genes

1. Protein-Coding Genes – Transcribed into mRNA, then translated into proteins.
2. tRNA Genes – Transcribed into transfer RNA (tRNA), which helps in translation.
3. rRNA Genes – Transcribed into ribosomal RNA (rRNA), which is a structural component of ribosomes.

mRNA, tRNA, and rRNA: Differences & Abundance

• mRNA carries genetic code from DNA to ribosomes making it the least abundant (~5%).
• tRNA brings amino acids to ribosomes and has moderate abundance.
• rRNA forms the structural and enzymatic core of ribosomes and is the most abundant (~80%).

Central Dogma: DNA → RNA → Protein

1. Transcription: DNA → mRNA.
2. Translation: mRNA → Protein.
3. Post-translation: Protein undergoes modifications and degradation.

Role of Hydrogen Bonding in DNA vs. RNA

• DNA: Double-stranded, held together by hydrogen bonds.
• RNA: Usually single-stranded, but can form secondary structures (e.g., tRNA loops).

Why RNA Likely Evolved First

1. RNA can store genetic information like DNA.
2. RNA has catalytic activity (e.g., ribozymes), which DNA lacks.

Ribozymes: RNA Molecules with Enzymatic Activity

• Catalyze biochemical reactions.
• Examples:
  • Self-splicing introns.
  • Ribosome's rRNA, which catalyzes peptide bond formation.

Gene Expression and Regulation:

Transcription, Translation, and mRNA Breakdown

1. Transcription – DNA is copied into mRNA.
2. Translation – Ribosomes synthesize proteins from mRNA.
3. mRNA Decay – mRNA is degraded to regulate protein production.

Factors Determining mRNA/Protein Levels

• Gene transcription rate.
• mRNA stability (short-lived vs. long-lived).
• Translation efficiency.
• Protein degradation rate.

Structural Differences Between DNA and RNA

• DNA is double stranded while RNA is single stranded.
• DNA contains deoxyribose while RNA contains ribose.
• DNA uses the bases A, T, C, G while RNA uses the bases A, U, C, G.
• DNA is more stable while RNA is less stable.

Why RNA is Less Stable Than DNA

1. 2'-OH Group in Ribose – Makes RNA more prone to hydrolysis.
2. Single-Stranded Structure – More susceptible to nucleases.

Why Rapid mRNA Degradation is Beneficial

• Allows for quick changes in gene expression.
• Prevents unwanted protein synthesis.
• Helps cells respond to environmental changes.

Identifying Intact vs. Degraded RNA on a Gel

• Intact RNA appears as distinct bands.
• Degraded RNA appears as a smear.

Heat Shock and HSP Proteins

• Heat shock: A stress response triggered by high temperatures.
• HSPs (Heat Shock Proteins): Chaperones that help refold proteins and prevent aggregation.

Chlamydomonas Heat Shock Response

• Increased mRNA abundance of HSP genes.
• HSP proteins accumulate to protect against damage.

Linking RNA Blots to Gene Expression

• Heat shock increases HSP gene transcription.
• RNA blot shows increased transcript levels during stress.

Gene Regulation and Omics Fields:

Constitutive, Induced, and Repressed Gene Expression

• Constitutive: Always expressed (e.g., housekeeping genes).
• Induced: Turned on in response to a stimulus.
• Repressed: Turned off when not needed.

Housekeeping Genes/Proteins

• Required for basic cellular functions (e.g., GAPDH, actin).
• Always expressed (constitutive expression).

Omics Fields: Definitions & Uses

• Genomics studies DNA sequences & mutations used to identify genetic predispositions.
• Transcriptomics analyzes mRNA expression used to measure gene activity.
• Proteomics studies proteins & modifications used to directly reflect cell function.
• Metabolomics examines small molecules & metabolites used to show real-time metabolic state.

Why Proteomics & Metabolomics Provides More "Meaningful" Data

• Genomics can tell what COULD happen.
• Transcriptomics can tell what is being transcribed.
• Proteomics & Metabolomics tell what IS happening at the functional level.

Transcriptome Profile of Chlamydomonas During Growth

• Gene expression changes to adapt to different growth phases.
• Early phases: High expression of growth-related genes.
• Late phases: Stress and adaptation genes activated.

How Genes Contribute to Non-Protein Molecule Synthesis

• Retinal (a light-sensitive molecule in vision) is not encoded by a single gene.
• Instead, its synthesis involves multiple enzymes, each encoded by different genes.
• Retinal is derived from β-carotene through enzymatic reactions.
• Genes encode enzymes that catalyze the biosynthesis of molecules such as:
  • Retinal where β-carotene (precursor) is converted into retinal by β-carotene oxygenase, encoded by specific genes.
  • Chlorophyll multiple enzymes encoded by genes like ChID, Chll, and ChIH are involved.
  • Steroid Hormones (Estrogen/Testosterone) are synthesized from cholesterol through enzymes like CYP19A1 (aromatase) for estrogen.

Post-Translational Regulation

• Control of protein activity after translation.
• Modifications change protein function, stability, or localization.

Examples of post-translational modifications

1. Phosphorylation – Adds phosphate groups which regulates enzymes in signaling pathways.
2. Ubiquitination – Marks proteins for degradation.
3. Methylation/Acetylation – Alters gene expression by modifying histones.
4. Glycosylation – Adds sugars to proteins, affecting cell signaling.

Genotype vs. Phenotype

• Genotype: The genetic makeup of an organism (DNA sequence).
• Phenotype: The observable traits influenced by genotype & environment.
• A mutation in the OCA2 gene can be used to exemplify genotype producing a blue eyes phenotype instead of brown,

Forward vs. Reverse Genetics

• Forward Genetics begins with a phenotype, then finds the gene responsible and uses random mutagenesis to generate mutations, then screens for phenotypic changes.
• Reverse Genetics starts with a known gene, then examines its role by mutating, silencing, or deleting it.

Basics of Reverse Genetics

• Common techniques used:
  • Gene knockout where a gene is deleted to see what happens.
  • RNA interference (RNAi) where gene expression is silenced.
  • CRISPR-Cas9 where precise gene editing takes place.
  • Scientists knock out the p53 gene in mice to study its role in cancer suppression, as an example.

Insertional Mutagenesis as a Forward Genetics Approach

• Randomly inserts DNA into a genome, disrupting genes.
• Useful in forward genetics as it helps researchers to study unknown genes based on observable traits.

Steps of an Insertional Mutagenesis Project

• Step 1: Generating a Mutagenized Population containing colonies of mutated cells, where each dot originated from the same species but has a different mutation.
• Step 2: Screening for Mutants with a Target Phenotype , such as screening non-motile cells to identifying flagella mutants
• Step 3: Determining the Site of Insertion, using Polymerase Chain Reaction (PCR) to amplify and identify the disrupted gene
• Step 4: Rescuing the Mutant Phenotype (Genetic Complementation) by introducing a functional copy of the disrupted gene to restore the phenotype.

Cycle 3:

Energy Types and Examples

• Potential Energy is stored energy due to position or structure, such as water behind a dam, a stretched spring.
• Kinetic Energy is Energy of motion, exemplified by A rolling ball, a moving car
• Chemical Energy is potential energy stored in molecular bonds, like ATP, and glucose.

Types of Systems in Thermodynamics

• A closed system exchanges energy but not matter with surroundings, as illustrated by a closed water bottle.
• An open system exchanges both energy and matter with surroundings like Living organisms, an open cup of coffee
• An isolated system exchanges neither energy nor matter modeled by The universe (theoretically), a perfect thermos (hypothetically)

The Laws of Thermodynamics

• First Law of Thermodynamics (Conservation of Energy) defines energy that cannot be created or destroyed, only transferred or transformed, for example food energy is converted into ATP, then into muscle movement.
• Second Law of Thermodynamics (Entropy Increases) occurs since In any energy transfer, some energy becomes unusable, increasing entropy (disorder), and the heat released by metabolism cannot be fully reused.

Four Levels of Protein Structure

• Primary structure involves the Amino acid sequence utilizing Peptide bonds found in an insulin sequence.
• Secondary structure is responsible for Local folding utilizing Hydrogen bonds found in Silk (β-sheets), or Keratin (a-helices)
• Tertiary structure creates a 3D shape of one polypeptide utilizing Hydrogen bonds, ionic bonds, disulfide bridges, and hydrophobic interactions found in Enzymes and Myoglobin.
• Quaternary structure is for the Assembly of multiple polypeptides utilizing the same structure as tertiary, exemplified by Hemoglobin, and Collagen
• Each amino acid has a unique R group that affects its polarity, charge, and interactions.

Why We Need to Eat

• To power cellular work with an energy source.
• To provide building blocks for macromolecules to serve as a matter source.
• To maintain order (low entropy), which overcomes the natural tendency toward disorder, according to the 2nd Law of Thermodynamics.

Energy in Living Systems

• An organism takes in low-entropy energy and releases high-entropy waste.
• Free energy (G) is the usable energy for work, defined with AG (Change in Free Energy) = G_final - G_initial.
  • Spontaneous reaction: has a Negative AG and releases energy.
  • A non-spontaneous reaction: has a positive AG and requires energy.
• Enthalpy (H) is the system's total energy and is Lower for more spontaneous (-AG).
• Entropy (S) is the measure of disorder and is Higher for more spontaneous (-AG).

Types of Reactions

• Exothermic Reactions release heat such as Combustion, and freezing water
• Endothermic reactions absorb heat such as Boiling water, and photosynthesis.
• Exergonic reactions release free energy (-∆G) like Cellular respiration
• Endergonic reactions require free energy (+∆G) like DNA replication, and protein synthesis.

Enzymes: Role in Reactions

• They speed up spontaneous reactions by lowering activation energy (Ea) and couple ATP hydrolysis to non-spontaneous reactions.
• AG is the change in free energy and is negative for exergonic reactions.
• Transition State is the High-energy unstable state before reaction proceeds
• Activation Energy (Ea) is the energy needed to reach the transition state
• Enzymes lower Ea, making reactions faster

How enzymes lower activation energy

1. Position substrates correctly.
2. Induce strain on bonds to make breaking easier.
3. Microenvironment stabilization (e.g., adjusting pH).
4. Temporary covalent bonding to stabilize transition states.

Enzymes and Evolution

• Without enzymes, reactions would be too slow for life to exist.
• Evolution favored efficient and specific catalysts.

Protein Folding

• Primary structure determines the final 3D shape.
• Folding depends on hydrophobic interactions, hydrogen bonds, and disulfide bonds.
• Disrupts hydrogen bonding and weakens hydrophobic interactions.

Energy Funneling Model of Protein Folding

• Proteins fold through multiple pathways toward the lowest energy state.

The Role of chaperones

• Assist in correct folding and prevent aggregation
• Example: HSP70 helps refold misfolded proteins under stress.
• Inside cells, proteins face crowding, interactions, and competing factors

Structure and Function of Chaperones

• They bind unfolded/misfolded proteins to prevent aggregation and utilize ATP to aid in folding.
• GroEL/GroES chaperonin forms a barrel-like structure for folding, as an example.

How Enzymes Work: The Catalytic Cycle

1. Substrate binds to active site (induced fit model).
2. Enzyme stabilizes the transition state.
3. Reaction occurs.
4. Products are released.
5. Enzyme is free to catalyze again.

• The enzyme changes shape slightly to fit the substrate more snugly.

Cytosolic Protein Synthesis and Ribosomes

• Cytosolic protein synthesis takes place on free ribosomes in the cytoplasm.
• Proteins synthesized in the cytosol stay in the cytoplasm or target specific organelles but do not go through the secretory pathway.

The Secretory Pathway

• It is ​​a system for modifying, packaging, and transporting proteins and their components are:
  • Rough Endoplasmic Reticulum (RER) which synthesizes and folds proteins.
  • Golgi Apparatus which Modifies and sorts proteins.
  • Vesicles - Transport proteins between compartments.
  • Plasma Membrane – Secretes proteins.
• Types of proteins that go through this pathway include membrane proteins, Secreted proteins (e.g., insulin), and Lysosomal enzymes.

Protein Targeting to the ER

1. Signal sequence (a short amino acid sequence) directs translation to the ER.
2. Signal recognition particle (SRP) binds to the signal sequence.
3. SRP receptor on the ER membrane binds SRP, docking the ribosome.
4. Protein is threaded into the ER and processed.

Simple vs. Facilitated Diffusion

• Simple diffusion is the movement of molecules down their concentration gradient without assistance that requires molecules to be small, non-polar, or hydrophobic.
• Facilitated diffusion is also the movement of molecules down in the gradient but requires help from transport proteins
• Simple diffusion does not utilize protein or energy and examples are O2, CO2, and steroids.
• Facilitated diffusion uses a channel or a carrier for transport and ATP is not needed, examples are Glucose, and ions.

Transport Against a Concentration Gradient

• Moving molecules against their concentration gradient (low to high concentration) requires energy (ATP or ion gradients).
• Passive transport occurs when AG is negative (spontaneous).
• Active transport requires AG > 0, meaning energy input is needed.

Transport Curves: Simple vs. Facilitated Diffusion

• Facilitated diffusion depends on transport proteins, which saturate at high substrate concentrations which is what causes a peak rate.
• Simple diffusion is not limited by transport proteins.

Structure of ABC Transporters

• ATP-Binding Cassette (ABC) Transporter moves molecules across membranes using ATP and are made of:
  • Transmembrane domain that creates a pathway for molecules
  • APT binding domain that binds and hydrolyzes ATP
  • A regulatory domain controlling activity in some ABC transporters

Cystic Fibrosis and CFTR

• Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride ion channel in epithelial cells and dysfunction leads to thick mucus in lungs and digestive issues.
• The AF508 mutation causes misfolding, leading to degradation before reaching the membrane.
• Researchers showed that AF508 CFTR is functional based on an In vitro test where it functioned when artificially inserted into membranes, which shows how there is a trafficking issue in CFTR.

Role of Chaperone Proteins (HSP90) in CFTR Folding

• HSP90 assists in proper protein folding.
• It helps wild-type and AF508 CFTR fold correctly and dysfunction of chaperones can increase CFTR degradation.

Fatty Acid Saturation and Membrane Fluidity

• Saturated fatty acids = Less fluid (more rigid).
• Unsaturated fatty acids = More fluid (more flexible).

Temperature and Membrane Fluidity

• Low temperature = Membranes become rigid.
• High temperature = Membranes become too fluid.

Maintaining Proper Membrane Fluidity

• Ensures proper protein function.
• Allows transport proteins to work.
• Supports electron transport in mitochondria.

Role of Desaturases in Fatty Acid Biosynthesis

• They introduce double bonds into fatty acids and increases membrane fluidity under cold conditions.

Bacterial Desaturase Expression vs. Temperature

• Low temperatures cause Increase desaturase expression for more unsaturated fatty acids for fluidity.
• High temperatures result in Decrease desaturase expression for more saturated fatty acids and prevents the membrane from becoming too fluid.

Cycle 4:

The Two Phases of Photosynthesis

• Light Reactions, in the thylakoid membrane, convert light energy into chemical energy, generate ATP and NADPH for the Calvin cycle, while releasing O2 as a byproduct.
• Calvin Cycle, in the stroma, uses ATP and NADPH to convert CO2 into glucose and does not require light directly.

Structure and Function of a Photosystem

• Antenna Complex, Reaction Center, and Primary Electron Acceptor are the core components of a photosystem.
• Antenna Complex: Captures light energy.
• Reaction Center: Converts light energy into chemical energy.
• Primary Electron Acceptor: Transfers excited electrons to the electron transport chain (ETC).

Definition of Photosynthesis

• Photosynthesis is a process where plants, algae, and some bacteria convert light energy into chemical energy, storing it in organic molecules.

Global Primary Productivity

• There is little photosynthesis in warm parts of the ocean due to the Lack of nutrients (especially nitrogen and iron) which limits phytoplankton growth.

Energy and Matter Flow

• Photosynthesis Converts CO2 + H2O into glucose + O2 where CO2 is oxidized into Glucose that is reduced.
• Cellular respiration converts glucose + O2 into CO2 + H2O, where Glucose is oxidized into CO2.

Autotrophs vs. Heterotrophs

• Autotrophs uses light or inorganic compounds for energy, or light in (photosynthesis) and matter from CO2 and are plants and cyanobacteria.
• A heterotroph consumes organic molecules for energy and the energy and matter both come from food, this is an example is animals and fungi.

Photosynthesis as an Endergonic Redox Reaction

• Requires light energy input which causes Water (H2O) to be oxidized to O2 and CO2 being reduced to glucose.

Structural Features of the Chloroplast

• Thylakoid Membranes are responsible for light reactions (ATP/NADPH production)
• Stroma is the Site of the Calvin cycle
• Granum is the Stack of thylakoids
• Chloroplast Genome is the site of encoding of proteins (including D1)

The Electron Transport Chain (ETC) and ATP Synthase

• The electron transport chain transfers electrons from water to NADP+, producing NADPH, and pumps protons (H*) into the thylakoid lumen, creating a proton gradient.
• ATP synthase uses proton gradient to generate ATP from ADP + P.

Redox Potential and Electron Flow

• Redox potential is a molecule's ability to accept or donate electrons.
• Chlorophyll increases a strong donor in redox potential upon photon absorption.

P680, P680, and P680+*

• P680 (ground state) a normal chlorophyll in PSII.
• P680* is the excited version by light, becomes a strong electron donor.
• P680+ is missing an electron, must be reduced by water splitting.

Negative Phototaxis in Chlamydomonas

• Sometimes, Chlamy is negatively phototactic because too much light can damage PSII, so cells move away from intense light.

PSII Damage and Repair

• High light intensity overproduces P680+, leading to D1 protein damage in the PSII, and constant D1 synthesized and replaced aids in repair.

Basic of the Calvin Cycle

• The Calvin cycle takes place In the stroma of the chloroplast and causes carbon dioxide (CO2) to be fixed into organic molecules.
• It allows plants to store energy from sunlight as sugars, and it has three stages.

1. Fixation - CO2 is attached to RuBP by Rubisco.
2. Reduction – Produces G3P using ATP and NADPH.
3. Regeneration – Regenerates RuBP to continue the cycle.

• The key molecules are:
  • Rubisco: fixes CO2.
  • RuBP: Carbon acceptor molecule.
  • PGA: 3-carbon intermediate.
  • G3P: make glucose.

Oxygenic vs. Anoxygenic Photosynthesis

• Oxygenic Photosynthesis requires light and utilizes PSI & PSII while producing O2 (from water)
• Anoxygenic Photosynthesis requires light and utilizes one photosystem without O2, and uses (H2S or other donors)

Evolutionary advantage of oxygenic photosynthesis

• Allowed for O2 production, leading to:
  • Aerobic respiration (more ATP production).
  • Ozone layer formation, protecting life from UV radiation.
  • Evolution of multicellular life.

Thermodynamics of Respiration as a Redox Process

• Cellular respiration involves electron transfer of a Glucose that is oxidized (loses electrons) to CO2 and Oxygen that is reduced (gains electrons) to H2O.
• C6H12O6+6O2→6CO2+6H2O+Energy(ATP)C_6H_{12}O_6 + 6O_2 →6CO_2 + 6H_2O + Energy (ATP)
• This process is exergonic (AG < 0), releasing usable energy.

Role of Mitochondria in Chlamy Cells

• They perform cellular respiration to generate ATP, interact with chloroplasts for energy balance, and help regulate redox homeostasis under low oxygen conditions.

Overview of Metabolism: Catabolism vs. Anabolism

• Catabolism is a chemical process that results in the Breakdown of molecules (e.g., glucose) with an Exergonic result of AG > 0.
• Anabolism is a chemical process that results in Building molecules (e.g., proteins, lipids) with an Endergonic result of AG < 0.
• Catabolism fuels anabolism by generating ATP and NADH.

ATP and NADH Ratios

• High ADP/ATP → Signals low energy → Stimulates ATP production.
• Low ADP/ATP → Signals high energy → Inhibits ATP production.
• High NAD+/NADH → Stimulates catabolic reactions.
• Low NAD+/NADH → Slows respiration.

Electron Transport Chain (ETC) and ATP Production

1. NADH donates electrons at Complex I.
2. FADH2 donates electrons at Complex II.
3. Electrons move through Complex III and IV.
4. O2 is the final electron acceptor, forming H2O.

Why Does FADH2 Oxidation Occur After Complex I?

• FADH2 skips Complex I, contributing less proton pumping than NADH and generates less ATP per molecule than NADH.

Proton Pumping and Chemiosmosis

• Complexes I, III, and IV pump H+ ions into the intermembrane space.
• ATP synthesis is utilized through the energy from electron transport and proton gradients.
• The movement of H+ back into the mitochondrial matrix through ATP synthase drives ATP production.

Why Use Proton Gradients in Chemiosmosis?

• Protons are small, abundant, and easy to pump.
• Membranes are impermeable to H+, allowing controlled energy release.

Role of Redox-Active Cofactors in the ETC

• Iron (Fe) is essential in cytochromes for electron transfer to provide metal cofactors.
• Without Fe, the ETC cannot function due to why Electrons move spontaneously down the ETC.
• Redox potential increases from NADH → О2, and Oxygen has the highest electron affinity.

Coupling Electron Transport with ATP Synthesis

• The proton gradient from the ETC drives ATP synthase in coupling.
• Uncoupling proteins (UCPs) allow protons to bypass ATP synthase, dissipating energy as heat in uncoupling.

Physiological Role of Regulated Uncoupling.

• Babies & hibernating animals use UCPs in brown fat for warmth during thermogenesis.

Uncoupling and Metabolism

• Higher UCP expression = More heat, less ATP storage.
• Increased metabolism = More calorie burning.

Chemical Uncouplers: 2,4-Dinitrophenol (DNP)

• Disrupts proton gradient, making ATP synthesis inefficient.
• Causes overheating and metabolic failure due to toxicity.

Metabolic Shifts Under Low Oxygen

• Switches to anaerobic glycolysis.
• Increases lactate production (fermentation).
• Reduces oxidative phosphorylation.

The Warburg Effect

• Cancer cells favor glycolysis, even with oxygen present.
• Several methods are utilized to remodel cellular respiration

1. Increase glucose uptake (more glucose transporters).
2. Increase glycolysis (high hexokinase activity).
3. Inhibit pyruvate dehydrogenase (PDH) using PDH kinase.

• The hypothesis for this effect is twofold: Rapid ATP Production in the Glycolysis, which is faster than oxidative phosphorylation Even though it's less efficient, is said to meet high energy demands. Secondly, to support Biosynthesis support as Glycolysis produces intermediates for nucleotides, lipids, and proteins.

Cancer Detection Using Metabolism

• Fluorodeoxyglucose (FDG), a radioactive glucose analog, is taken up by cancer cells.
• High glycolysis = Higher FDG uptake, visible in PET scans.

Cycle 5:

Basics of Measuring Respiration in Isolated Mitochondria

• Oxygen electrode (Clark electrode): Measures O2 consumption.
• Buffer solution: Maintains optimal pH and ion concentrations.
• Substrates (e.g., NADH or succinate) to provide electrons for the electron transport chain (ETC).
• ADP & Pi: Necessary for ATP synthesis (drives respiration).
• Uncouplers (e.g., DNP) to separate electron transport from ATP synthesis.

Changes in Respiration Rate by Adding Different Molecules

• NADH addition Increases O2 consumption because it Fuels ETC by donating electrons.
• ADP + Pi Furthers Increases O2 consumption by Stimulating ATP synthesis, increasing proton flow.
• Uncoupler (DNP, FCCP) Maximizes O