BCH - Exam 3

Biological cell membrane

• Thin

• Semi-permeable

  • Control movement of substances into and out of cell

  • Selective for different substances

• Phospholipid bilayer with proteins embedded and sugars associated

• Define borders of cells, tissues, and organelles

Membrane composition

Phospholipids, proteins, cholesterol, and carbohydrates (sugars)

• Different cells & organelles have different amounts of each type of constituent and this is related to function

Properties of lipids

• Significant # of C-H bonds

• Insoluble in water

• Amphipathic: hydrocarbon (hydrophobic) tail + hydrophilic head

Integral membrane

Protein whose amino acids are inserted into membrane e.g., transmembrane protein

Peripheral membrane

• Associate proteins do not span membrane

• Proteins often in cytoplasm and associated with membrane upon PTM

Fluid Mosaic model

• Membranes are fluid

• Lipids form a bilayer embedded primarily with proteins

• Components "float" around in 3D space

• Evidence: Frye and Edidin experiment showed protein mixing when cells fuse

Fatty acid composition

• Carboxylic acids (head) + long-chain hydrocarbon side groups

• Even number of carbon atoms

• Most common have 14-20 carbons

• Rarely found "free" in nature

• Biosynthesized by connection of C2 units

• Occur in esterified form → modified to form “more complex” lipids

Saturated fatty acids

• Only single C-C bonds

• Less fluid → higher melting temperature

• Longer chains = more solid

Unsaturated fatty acids

• Contain one or more C=C (double) bonds

• More fluid → lower melting point

• Cis vs. Trans configurations affect properties

  • “kink” in cis configurations → increases distance between H-bonds

Factors affecting fluidity of fatty acids

• Carbon chain length

  • long chain = more solid/rigid = lower fluidity = higher melting temp

• Number of cis double bonds

  • fewer cis bonds = less kinked = more solid/rigid = lower fluidity = higher melting temp

Δ nomenclature of FA

ω nomenclature of FA

Micelle

Unilayer assembly of lipids that have a hydrophobic core → transport of hydrophobic substances

Membrane lipids

• Phospholipids

• Sphingolipids

• Cholesterol

Phospholipids

• Amphipathic:

  • Polar hydrophilic head: phosphate & alcohol

  • 2 hydrophobic fatty acid tails

• Head & tail joined by phosphodiester linkage

• Glycerophospholipids = glycerol backbone

• Sphingophospholipids = spingosine backbone

• Major constituents of biological membranes

Glycerophospholipids

• Major lipid component of biological membranes

• Glycerol backbone: 3 carbons & 3 OH

  • 2 carbons esterified to fatty acid tails

• Bonded to a fatty acid by ester link & polar head group via a phosphate group

• Characterized by variable head groups (different molecular size & charges)

Largest glycerophospholipid

Phosphatidylinositol 4,5-bisphosphate (PI)

Smallest glycerophospholipids

Phosphatic acid (PA)

Neutral glycerophospholipids

• Phosphatidylethanolamine (PE)

• Phosphatidylcholine (PC)

Negative-charged glycerophospholipids

• Phosphatidylserine (PS)

• Phosphatidylinositol 4,5-biphosphate (PI)

• Phosphatidylglycerol (PG)

• Phosphatidic acid (PA)

• Cardiolipin

Shape of glycerophospholipids

• Head group (size and charge) + fatty acid (saturated vs unsaturated) dictates “shape” of a lipid

• Small head, unsaturated tails = cone, negative curvature

• Cylindrical lipid (head = tail size) = flat membrane

Distribution of lipids between membrane leaflets

• Inner leaflet of bilayer contains higher % of negatively charged glycerophospholipids (PS and PI) than outer leaflet (neutral lipids)

• Phospholipid asymmetry → protein folding

Sphingolipids

• Backbone: sphingosine

• Can be phospholipids or not

• Derivatives of 18 carbon unsaturated, amino alcohol sphingosine (relatively long)

• Fatty acid linked to amino alcohol sphingosine via an amide bond to form a ceramide

• Very stable, rigid & provides insulation → common in neurons

• Ceramide as a common precursor

  • Cerebroside: ceramide + glucose head group

  • Ganglioside: ceramide + oligosaccharide head group

Cholesterol

• Unique tetracyclic structure

• Helps membrane flexibility → keeps membranes from solidifying when cold and breaking apart when hot

  • Stiffens & maintains fluidity of membrane lipids

• Weakly amphipathic because of hydroxyl group

• Bulky, rigid structure disrupts regular fatty acid chain packing in membranes

• Enriched in lipid rafts

Membrane rafts

• Membranes can have “domains”

• Lipid rafts: type of membrane domain (enriched with cholesterol)

  • Contain tightly packed lipids

  • Enriched in saturated lipids

• Function: cell signaling and sorting of proteins into organelles

Cardiolipin

• Double glycerophosphate backbone + 4 fatty acid tails

• Conical in shape

  • More IMM = more ETC proteins = more energy!

• Found in inner mitochondrial membrane

• Both outer & inner mitochondrial membranes are bilayers

GPCR

• 7 transmembrane domains

• C-terminus intracellular (cytoplasmic) → binds G-proteins

• N-terminus extracellular

“Inside positive” rule

• Negative charges of phospholipids on inner leaflet of membrane interact via charge-charge interactions with +ve charged amino acids on cytoplasmic side of transmembrane proteins

• Protein translocon (spans ER membrane) → passes proteins

Passive transport

No energy required (ΔG < 0)

• Non-mediated transport (simple diffusion)

• Mediated (facilitated diffusion): diffusion of solutes accelerated by membrane proteins (pores, carriers, permeases)

Non-mediated transport

• Simple (passive) diffusion

• Slow & down concentration gradient

• Easier for hydrophobic (non-polar) than for hydrophilic molecules

• Results in equal concentrations on both side of membrane

• For non-polar and uncharged molecules e.g., gases (O2 & CO2)

Mediated (facilitated) transport

• Passive transport

• Accelerated diffusion for molecules that cannot do simple diffusion (fast enough)

• Important for polar and charged molecules e.g., ions, glucose, water

Molecules that facilitate diffusion (mediated transport)

• Transmembrane protein pores (aquaporin) → fast and selective (Na, K, Cl)

• Carrier molecules (ionophores) → highly selective

• Permeases (GLUT transporters) → generally slower, but more specific than protein pores

  • also transmembrane

Aquaporins

• Water channel proteins

• Structure = function → hydrophilic inside channel & hydrophobic outside channel

• Crucial in cells like erythrocytes

• Rapid transport of water → maintain osmotic balance & ion gradients

Glucose Transporters (GLUT)

• Carrier protein (facilitated diffusion)

• Facilitate glucose movement down concentration gradient (passive: high → low)

• Transport can occur in both directions depending on concentration gradient of glucose

Active mediated transport

• Energy/ATP required from ion gradients (ΔG > 0)

• Crucial for maintenance of membrane potentials

• Movement against concentration gradient


• Na/K ATPase

• Na-Glucose Symport

Na+/K+ ATPase

• Active transport of ions using ATP hydrolysis

• Antiport: 3 Na+ ions out & 2 K+ ions in

• E1 conformation: open on inside of cell, closed on outside, and has affinity for Na+

• E2 conformation: open on outside of cell, closed on inside and has affinity for K+

Mechanism of Na/K ATPase

1. E1 state binds intracellular ATP via intracellular subunits and binds 3 Na+

2. Transporter is phosphorylated → conformation change (“high-energy”)

3. Protein adopts E2 conformation → 3 Na+ released out of cell (through membrane)

4. E2 binds 2 K+ from outside cell

5. Phosphate group on protein (transporter) is hydrolyzed → conformational change to E1 (binds ATP)

6. 2 K+ released into cell

Na-Glucose symport

• Secondary active transport (does not use ATP directly)

• Generates potential energy in the form of ion gradients

• Symport: Na+ & glucose transported in same direction

Cell signaling

• Ability of a cell to receive, process, and transmit signals

• Occurs between: cell & environment, neighboring cells and within the cell itself

Signal transduction

Process by which a chemical or physical signal is transmitted through a cell as a series of molecular events

Stages of cell signaling

1. Reception: cell detects stimulus (molecule) by
receptor

   • Intracellular: if ligand is trapped inside cell or is small/hydrophobic enough to pass through membrane

   • Extracellular: on cell surface if ligand is not permeable across membrane

2. Transduction: signaling molecule binding induces receptor protein change

3. Response (effect):
receptor change triggers specific cellular change

4. Resetting/Recycling (optional): cell and receptor return to original state

Types of receptors

• Cytokine receptors: IL-2R

• Receptor tyrosine kinase: insulin receptor, epidermal growth factor receptor (EGFR),

• GPCR: acetylcholine signaling, 𝛼₁-adrenergic receptor

• Channel proteins (ligand-gated ion channel): acetylcholine

Receptor Characteristics

• Proteins made of amino acids

• Amphipathic:

  • At least one hydrophobic transmembrane domain


  • Hydrophilic extracellular domain for ligand binding

  • Hydrophilic intracellular domain for effector binding

Receptor Tyrosine Kinases (RTKs)

• Single transmembrane domain

• Built-in cytoplasmic enzyme (kinase) domain → phosphorylate tyrosine amino acids to form recognition sites for scaffolding or effector proteins

• Must dimerize to be functional

• Kinase domains of each RTK monomer cross-phosphorylate cytoplasmic domain on other receptor unit

G-protein coupled receptors (GPCR)

• 7 transmembrane domains

• N-terminus always extracellular

• C-terminus always intracellular → binds G proteins for signal transduction

• G proteins bind GDP or GTP

Channel Proteins


• Form hydrophilic membrane pores

• Narrow & highly selective → gated (unlike pores = open)

• Ion channels:

  • Voltage-gated channels

  • Mechanically gated channels

  • Ligand-gated channels: respond to ligand-binding

Endocrine ligands

• Long range

• Signals travel from distant cells via bloodstream

• Slow, long-lasting response

Paracrine ligands

• Short range

• Local signals between nearby cells

• Quick, short-lived response


Autocrine ligands

• Very short range

• Cell produces and responds to its own signals

Ligand classification by effect

• Hormones: endocrine glands; involved in homeostasis

• Growth factors: endocrine, paracrine or autocrine; stimulate growth

• Cytokines: paracrine or autocrine; immune response

• Neurotransmitters: paracrine; stimulate neurons

Types of transducing (effector molecules)

• When receptors bind their ligands, they “change conformations” and activate intracellular processes (protein activation)

• Enzymes: kinases, phosphatases, hydrolyases

• Second messenger molecules: cAMP, inositol triphosphate, diacylglycerol

Post-Translational Modifications

• Phosphorylation & lipid addition (isoprenylation) to proteins

• Can:


  • Switch proteins on/off


  • Change protein location

  • Target proteins for degradation

Kinase

• Catalyzes transfer of phosphate group from ATP to a molecule

• Can elicit an ON or OFF effect

Phosphatase

Removes phosphate group from its substrate without ATP

Phosphorylation

• Covalent, dynamic modification

• Very common in signal transduction pathways

• Involves transfer of phosphate group from ATP to hydroxyl group on amino acid

• Adds negative charge to protein

• Does not involve water addition or loss → not a hydrolysis reaction (unlike GTPase)

Isoprenylation

• Addition of hydrophobic molecules (fatty acid) to a protein

• Farnesylation of Ras proteins (GTPase protein)

  • Ras proteins only functional in membrane (ineffective in cytoplasm)

  • C-terminal tail w/ lipid attached enters

GTPases (G-proteins)

• Bind to and hydrolyze guanosine triphosphate (GTP) molecules

• Also known as guanine nucleotide-binding proteins (G-proteins)

• Hydrolase enzymes → bind to and hydrolyze GTP molecules to GDP

• Ras proteins

• Guanine Nucleotide Exchange Factors (GEF): GDP → GTP (turn GTPase ON)

• GTPase Activating Protein (GAP): GTP → GDP (turn GTPase OFF)

Second messenger molecules

• Small molecules that are enzymatically generated and transmit signals within a cell

• Inositol phosphate

• Diacylglycerol

• cAMP: synthesized from ATP by adenylate cyclases

cAMP

• Intracellular signaling molecule

• Activates protein kinase A (PKA) by binding to regulatory subunits of PKA relieving inhibition

• PKA phosphorylates (activates) transcription factors to promote gene expression

• Adenylate cyclase → cAMP → PKA → CREB (phosphorylated in transactivation domain)

Inositol phosphates and diacylglycerol

• Intracellular signaling molecules

• IP₃

  • Binds to intracellular calcium channels to release calcium

  • Formed by phospholipase: cleaves head group from PI → DAG in membrane & IP2 in cytoplasm

  • Derived from phosphatiyl inositol (glycerolipid)

Signal integration

• Processing multiple simultaneous signals
to coordinate a unified cellular response

  • Increased signal precision & efficiency

  • Increased diversity in response to signal

• Two or more pathways downstream of common receptor

• Converging pathways: two phosphorylation events needed to activate an enzyme requiring two different pathways

• Opposing pathway effects

Signal amplification

• Increase in intensity of a signal through networks of intracellular reactions

• Involves enzymes and production of second messenger molecules

SH2 domain effectors

• Src homology 2 domains (SH2 domains) bind phosphorylated tyrosines on RTK

• Serve as scaffolds for other effector proteins e.g. bring nucelotide exchange factor proteins (GEF) close to GTPases to facilitate nucleotide loading (GTPase activation)

  • SOS (GEF for Ras protein) bound to Grb2 (SH2 domain) → activates GTPase in membrane

Insulin signaling

• Insulin receptor (receptor tyrosine kinase)

• Phosphotidylinositol-3-kinase phosphorylates PIP2 → PIP3

• PH domain binds PIP3 → upregulates expression of GLUT4 transporter on cell surface → increase glucose uptake by cells, lowering blood sugar levels

Epidermal Growth factor signaling

• Paracrine

• EGF binds Epidermal Growth factor receptor (EGFR) → signaling events that allow for cell cycle progression, cell growth

• Effectors: PI3K, GTPase, Phospholipase C

G-proteins

• Found in cytoplasm

• Heterotrimers: Gα (binds GDP or GTP), Gβ, Gγ

  1. In inactive state, Gα bound to GDP

  2. GPCR binds ligand → receptor changes conformation → allosteric change in Gα → GDP replaced by GTP

  3. GTP activates Gα causing it to dissociate from GβGγ (remain linked as dimer)

  4. Activated Gα activates another effector

Gα subunit

• Gα: inhibits adenylate cyclase → cAMP decreases → PKA decreases

• Gαs: stimulates adenylate cyclase → cAMP increases → PKA increases

• Gαq: stimulates phospholipase C (PLC) → converts phosphatidylinositol (PI2) into diacylglyercol (DAG) and inositolphosphates (IP3)

𝛼₁- adrenergic receptor

GPCR: leads to muscle contraction by increasing calcium

1. 𝛼₁- AR binds to Gα_q

2. Gα_q activates PLC

3. PLC converts PIP2 → IP3 + DAG

Acetylcholine signaling

• Neurotransmitter = paracrine signaling molecules

• Signaling pathway:

  • Ligand gated ion channel: ion diffusion across membrane → rapid depolarization

  • GPCR

Interleukin-2 signaling

• Autocrine: expressed by activated T cells & stimulate cell proliferation

• IL-2 binds to portions of IL-2 receptor (IL-2R) → receptor forms trimer (oligomeric) → kinases associate with trimeric complex to stimulate signaling

  • IL-2R is not a RTK!

Signaling Termination


• Reverse activating modifications e.g., kinase vs. phosphatase

• Receptor internalization (endocytosis via β-arrestin) and degradation

Ligand-Receptor Pharmacology

• Characterized by:


  • Binding rates (on/off rates)

  • Dissociation constant (Kd)

  • Receptor affinity

• Ligands can switch signaling on/off & moderate signaling intensity

K_on rate

On-rate: how readily ligands bind to receptors

K_off rate

Off-rate: how readily ligands unbind from receptors

Dissociation constant (K_d)

• Concentration of ligand required to occupy 50% of receptor binding sites

• K_d is inversely related to binding affinity

  • High K_d = low receptor-binding affinity

  • Low K_d = high receptor-binding affinity

Ligand binding efficacy

• Agonists: always elicit a biological response (ON or OFF)

• Inverse agonists: elicit OFF response

• Antagonists: elicit NO biological response

  • Can inhibit agonists (biological response elicited)

Restriction endonuclease enzyme

• Cut DNA at specific target sequences

• Create “blunt” or "sticky” ends (short, single-stranded overhangs) for DNA manipulation

• If two molecules have these “sticky ends” complementary overhangs, they can base-pair and stick together → use DNA ligase to seal two molecules together and join gaps in DNA backbone

Challenges of gene isolation and cloning

• Genes are not discrete entities

• Difficult to purify specific genes from complex DNA mixtures

Plasmid

• Small, circular, self-replicating extrachromosomal DNA molecules

• Naturally occur in bacteria

• Advantages/uses of inserting gene of interest into plasmid:

  • Protect genes from degradation

  • Replicate genes in bacteria

• We can stick DNA into plasmids → put plasmid into bacteria to produce lots of DNA

• Cannot be inserted into humans:

  • Don’t contain right transcription regions for humans

  • Would be rapidly degraded

Recombinant DNA

• DNA molecules formed by laboratory methods

• Normally made by bringing together multiple pieces of DNA from different sources and incorporating them into one piece of DNA

• Possible because while DNA differs between species in terms of sequence, structure of DNA is identical

• “Recombinant” piece of DNA can then be inserted into cells for various uses

  • plasmid propagation

  • gene therapies

  • industrial scale production of proteins

Transforming bacteria with our plasmid containing gene

• Once plasmid is inside bacteria → culture bacteria and generate billions of copies

• How do we know the plasmid is in the bacteria?

  • Selection genes in plasmid

  • Antibiotic resistance

Features of a plasmid

• ORI site (origin of replication)

• Multiple cloning sites (restriction enzyme site)

• Selectable markers (antibiotic resistance gene)

• Promoter site

Genomic DNA libary

Contains entire genome of organism, including coding (exons) & non-coding (introns) regions

cDNA library

Collection of cDNAs made from mRNA, representing only expressed genes (exons)

Making a genetic library

1. Digest (cut up) DNA into fragments using restriction endonuclease enzymes

2. “Dilute” fragments into solution of digested plasmid → provides

   conditions where one DNA fragment is ligated into one plasmid

3. Dilute plasmids and “transform” them → one plasmid into one organism

4. Genomic DNA library generated

Polymerase Chain Reaction

• DNAP: duplicates DNA & needs primer

• PCR used when you know which gene is needed from genome

  • Amplifies specific DNA segments

  • Uses gene-specific primers

• Requires:

  • ssDNA template with sequence of interest/gene

  • Specific primers with complementary sequence to gene

  • DNA source

  • dNTPs, divalent cations (Mg²+), ATP, free 3’OH end

  • Heat-stable DNAP from bacteria

1. Denaturation: dsDNA separated using heat (H-bonds broken, NOT phosphodiester bonds/DNA backbone)

2. Annealing: heat reduced → primers specifically annealed to gene (target sequence)

3. Extension: heat-stable DNAP extends primers → synthesizes new DNA strands

Agarose gel electrophoresis

• Used to separate/isolate DNA fragments by size (# of base pairs)

  • not applicable to proteins

• Smaller fragments move faster through gel

• -ve charge of DNA allows movement through gel towards positive terminus of gel

Reverse transcription of mRNA to make cDNA

• mRNA (starting material)

• Extract mRNA because it contains lots of A’s on its 3’ tail (poly A tail) → use primer rich in ‘T” to bind to tail

• Primers:

  • Oligo-dT primers: bind to poly-A tail of mRNA; non-specific (good for whole transcriptome → make cDNA library of all mRNA)

  • Gene-specific primers (with complementary sequence): bind to a particular mRNA sequence (if targeting a specific gene)

cDNA

• Complementary DNA synthesized from mRNA during reverse transcription

• Contains only exons

How do we isolate a plasmid of interest from bacteria?

Alkaline lysis: isolate plasmid and not bacterial genomic DNA

1. Add alkaline-OH groups (makes solution alkaline) + detergent (SDS) to bacterial cell preparations

2. Detergent solubilizes (disrupts) cell membrane & alkaline-OH unzips DNA (disrupts hydrogen bonding between DNA bases converting dsDNA → ssDNA )

3. Neutralize the pH → easy for small circular plasmid DNA to re-nature (plasmid isolated)

4. Impossible for larger genomic DNA to properly anneal → precipitates out of solution

5. Filter out large precipitates (genomic DNA) using a filter and collect/concentrate plasmid DNA

How do we know what the inserted DNA sequence in the plasmid is?

Sanger sequencing

• Determines DNA sequence inserted into plasmids

• Uses tagged ddNTP (dideoxynucleoside triphosphates) → terminates DNA strand elongation → length of fragments indicates sequence

  1. Add template DNA; primer; DNAP; dNTPs; ddNTPs

  2. DNAP extends primer by adding dNTPs

  3. Occasionally, ddNTP is incorporated instead of dNTP

  4. Once ddNTP is added, no more nucleotides can be added → synthesis terminated

  5. Creates fragments of different lengths, each ending in a fluorescently labeled ddNTP

  6. Gel electrophoresis: fragments separated by size (smallest move fastest)

  7. Order of fluorescent colors indicates sequence of bases

Sources of recombinant genes

• Genomic DNA

• cDNA synthesized from mRNA

Isolating a gene

• From mRNA: reverse transcribe to cDNA → PCR with gene-specific primers → gel electrophoresis → purify band

• From genomic DNA: PCR with specific primers → gel electrophoresis → extract and purify band

• From a library: screen cDNA/genomic library with a probe → identify and isolate clone

• Via restriction digest: if restriction sites flank gene → cut with enzymes → purify via gel electrophoresis

Vector

• Piece of “engineered” DNA that functions as a vehicle to carry foreign DNA molecules into another cell

• Can be designed specifically for human cells (could contain human promoters)

• Insert healthy gene into human cells using a viral vector (contains necessary genes to insert own DNA into host cell DNA)

Recombinant proteins

• Host selection considerations crucial for amount of protein & correctly folded modified protein

  • Bacteria: produce more protein, less proper modification

  • Eukaryotic cells: produce less protein, more properly modified

• Require: transcription start site, translation start site, ORI site

• Inducible expression is advantageous so include an operon in bacterial systems

Isolating recombinant proteins

• Column-based chromatography

  • Size exclusion: most common method to separate proteins

  • Ion exchange

  • Affinity

• Electrophoresis: separates proteins by size/charge

  • SDS-PAGE for size-based separation

Size exclusion chromatography

• Column contains a stationary phase made of beads with tiny pores

• More interaction between small proteins and pores → slower progress of small proteins through column

  • Larger proteins elute first

• Fractions collected separately

• Molecular weight of proteins in each fraction investigated by SDS-PAGE

Ion exchange chromatography

• Column contains stationary phase made of beads with charge

• More interaction between opposite charged proteins and stationary phase → proteins retained while others pass through

  • To remove retained proteins wash with salt or pH gradient buffer

• Salt (NaCl) competes with proteins for binding sites on resin → increasing concentration causes more proteins to elute

• Downside: salt and pH can alter protein structure

Affinity chromatography

• Column contains a stationary phase made of beads with a molecule that protein of interest is known to bind e.g. ligand, antigen, antibody

• Protein is retained in stationary phase by binding to specific binding molecule

• To remove retained proteins, need to outcompete stationary phase for binding to your protein → add competing molecules

  • high concentration of ligand (to compete with the bead-bound one)

  • add salt or change pH gradient (to weaken binding)

• Eluted proteins may contain ligand when eluted

• Downside: salt and pH gradient can alter protein structure

PAGE electrophoresis

• Poly-acrylmide gel electrophoresis

• Used to perform analysis of proteins (know if protein is correct)

• Separate proteins based on size and charge

• Small, charged molecules move faster through gel than larger uncharged ones