BIOL 201 - Cell Bio & Metabolism

First Law of Thermodynamics

• in an isolated system, the total amount of energy remains constant over time

• energy is neither created nor destroyed, just converted from one form to another

Second Law of Thermodynamics

• an isolated system always tends towards disorder

metabolism

• science of energy conversions

• organisms devote 20% of genome to this

• high-quality → low-quality energy

caveats of energy

1. organisms store energy during growth

2. organisms store energy temporarily during activity

inequality of energy

• “Low quality’ = HEAT (random kinetic energy)

  • less order

• “High quality” = Potential energy of mass on a pull

  • more ordered/structured

• Joule’s Experiment

• Organisms convert high-quality → low-quality energy

Gibbs free energy

• available energy to an organism

• H – “unavailable energy”

• G = H – TS

• Released by ATP hydrolysis

  • Cells capture it to create order

• the change in this determines whether a process occurs spontaneously

Enthalpy

• total energy in a system

• H

Entropy

• disorder in a given system

• S

ATP binding

• free energy can temporarily distort the 3D structure of proteins

• induces strain

• protein relaxes back into native configuration → chemical reaction forced to occur

change in free energy

• determines whether a process occurs spontaneously

• ΔG = G_final – G_initial

• ΔG = ΔH – TΔS

  • for “isolated system”, ΔH = 0

  • entropy always increases; ΔS > 0

• ΔG < 0 for a spontaneous process

• values are standardized (ΔGº’)

  • T = 298K, P = 1atm, pH = 7.0, all concentrations = 1M

  • standard free energies are additive → “coupled” reactions

Redox reactions

• Reduction = gaining an electron

• Oxidation = losing an electron

• provide a basis for energy transduction

• fuels are oxidized by metabolic enzymes

NAD⁺

• principal electron acceptor in metabolic redox reactions

• GAPDH brings this into position to be reduced to NADH

• may be useful in a wide range of therapies

  • ex: tuberculosis drug Isoniazid → active form binds NADH, inhibits cell wall synthesis enzyme

• FAD used when available free energy can’t reduce this

• requires ΔGº’ = 52.6 kcal/mol to capture e^-

FAD

• used when available free energy can’t reduce NAD⁺

• less ΔGº’ required than NAD⁺ → NADH (43.4 kcal/mol vs. 52.6)

• reduced to FADH₂

ATP

• “energy currency” of the cell (like a $20)

• needs ΔGº’ = -7.3 kcal/mol to be hydrolyzed (20 k_BT)

• big enough to do something with but small enough to avoid too much waste

thermal energy

• serves as baseline for cellular energy scales

• 〈E〉= 3/2k_BT

• k_B = Boltzmann’s constant

• k_BT = 0.6 kcal/mol

  • average amount of energy an H₂O molecule has when it collides

• ATP hydrolysis ≈ 20 k_BT (physiological conditions)

Brownian motion

• the random motion of particles suspended in a medium

  • molecules continuously undergo small, random fluctuations

• “random walk” → at any given moment, molecule shifts left OR right

• as # “steps” increases, particles start to diffuse away from each other

• molecules diffuse w/ characteristic diffusion coefficient (D)

  • smaller molecules = “faster” diffusion, larger D

  • Diffusion coefficient (D) describes spread of population of molecules

  • diffusion is mostly ACTIVE (a bit thermal, but it’s negligible in cells)

• explores a lot of space, but on average gets nowhere

  • Mean Squared-Displacement: 〈x²〉= 2Dt

mechanical energy

• energy that is possessed by an object due to its motion or due to its position

  • sum of kinetic and potential

• cells and subcellular structures feel and produce mechanical forces

• 1 N (newton) = 1kg x 1 m/s²

• cellular forces measured in pN (piconewton) to nN (nanonewton)

  • 1 pN = 10^-12 N

  • k_BT = 4.1 pN nm

  • ATP hydrolysis ≈ 20 k_BT ≈ 80 pN nm (a tiny amount of work)

electromagnetic energy

• the various energies that travel as wavelengths through space at the speed of light

• photons absorbed and emitted (electric + magnetic fields)

• electrostatic potentials

  • surfaces of protons contain many charged residues

  • Coulomb’s Law: electrical force between two charged objects is directly proportional to the product of the quantity of charge on the objects

  • moving 2 opposite charges from 0.3 nm to 0.15 nm apart

  • E = 2.3 k_BT (pretty small) → need a large surface area for 2 proteins to stick together

• a little stronger than thermal energy

photons

• elementary particle that is a quantum of the electromagnetic field

• E (energy) = hν

  • h = Planck’s constant

  • ν = frequency in Hz

  • visible particle ≈ 2 eV = 80 k_BT

• k_BT = 25 meV (milli-electric Volts)

• absorbed to ultimately produce ATP in photosynthesis

breaking bonds

• non-covalent bonds (ex. H-bonds)

  • 2-12 k_BT → varies because of random motion of thermal energy

• electrostatic bonds

• probability of breaking: P = e^–E/k_BT

  • ex. P = e^-3 = 0.05 → 5% of breaking

• 10¹¹ collisions per second

transition state

• short-lived configuration of atoms at a local energy maximum (highest potential energy) in a reaction-energy diagram

• catalysts lower this value → less ΔG required to get over this point

  • energy obtained from random collisions → if value is lower, then higher probability to get over it

• products have LOWER ΔG than reactants (overall negative = spontaneous rxn)

covalent bonds

• chemical bond that involves the sharing of electrons to form electron pairs between atoms

• very stable

• E ≈ 100 k_BT

• probability of breaking: P = e^-100 = 3.7×10^-44

  • by itself, a collision will break this every 10²⁴ years

cytoplasm

• an active material in the cell

• far from equilibrium because of ATP hydrolysis

• has different consistencies depending on the size of the particle in it

  • for ions: like water

  • for organelles/macromolecular complexes: like glass

• carbon/ATP depletion causes transformation into glass consistency (molecules frozen)

1. very CROWDED

   • contains ions & H₂O (0.1nm), sugars, amino acids, proteins & DNA & RNA(10-100 nm), organelles (1µm), etc.

   • as packed as protein crystals (20-60% protein by weight)

2. rough-and-tumble place

   • proteins in constant motion → constantly smashing into each other

   • Brownian motion → “random walk”, 1-D: 50% step right, 50% step left

   • as # “steps” increases, particles start to diffuse away from each other

   • Diffusion coefficient (D) describes spread of population of molecules

   • cells have to fight to maintain spatial organization

   • collisions distort structure of individual proteins → conformational changes

   • large organelles/proteins move in place, tiny ions/particles basically move freely

3. viscous

   • inertia = resistance of an object to any change in its state of motion

   • viscosity = measure of a fluid’s resistance to flow

   • Reynold’s Number: Re = inertial forces/viscous forces = ρνL / µ = (density)(velocity)(Length) / (dynamic viscosity)

     • cells, organelles, proteins are in LOW Re environment (~10^-4)

     • inertia is completely negligible here

4. elastic

   • elasticity = tendency of an object to return to its original shape after deformation

5. meshwork

   • long filamentous proteins → actin filaments

   • organelles, polymers, other structures define “pore size” → this is why larger macromolecular complexes are “trapped”/unable to move on their own

inertia

• resistance of an object to any change in its state of motion

viscosity

• measure of a fluid’s resistance to flow

Reynold’s number

• dimensionless quantity that helps predict fluid flow patterns

• Re = inertial forces/viscous forces = ρνL / µ = (density)(velocity)(Length) / (dynamic viscosity)

elasticity

• tendency of an object to return to its original shape after deformation

glycolysis

• Stage 1 of metabolism

• converts glucose (C₆H₁₂O₆) into pyruvate

• harvesting electrons

• yields NET production of 2 ATP per 1 glucose

1. Phosphorylate glucose by adding P_i from ATP

   • Creates glucose 6-phosphate → goes on to form other metabolites

   • Catalyzed by hexokinase enzyme

2. Change glucose ring → fructose ring

   • hexameric → pentameric

   • catalyzed by phosphoglucose isomerase

   • fructose 6-phosphate → used for downstream processes like making glycolipids

3. Phosphorylate again w/ another ATP

   • produces fructose 1,6-bisphosphate

   • catalyzed by phosphofructokinase

4. Formation of dihydroxyacetone phosphate

   • catalyzed by aldolase

5. Split into 2 molecules

   • 2 x glyceraldehyde 3-phosphate (G3P)

6. 2 NAD⁺ used to make 2 NADH

   • Oxidizing G3P with glyceraldehyde 3-phosphate dehydrogenase

   • 1,3-bisphosphoglycerate formed

7. Dephosphorylation

   • 2 ADP → 2 ATP produced

   • 3-phosphoglycerate formed by phosphoglycerate kinase

8. 3-phosphoglycerate mutated → 2-phosphoglycerate

   • catalyzed by phosphoglycerol mutase

9. Condensation → H₂O produced

   • done by enolase

10. Dephosphorylation

    • 2 ADP → 2 ATP produced

    • pyruvate formed

    • catalyzed by pyruvate kinase

pump priming

• generates useful metabolite

• increases free energy of reactants

  • larger ΔG, smaller activation barrier to get over transition state

• NOT catalysis

regulation of glycolysis

• huge enzymes like “disassembly line”

• cells must monitor glycolytic flux by conformational switches to enzymes modulated by allosteric activators and inhibitors

• cancer cells → increased flux

  • mutations in glycolytic enzymes found in tumors → block product inhibition of enzyme

  • cause is DNA damage, NOT misregulation of glycolysis

• steps w/ big –ΔG effectively irreversible

• PFK1 → gatekeeper

  • also catalyzes step 3 (phosphorylation of fructose 6-phospate → fructose 1,6)

  • once this step is done, you can’t go back

PFK1

• phosphofructokinase → controls glycolytic flux

• tetramer → 4 individual polypeptide chains linked together

• switches between active and inactive states by conformational change

  • tense = inactive, relaxed = active

  • 2 substrate binding sites open in active state

  • inhibitors at inactive state

• allosteric enzyme

  • enables cells to have control over switching glycolysis on/off

• inhibited by its products → ATP and citrate

  • ATP also a substrate → as its concentration increases, it becomes an inhibitor

• activated directly by AMP, indirectly by excess fructose-6-phosphate (starting material for step 3)

  • AMP = starting material for ATP (adenosine monophosphate)

  • PFK2 starts phosphorylating when there is excess → produces fructose 2,6-bisphosphate which acts as direct activator for PFK1

MWC model

• explains allostery: when allosteric enzyme switches between active/inactive conformation, ALL 4 subunits must switch at once

• one ligand binds in inactive state → rapid switch of molecule to active state

  • then all other ligands bind, enzyme performs rxn

• as soon as one ligand is lost → rapid switch to inactive state

  • all other ligands dumped immediately

• S-shaped sigmoid curve

• used to find hemoglobin

mitochondria

• eukaryotic organelle

• double membrane structure

• form complex network of tubes

• originated from bacteria → Endosymbiont hypothesis

  • aerobic respiration was the driver of this

  • evolutionary advantage

  • helped cells produce more power per gene

• make their own ribosomes, segregate their own DNA (mtDNA), etc.

• have their own genetic code & different codon usage

  • ex. UGA = Stop for standard code, Trp for mitochondria

  • determines protein length

• function influences lifespan

  • ex. mice w/ homozygous mutation die very young

  • ex. mutations in mitochondrial proteins like ETC increased lifespan in C. elegans

• mutations affect 1/5000 live births

  • can cause problems in all tissues/cell type of body

  • morphology varies

Endosymbiont hypothesis

• ancestral cell w/ nucleus & eukaryotic plasma membrane

• engulfed a bacteria capable of oxidative phosphorylation w/ ATP synthase, bacterial DNA, bacterial plasma membrane

• bacteria became mitochondria w/ mitochondrial matrix and unique genome (mtDNA)

• controversial hypothesis!!

  • alternative: aerobic proto-eukaryote enlarged & engulfed its respiratory surfaces (ATP synthase complexes)

  • central counter: host was already aerobic

  • DNA sequence analysis proved these wrong → chicken liver mitochondria closer to E.coli than bovine erythrocyte

• mitochondria increased power per gene

mitochondrial proteins

• built from a combination of nuclear DNA and mtDNA

  • small % of mitochondrial proteome is built from the mitochondrial genome

• ribosome: built from some proteins synthesized in cytoplasm and imported, combined w/ locally synthesized proteins

• several translocases work as channels to bring cytosolic proteins into mitochondrial matrix

mtDNA inheritance

• modeled from petite mutation in yeast (inhibits growth)

• 2 haploid (1n) parents w/ wild-type nuclear genes → one w/ normal mitochondria, one w/ petite

  • mating by cell fusion → diploid (2n) zygote

  • yeast sporulate → even segregation of nuclear DNA

  • mitochondrial segregation is RANDOM

• petite cells occur by chance due to random segregation of mtDNA

mitochondrial fission

• the process by which mitochondria divide or segregate into two separate mitochondrial organelles

• GTPase called DRP1 oligamerizes in ring shape on mitochondrial membrane and squeezes it apart

• mutants have opposite phenotypes to fusion

• continuous and ongoing at all times

• promotes equal segregation of mitochondria into daughter cells

  • as cells enter mitosis, they fragment mitochondria into as many small pieces as possible

  • DRP1^-/- cells segregate mitochondria asymmetrically

• often occurs at point of Endoplasmic Reticulum-mitochondrial contact

  • triggers recruitment of DRP1

mitochondrial fusion

• merging of two or more mitochondria within a cell to form a single compartment

• 2-step process because of the double membrane:

  • Mitofusins (MFN1/2) form trans-dimer extending from one mitochondria to another

  • conformational change in MFN1/2 brings outer membranes together

  • Inner membrane: 2 copies of OPA1 dimerize → undergo conformational change to smash inner membranes together

• mutants have opposite phenotypes to fission

• continuous and ongoing at all times

mitochondrial repair

• damage accumulated to mtDNA and protein contents

  • reactive oxygen species → byproduct of metabolism, toxic waste!!

• fission & fusion promote mixing of mitochondrial contents & enable “rescue”

  • defective mitochondrion fused w/ working mitochondrion → now able to produce proteins to rebuild machinery → undergoes fission again to create “rescued” mitochondrion w/ its own genome

mitophagy

• the process of cells degrading their mitochondria

• clears out dysfunctional mitochondria and controls number

• eaten by large multi-protein organelle autophagosome

  • spits out nucleic & amino acids for rebuilding

• PINK1 → identifies/targets damaged mitochondria

• Parkin → tags mitochondria for degradation

  • Ubiquitin ligase, recruits autophagosome

• Autosomal Recessive Early-Onset Parkinson’s Disease linked to misregulation of this process

  • PINK1 & Parkin mutations - dysfunctional mitochondria not degraded

  • tolerated for decades, then triggers disease later in life

citric acid cycle

• Stage II of metabolism

• pyruvate enters cycle as C2 (2 carbon) acetyl group on acetyl-CoA

  • bound to CoA (Coenzyme A) in 3-step process

  • 1. Decarboxylation

  • 2. Oxidation (NAD⁺ → NADH)

  • 3. Transfer of acetyl group

  • performed by huge pyruvate dehydrogenase complex

  • ΔGº’ = -80kcal/mol, basically irreversible rxn

• Krebs measured O₂ consumption of pigeon muscle w/ manometer (U-shaped pressure thingy)

  • addition of citrate increased rate of consumption

1. C2 acetyl group added to oxaloacetate (C4 carrier) by citrate synthase

   • citrate formed

   • oxaloacetate binding to citrate synthase creates binding site for Acetyl-CoA (INDUCED FIT)

2. Citrate converted → cis-Aconitate → isocitrate (less stable form)

   • done by aconitase

   • H₂O out, then H₂O in

4. C6 isocitrate converted → ⍺-ketoglutarate

   • done by isocitrate dehydrogenase

   • also converts NAD⁺ → CO₂ + NADH + H⁺

5. ⍺-ketoglutarate converted → C4 Succinyl-CoA

   • done by ⍺-ketoglutarate dehydrogenase (same steps as pyruvate dehydrogenase)

   • also converts NAD⁺ + HSCoA → CO₂ + NADH + H⁺

6. Succinyl CoA converted → Succinate

   • done by succinyl-CoA synthetase

   • also converts GDP + P_i + H₂O → GTP + HSCoA

7. Succinate converted → Fumarate

   • done by succinate dehydrogenase

   • also converts FAD → FADH₂ (less energy to reduce than NAD)

8. Fumarate converted → Malate

   • done by fumarase

   • H₂O comes in

9. Malate converted back to oxaloacetate

   • done by malate dehydrogenase

   • also converts NAD⁺ → NADH + H⁺

substrate channeling

• enabled by large metabolic complexes

• passing of the intermediary metabolic product of one enzyme directly to another enzyme or active site WITHOUT its release into solution

• increase reaction rates and preserve orientation

• ex. ETC

radioactive carbons

• Carbon-14 allowed scientists to track fates of metabolites in citric acid cycle

• learned that the 2C that enter cycle do NOT leave the first time around (from CO₂ produced by steps 4 & 5)

  • carbons cleaved off from bottom to form CO₂ → next carbons in molecule move down

  • symmetry of molecules makes succinate dehydrogenase unable to distinguish orientation after step 5

• 0% radioactivity 1st time → 50% radioactivity 2nd time, etc.

citric acid cycle enzymes

• major oncogenes

  • mutations in isocitrate dehydrogenase produce “oncogenic metabolite”

  • produces 2-hydroxy-glutarate instead of ⍺-Ketoglutarate

  • inhibits histone demethylation → change in gene expression → cancer

• TCA metabolites → source of biosynthetic precursors & cofactors

  • fatty acids, sterols, glutamate, other amino acids, purines, etc…

  • exit of intermediates from citric acid cycle

Electron Transport Chain

• Stage III of metabolism

• converts NADH reduction into a proton gradient

  • intermembrane space → positively charged

  • cytosolic matrix → negatively charged

• iron clusters & other “prosthetic groups” pass e^- downhill

  • decentralized electron clouds

  • Hemes, Fe-S clusters

• Complex I: NADH-CoQ reductase

  • NADH → NAD⁺ + H⁺ sends 2 e^- in

  • e^- pass through Fe-S clusters (different e^- comes out each time)

  • 4H⁺ move against concentration gradient SEPARATE from e^- transfer → transverse helix pump

  • e^- passed to ubiquinone (CoQ) → lipid-like carrier

  • 2H⁺ + 2e^- + CoQ → CoQH₂

  • CoQH₂ transfers e^- to complex III

• Complex II: from citric acid cycle (succinate-CoQ reductase)

  • transfers e^- to CoQ from succinate

  • NOT near other complexes!!!

• Complex III: CoQH₂-cytochrome c

  • e^- transferred by CoQH₂ to cytochrome C

  • 2 e^- hit Q₀ site →1 goes up to Cyt c, other goes down, interacts w/ Q_i site

  • Cyt c dissociates, e^- released, new Cyt c binds to complex

  • unloaded CoQ leaves, new CoQH₂ comes in → 2nd e^- that goes down joins the 1st one in Q_i site, makes another CoQH₂ w/ 2H⁺

  • TOTAL: 4e^- IN, 2H⁺ IN, 2CoQH₂ IN → 2e^- OUT (Cyt c), 1 CoQH₂ OUT, 4H⁺ pumped ACROSS

• Complex IV: Cytochrome c oxidase

  • passes e^- from Cytochrome C → O₂

  • e^- pass down thru prosthetic groups (e^- carriers), meet with O₂ + 4H⁺ → 2H₂O

  • 1e^- per Cyt C, 4 needed to produce 2 H₂O out of 1 O₂

  • 4H⁺ also pumped across gradient

• activity level measured w/ changes in pH

• without O₂ → TCA cycle & ETC STOP b/c NAD⁺ is depleted

• ΔGº’ = 52.6 kcal/mol

electron carrier

• iron clusters and other groups that pass electrons downhill

  • Hemes, Fe-S clusters (ex. in Complex I of ETC)

  • have decentralized electron clouds

• reduction potential (readiness to gain e^-) increases down the chain

  • ΔG decreases

• electrons moved to higher reduction potential groups all the way to O₂ in ETC

proton translocation

• in Complex I of ETC

• 4 separate gated proton channels for 4H⁺ moving against concentration gradient

• separate from electron transfer

• channels have residues with regulated pKa

  • Ka = acid dissociation constant (affinity for protons)

  • pKa = -log₁₀ Ka

  • lysine residues in Complex I bind and release protons in response to CoQ reduction

  • gate = 2 Lys connected to half-channels in membrane

• transverse helix (t-helix) couples changes at CoQ site to conformational changes in pKa of Lys

  • sliding left: CoQ oxidized, H⁺ caught → right: CoQ reduced, H⁺ dropped → repeat

  • PUMP motion

• 300x a second!!

supramolecular assemblies

• parts of the structure are held together by very strong interactions, but not necessarily by covalent bonds

• ex. Electron Transport Chain → complexes are adjacent, molecules do not have to diffuse far

electron shuttle

• how cells regenerate cytoplasmic NAD⁺ pools

• 2 transport molecules → transport intermediate metabolites (malate & aspartate) across inner mitochondrial membrane in opposite directions

  • malate: cytosol → matrix

  • aspartate: matrix→ cytosol

• enzymes couple creation of malate from oxaloacetate to oxidation of NADH → NAD⁺

  • malate travels across membrane → is reduced back to oxaloacetate → NAD⁺ becomes NADH again

• oxaloacetate created from aspartate by transaminase in cytosol, turned back into aspartate in matrix

ATP synthase

• molecule that harnesses proton-motive force from inner mitochondrial membrane to make ATP

• H⁺ want to flow from high concentration → low concentration side: energy can be harvested!

• Split into Fragment 0 & 1 (F₀ & F₁)

• F₀:

  • subunit a → proton half-channels I & II

    • H⁺ flow up through half-channels

  • c ring → made of 10-14 identical c subunits

    • H⁺ flow from half-channel I → bind to negatively-charged residue in c subunit

    • ROTATES 360º until H⁺ reaches half-channel II → flows up & out

    • rotation drives enzymatic activity in F₁ → ATP synthesis

  • stator → made of 2 b subunits & δ subunit

    • holds ⍺ & β subunits in the head stationary while c ring rotates

• F₁:

  • 𝛾 subunit → non-symmetric shape, rotates!!

    • sticks up from c ring to head group

    • rotation physically pushes β subunits thru 3-stage cycle of reaction where they change conformations

    • 130 rotations per second = 1000 H⁺ = 300 ATP per synthase per second!

  • β subunits → 3 configurations

    • O config = OPEN → ADP + P_i can pop IN and OUT

    • L config = LOOSE → ADP + P_i trapped but NON-reactive

    • T config = TIGHT → ADP + P_i converted to ATP (REVERSIBLE)

    • 12 H⁺ needed for 360º rotation = 3 ATP

• ancient innovation → same structure found in bacteria, yeast, chloroplasts, etc.

• can run backwards to generate proton gradient FROM ATP

• ΔGº’ = 3 × 7.3 kcal/mol (50% efficient from ETC)

c ring rotation

• a subunit has Arg-210 residue (like Lys, can be protonated/deprotonated)

  • carries positive charge → interacts w/ negative residue in a c subunit

• proton comes up through half-channel I → binds w/ same negative residue and displaces Arg-210 to next c subunit

  • Arg-210 undergoes conformational change → kicks out fully rotated H⁺ in next subunit to bind w/ negative residue

  • H⁺ leaves out of half-channel II

  • c ring rotates always in ONE direction!!

  • Arg-210 back in original position → REPEAT process

• “Brownian Ratchet” → rotation driven by HEAT/random thermal collisions

  • must be “rectified” to avoid breaking 2nd Law by H⁺ concentrations on sides of membrane

  • Arg-210 = pawl of ratchet

ATP/ADP antiporter

• integral membrane protein that uses secondary active transport

  • uses energetically favorable movement of one molecule down its electrochemical gradient

• how ATP escapes mitochondrial matrix

  • lets ATP out while letting ADP in

chloroplast

• double membrane organelle

  • outer membrane = highly permeable to small molecules

  • inner membrane = less permeable, requires transporters

• intermembrane space = stroma (like cytoplasm)

• stroma contains thylakoids (membranous structure) → form stacks called grana

  • enclose distinct space called lumen

photoelectric effect

• emission of electrons from a material caused by electromagnetic radiation

• ex. ejection of electrons from a metal when light falls on it

• photosynthesis takes advantage of this!

chlorophyll

• photosynthetic pigment that absorbs light energy

• has porphyrin ring structure → central ion is Mg²⁺, has hydrophobic tail

• extensive system of conjugated double bonds around Mg²⁺ where e^- move

• when light (photon) strikes it → excites e^- → they delocalize over bonds surrounding Mg²⁺

  • excited state is unstable

  • excitation energy can be lost in 3 ways:

    1. as heat + fluorescence

    2. transferred to neighboring pigments through resonance energy transfer

    3. eject and transfer e^- w/ high energy to a nearby e^- acceptor → ground state achieved by acquisition of low energy e^- from nearby e^- donor

• look green because mainly blue and red wavelengths of visible light are absorbed while green is reflected

• largely found in thylakoid membrane in photosystems

photosystems

• where light reactions occur during photosynthesis

• 2 of them are coupled in thylakoid membrane → light harvested twice

• Z scheme of redox potential

• evolved from cyanobacteria (produced first O₂ in atmosphere = Great Oxidation Event) → algae → plants

• has a Light Harvesting Complex (LHC) → consists of hundreds of bridging chlorophylls

• has a reaction center w/ special pair of chlorophylls

• light energy captured by chlorophylls in LHC → funneled to special pair in reaction center

  • capture light of different wavelengths → absorbed energy transmits one-by-one thru resonance energy transfer

• energy excites e^- → ejects from special chlorophyll in reaction center to e^- carrier

light reactions

• 3 major complexes: PSII, Cyto bf, PSI

• 5 electron carriers: H₂O, plastoquinone (Q), plastocyanin (pC), ferredoxin (Fn,) NADPH'

• 2 key enzymes: Water-splitting complex (O₂-evolving), ferredoxin-NADP⁺ reductase (FNR)

• Stage 1:

  • Light absorption, generation of high-energy e^-, O₂ formation

• Stage 2:

  • During e^- transport, H⁺ are pumped into thylakoid membrane → proton-motive force formed

• Stage 3:

  • ATP synthesis

Photosystem II

• discovered second, but comes first

• light energy funneled from LHCs → reaction center ejects excited electron from P680 chlorophyll → transferred to Q

• Q picks up 2H⁺ and is reduced to QH₂

• ionized P680 extracts 4e^- from water-splitting complex, one at a time from 2H₂O → oxidized to form O₂ → released into lumen → atmosphere (all O₂ has been generated this way)

  • 4H⁺ also released to lumen → contribute to proton gradient across thylakoid membrane → produces ATP

  • NET: 2H₂O → 4H⁺ + 4e^- + O₂

• P680 = ONLY molecule on Earth that can extract e^- from H₂O

Cytochrome bf complex

• functional equivalent of cytochrome c reductase (Complex III) in mitochondria

• Reduced QH₂ passes its 2e^- to plastocyanin (pC) in 2 steps (Q cycle)

  • increases efficiency of proton pumping from stroma to lumen for ATP synthesis

  • NET: 2QH₂ + 2pC_oxidized + 2H⁺_stroma → QH₂ + Q + 2pC_reduced + 4H⁺_lumen

Photosystem I

• discovered first, comes last

• pC passes e^- to P700 (special pair chlorophyll) → energy of e^- boosted by light energy (photons)

• boosted e^- move within reaction center → ferredoxin (Fn)

• Fn transfers e^- w/ high energy → NADP⁺, which picks H⁺ to form NADPH

  • requires action of ferredoxin-NADP⁺ reductase (FNR)

cyanobacteria

• PSII with water-splitting complex + PSI

  • extract and transfer e^- from H₂O to make ATP & NADPH for CO₂ fixation into sugar

  • produced the oxygen that fundamentally transformed Earth's atmosphere → Great Oxidation Event ~ 2.0-2.5 bya

  • then transferred by endosymbiosis → algae → plants

• 3 key innovations:

  1. P680 chlorophyll in PSII → when oxidized has greater affinity for e^-

  2. Special H₂O splitting complex in PSII

  3. 2 light-harvesting photosystems in tandem

RuBisCO

• constitutes >50% of total chloroplast proteins → most abundant on earth!!

• 8 large and 8 small subunits

• active in presence of CO₂, Mg²⁺, light

• when light intensity increases, Rca (Rubisco activase) removes inhibitor → promotes conformational change in ATP-dependent manner to activate

  • action of Rca is under regulation of Thioredoxin (Tx) in light-dependent manner

• catalyzes CO₂ fixation: CO₂ (1C) + RuBP (5C) = Intermediate (6C) → 2x 3-Phosphoglycerate (3C)

• happens in chloroplast stroma

Calvin Cycle

• fixes CO₂ → glyceraldehyde-3-phosphate (G3P)

• 6 CO₂ → 12 PGA (3C) → 12 ATP used to make 12 1,3-bisphosphoglycerate (3C) → 12 NADPH used to make 12 G3P (3C)

• 2 G3P used to produce sucrose

• 10 G3P recycled → interact w/ 7 enzymes → 6 Ribulose 3-phosphate (5C) → 6 ATP used to convert to 6 RuBP (5C) → REPEAT!

• 18 ATP TOTAL, 12 NADPH TOTAL NEEDED

sucrose production

• converted from G3P primary at night (nocturnally)

• basically the reverse of glycolysis

• 2 G3P (3C) → 1 Fructose 1,6-bisphosphate (6C) x2

• 1 of of the fructose molecules stays as is

• other fructose → glucose 1-phosphate (6C) → UDP-glucose (UTP needed)

• UDP-glucose + Fructose 6-phosphate = 1 sucrose 6-phosphate (12C), UDP OUT → Sucrose (12C)

sucrose

• main sugar transported within plants

• provides constant energy supply to non-photosynthetic tissues (roots, fruits, etc.)

photorespiration

• catalyzed by RuBisCO (also an oxygenase) when CO₂ is low

• O₂ binds to RuBP → PGA + Phosphoglycolate + H₂O → Glycolate x2 → CO₂ OUT, Glycerate produced → ATP used up to convert back to PGA

• produces toxic 2-phosphoglycolate that needs to be converted back to PGA (a COSTLY process)

C3 plants

• major plants that fix CO₂ in all mesophyll cells of leaves

• no special features to combat photorespiration

  • not as efficient at fixation

• ex. rice

C4 plants

• CO₂ fixation is compartmentalized in these plants

  • ex. maize

• enhances fixation and reduces photorespiration

• PEP carboxylase takes up CO₂ → low concentration in mesophyll

  • used to convert to oxaloacetate → NADPH needed → Malate → bundle-sheath cells

  • bundle-sheath cells are deeper in leaves → high CO₂ concentration → Calvin Cycle

  • Malate → NADP⁺ used → Pyruvate → Mesophyll → Phosphoenolpyruvate → + CO₂ → Oxaloacetate → REPEAT

• projects trying to change C3 crop composition to this to be more efficient

chloroplast genetic systems

• most prokaryotic genes were transferred → nucleus during evolution

• 2 separate systems to make functional chloroplast

• ~4500 genes → nuclear genome → transcribed in nucleus → translated by cytoplasmic ribosomes → imported in chloroplasts

• ~90 protein coding genes still maintained in chloroplasts → transcribed & translated WITHIN organelle

  • >300 chloroplast genomes sequenced

  • all contain ~45x rRNAs + tRNAs and ~90x protein-coding genes → encode proteins found in PSI, PSII, Cyto bf, ATP synthase, RuBisCO, proteins involved in gene transcription & protein translation

chloroplast fission

• FtsZ: tubulin-like protein

  • self assembles into dynamic ring of protofilaments (Z-ring) beneath inner membrane of chloroplasts

  • Z-ring acts as scaffold for recruitment of other cell division proteins → generates contractile force → membrane constriction → division

• Dynamin: GTPase protein responsible for endocytosis in the eukaryotic cell

  • comes from cytosol → forms dynamin ring for outer membrane fission

• chloroplast fusion unclear → linked w/ tubular channels called stromules

animals that photosynthesize

• Elysia timida (sea slug) → has chloroplasts on its back

• Scientists created plant-animal hybrids (in hamster cells) w/ chloroplasts

  • maintained electron transport activity in cultured cells for 2 days after incorporation

cell-cell adhesion

• physical connection

• molecules bind to one another and to intracellular proteins

• mediated through membrane proteins called cell adhesion molecules (CAMs)

• strengthened by adding up many weak interactions

  • cell alters adhesion by deciding how many interactions are on its surface

• sorts out cells of different types into clusters

  • H.V. Wilson sponge experiments: mixing cells of two different species makes them grow separately, adhere only to cells of their own species

cell-matrix adhesion

• cells sticking to a non-membrane surface (the ECM)

• adhesion receptors or CAMs stick to ECM and get information from it

  • ECM released by other cells to send info

cell adhesion molecules

• membrane proteins that mediate cell adhesions

• cells express a wide, diverse range

  • many classes (ex. Cadherins, Integrins, Claudin, etc.) that each perform a specific function

  • each class has many individual molecules that share similar structures

• Ex. Cadherins

  • homotypic interactions: extracellular domain binds to similar extracellular domain of another cell

  • calcium binding sites

• Ex. Integrins

  • can function as both cell-cell and cell-matrix molecules

homotypic adhesion

• adhesive interactions between cells of the same type

heterotypic adhesion

• adhesive interactions between cells of different types

trans interactions

• intercellular/adhesive

• CAMs on one cell bind to the CAMs on an adjacent cell

• usually combined with cis interactions

cis interactions

• lateral/in the same cell

• monomeric CAMs on one cell bind to one or more CAMs in the same cell’s plasma membrane

cadherins

• calcium-dependent cell-cell adhesion molecules

• removing calcium → cells no longer adhere

• connect to cytoskeleton via adapter proteins that bind to intracellular domain or to other proteins (ex. Catenins, Vinculin, VASP, ZO1) → eventually bind to actin

adapter proteins

• physically link one protein to another protein by binding to both of them

• directly / indirectly (via additional adapters) connect cell-adhesion molecules or adhesion receptors to elements of the cytoskeleton OR to intracellular signaling proteins

extracellular matrix

• a complex combination of proteins and polysaccharides that is secreted and assembled by cells into a network in which the components bind to one another

• often involved in holding cells and tissues together

• mostly made up of Type IV collagen fibrils, but also Laminin, Entactin, Perlecan

  • Connected to intermediate filament cytoskeleton via Laminin

  • Collagen acts as intermediary between connected cells

collagen

• makes up the ECM (type IV)

• Type I, II, III, IV

• 25% of protein mass of human body

• triple-helix structure like a rope

• built from propeptides that are cleaved and cross-linked

  1. Protein peptides form from Rough ER

  2. Processed in various ways

  3. 3 wind around each other to form procollagen

  4. Transport to Golgi complex

  5. Processing in Golgi; lateral association

  6. Export out of cell

  7. Propeptide cleavage

  8. Higher order fibril self-assembly and cross-linking

• sends important information to the cell that it responds to, changes shape or direction of migration

• scurvy (vitamin C deficit) defect results indirectly from lack of this

  • Vitamin C is necessary cofactor for an enzyme that hydroxylates propeptides (strong interactions → higher annealing temp)

heterogeneous polymers

• characteristic of Collagen IV networks

• globular domains at the ends of triple helices bind to other collagen molecules in specific ways

  • C-terminal end forms dimer w/ C-terminal end of another collagen

  • N-terminal end forms tetramer w/ 3 other N-terminal ends

  • combine in endless series to get network of collagen that attracts other ECM components

laminin

• multi-adhesive ECM protein

• triple-helix and cross structure

• has different globular domains from various peptides that can either attach to each other heterogeneously like Type IV collagen or attach to other trans-membrane proteins (integrins, collagen, other cellular receptors)

integrins

• cell-matrix adhesion molecules

• straighten upon activation

• cell controls ability of laminin binding to them

  • Talin signal binds to intracellular domain, Kindlin binds to Talin

  • Activates extended conformation change that is able to bind to laminin

desmosome

• a trans-membrane structure by which two adjacent cells are attached

• formed from protein plaques in the cell membranes linked by intermediate filament cytoskeleton

  • forms trans-cellular network of “cables”

• disruption compromises epithelial tissue integrity

focal adhesion complexes

• integrins cluster into these rather than being uniformly spread

• act like the “feet” of the cell → important for cell movement

• attach to actin filaments (dynamic force-generating cytoskeleton)

• connect to signaling pathways (intracellular) through intermediate proteins that interact w/ other receptors receiving non-adhesion info

• have a layered structure

  • membrane-apposed integrin signalling layer w/ integrin cytoplasmic tails, adhesion kinase, paxillin

  • intermediate force-transduction layer w/ talin and vinculin

  • uppermost actin-regulatory layer w/ zyxin, vasodilator-stimulated phosphoprotein, a-actinin

  • integrin/ECM layer? actin layer?

endocrine signaling

• type of chemical signaling between cells

• endocrine glands secrete hormones that affect distant target cells through blood/circulatory system

• ex. epinephrine (adrenaline) secreted by the adrenal glands, insulin secreted by pancreas

chemical signalling

• type of communication between cells

• signals are received by cell surface receptors

  • usually chemicals that are soluble so they can be transported through blood

  • receptors help signals cross lipid bilayer and transduce to interior of cell

• signals change internal state of cell:

  • modification of cellular metabolism, function, movement

  • OR modification of gene expression, development

• cellular response can be short-term OR long-term

  • short-term = rapidly executed, rapidly inactivated, NOT “all or nothing”

  • long-term = time frame is less critical, “decision” is more critical, developmental, signals can determine cell fate

• property of ligand binding to receptors according to chemical equilibrium

• competitive balance between stimulation and inhibition

ligand binding

• binding to receptors according to chemical equilibrium

• R + L ←→ RL

  • R = receptor, L = ligand, RL = ligand bound to receptor

  • reversible chemical rxn

• K_d = ([R][L]) / [RL]

  • K_d = concentration of unbound [L] at which half of the total molecules of R are associated with L

• as ligand concentration increases, there is a higher fraction of surface receptors with a bound ligand

  • but the physiological response of cells is often more sensitive than anticipated

• signalling pathways = series of REVERSIBLE chem rxns → binding drives rxn towards HIGH PKA activity

  • [GPCR] + [hormone] ←→ k_bind[GPCR][hormone] / k_release[GPCR:hormone] ←→ [GPCR:hormone]

adrenaline

• hormone that triggers short-term responses

• in cardiac muscle: increases contraction

• in liver: converts glycogen to glucose for release into bloodstream, inhibits glycogen synthesis

• in skeletal muscle: converts glycogen to glucose

• used as vasoconstrictive medicine (EpiPen)

G-protein coupled receptors

• large family of receptors that respond to many hormone signals

• have 7 trans-membrane domains

• loops on exoplasmic face and cytosolic face are specific for a particular ligand to bind on

• ligand binding changes overall conformation of receptor → changes ability to bind other proteins

• hormone binding recruits the G-protein

  1. binding of hormone induces conformational change in receptor

  2. activated receptor binds to G_⍺ subunit

  3. Binding of GTP to G_⍺ triggers dissociation of G_⍺ both from receptor and from G_β𝛾

  4. Hormone dissociates from receptor; G_⍺ binds to effector molecule in GTP-bound state & activates it

  5. Hydrolysis of GTP → GDP causes G_⍺ to dissociate from effector and reassociate with G_β𝛾

• not only for hormones!

  • subfamily that has different specificities for odorants (smells)

  • have own specialized G_olf G-protein

  • ~400 genes in humans, ~1200 in mice, dogs, etc.

G-proteins

• trimeric GTPases that transduce hormone signals

  • ⍺, β, 𝛾 subunits tethered to membrane through attached lipid

• GDP-bound “off” state: subunits are close together

• GTP-bound “on” state: ⍺ subunit dissociates from other subunits

• automatic off switch → subunits have enzymatic activity to convert GTP back to GDP

• dissociate within seconds of ligand binding

• β𝛾 subunits can also activate effector molecules (ex. K⁺ channel)

• some can inhibit effector molecules while others activate them (competitive balance)

  • ex. stimulatory hormones epinephrine, glucagon, ACTH v.s. inhibitory hormones PGE₁ & adenosine

Fluorescence resonance energy transfer

• biophysical technique to determine the proximity of two molecules

• attach fluorescent molecules w/ different absorption & emission spectra to the two proteins to see if they are touching

• shine excitation light on one fluorescent protein and see if it reflects light back or transfers to another fluorophore with an absorption spectrum that is equal to the 1st fluorophore’s emission spectrum (ex. CFP and YFP)

  • second fluorophore will emit light in a different emission spectrum if in close proximity to 1st fluorophore

cAMP

• made by adenylyl cyclase (common effector of activated G-proteins)

  • stimulatory hormone can be epinephrine, glucagon, ACTH

• second messenger

• activates Protein Kinase A (PKA)

Protein Kinase A

• activated by cAMP

• a protein that regulates activity of other proteins by covalently adding phosphate groups to them

• consists of 4 subunits (2 regulatory, 2 catalytic)

  • when activated → catalytic sites begin phosphorylating other proteins

• directly controls molecules of glycogen metabolism

  • stimulation of glycogen breakdown

  • inhibition of glycogen synthesis

signal amplification

• due to multi-step activation

• ex. single epinephrine signal (10^-10 M) → many adenylyl cyclases → cAMP (10^-6 M) → PKA → many activated enzymes → more product

• response of cells is more sensitive than anticipated!!

second messengers

• common feature of signaling pathways

• intracellular signaling molecules released by the cell in response to exposure to extracellular signaling molecules

• different effector molecules will release different ones

  • ex. IP₃ and DAG are produced by stimulation of Phospholipase C (effector)

IP₃

• second messenger

• binds to Ca²⁺ channel in the ER

  • allows calcium to be released from internal stores → cytoplasm

• activates Protein Kinase C

  • binds to other second messenger DAG → phosphorylation of substrates

nuclear hormone receptors

• intracellular receptors for steroid hormones

• hormones can diffuse through lipid bilayer membrane into the cell

• control gene expression and other processes