Exam 1 (Ch.1, 2, 3, 3+5, 4, 12.3)

Basic Properties of Cells

• Are alive

  • Acquire and use energy

  • Reproduce (cell division)

  • Move

  • Respond to stimuli

  • Self-regulate (e.g. when to divide)

Eukaryotes

• Can be both unicellular & multicellular

• Has membrane bound nucleus (which carries DNA)

• Has membrane-bound organelles

• Big

• Cellular differentiation → specialization

• Ribosome present

• i.e. Animals, plants, fungi, protists

• Has plasma membrane, cellulose in cell wall, cytoplasm, cell wall, flagella, lysosomes

• Cell division by mitosis

Prokaryotes

• Unicellular (single-celled)

• No nucleus (which carries DNA)

• No membrane-bound organelles

• Small

• Ribosome present

• i.e. Bacteria & archaea

• Has plasma membrane, pilus, flagella, cytoplasm, peptidoglycan in cell wall

• Cell division by binary fission

• No histone proteins w/ DNA

Ribosome

• Present in both prokaryotes and eukaryotes

• Non-membrane bound organelle

• Made by proteins

  • RNA & protein

  • Rough ER → ribosome on ER

Cells evolution

• Last common ancestor → 3 cell types

  • Prokaryotes: Bacteria, Archaea

  • Eucarya: Eukaryotes

Electron microscope can measure …

1 nm-100nm

Standard light microscope can measure …

1 um-100 um

Human vision can measure …

1 mm and above

Proteins

• Made up of enzymes

• Are polypeptides → chains of amino acids

  • Have backbone and R groups

• Functions

  • Transport

  • Motors (help in movement)

  • Cell signaling (enzymes or hormones)

  • Gene expression (RNA pol)

  • Structural proteins (present on cell membranes)

How does protein shape determine function?

• Unique shape/structure determined by unique amino acid sequence

• R groups → amino acid properties

• 3 things determine shape

  • 1) # of amino acids

  • 2) Types of amino acids

  • 3) Organization of amino acids

4 Levels of Protein Structure

1. Primary → amino acid sequence (number, types, organization of them) → determines protein shape

2. Secondary → backbone interactions (non covalent interactions in backbone determines secondary structure)

3. Tertiary → R group interactions

4. Quaternary → R group interactions

Covalent Bonds

• Strong bonds

• Shared electrons

• Non-polar covalent bonds: electrons are equally shared; no partial charges

• Polar covalent bonds: one atom has partial neg charge, other has partial pos charge due to unequal distribution of charge

  • contain 1 or more electroneg atom (O, N, S)

• Formation of covalent bond is accompanied by release of energy which must be reabsorbed at some later time

  • large energy needed to cleave C-H, C-C, or C-O covalent bonds

  • stable under most conditions b/c thermal energy of a molecule = too weak to break a covalent bond

Polar molecules are …

Hydrophilic b/c can react w/ water

• easily dissolves in water

Non-covalent bonds

• Weak bonds in cell due to presence of water, but strong within core of protein (where water is often excluded and where they are collected together)

• Ionic bonds: attraction between + and - ions (attraction between fully charged component)

  • dissolves in water very easily

• Hydrogen bonds: attraction of partial charges on polar molecules (need partially (+) charged hydrogen atom in molecule)

  • weak, easily broken

  • form between a bonded electroneg atom (N,S) w/ a partial neg charge AND a bonded hydrogen atom that bears a partial pos charge

  • strength = additive

• Hydrophobic interactions: association of nonpolar molecules (= hydrophobic)

  • clumping together minimizes exposure to polar molecules

• van der Waals forces: weak attractive forces when atoms are very close/ due to close proximity

Nonpolar molecules are …

• Hydrophobic b/c no charged regions that would attract them to poles of water molecule

  • when mixed w/ water, they clump up together which minimizes their exposure to the polar surroundings

Protein Secondary Structure

• Local folding of the protein backbone which are stabilized by hydrogen bonds → backbone interacts w/ backbone

  • Hydrogen bonds are mainly between backbone atoms

  • R groups “stick out” from the backbone

• 2 types

  • 1) α helix: protein backbone coils into a spiral/spring w/ R groups sticking outward

  • 2) β pleated sheet: folded/pleated strands

Protein Tertiary Structure

• Final 3D shape of one polypeptide chain (folded shape of 1 polypeptide) → R groups interact w/ R groups

• Types of R group interactions that are non-covalent

  • van der Waals/hydrophobic interactions

  • H-bonds

  • Ionic bonds

• Types of R group interactions that are covalent

  • Disulfide bond between R groups of two cysteine groups

How to identify side chain?

• Look for the central carbon and identify the group attached that’s not:

  • amino group (NH₂)

  • carboxyl group (COOH)

  • hydrogen (H)

Different types of amino acids that are determined by R groups

• Polar charged: clearly written (-) or (+) on side chain → name includes acid if (-) side chain

• Polar uncharged: all oxygens on side chains are uncharged/no explicit written charge

• Nonpolar: side chains are primarily carbons and hydrogens; have no oxygen

• Side chains w/ unique properties

  • Glycine: has tiny H as side chain, making it very flexible

    • hydrophobic/hydrophilic

  • Cysteine: has side chain containing -SH that can form disulfide bonds which help stabilize intricate shapes of proteins (especially those present outside cells)

    • covalent

  • Proline: has a side chain that can loop to the backbone, making the protein bend/less flexible → turns = rigid

    • hydrophobic

How does the charge of side chains change depending on pH? (Specific to polar charged amino acids)

• The side chain can gain or lose H⁺

  • at low pH (<7) → lots of H⁺ → groups tend to gain H⁺

  • at high pH (>7) → less H⁺ → groups tend to lose H⁺

• Make sure to compare with original side chain before it gains/loses H⁺

Properties of side chains: Polar uncharged amino acids

• Hydrophilic side chains tend to have partial (+) or (-) charge allowing them to:

  • participate in chemical rxns

  • form hydrogen bonds

  • associate w/ water (can form hydrogen bonds w/ water)

• Often reactive

Properties of side chains: Nonpolar amino acids

• Hydrophobic side chains consists almost entirely of C & H atoms (lack O & N)

• Tend to form inner core of soluble proteins, buried away from aqueous medium

• Play important role in membranes by associating w/ lipid bilayer

• Can’t form electrostatic bonds or interact w/ water

For disulfide bonds between cysteines, bonds are …

• Broken in reducing environment/after reduction

  • S-S bond breaks

• Made in oxidizing environment/after oxidation

  • S-S bond forms

  • so, disulfide bonds play more important role in structure in proteins that function in an oxidizing environment

Soluble proteins

• Soluble in water

• Inside of cell compartments, it’s mostly water

  • cytoplasm/cytosol

  • inside of nucleus

  • mitochondrial matrix/intermembrane space

• B/c of this …

  • Surface amino acids = hydrophilic & polar so they can associate w/ surrounding water and contribute to proteins solubility in aqueous solution

  • Core amino acids = hydrophobic & nonpolar, often tightly packed together

• Fatty acid tails are hydrophobic → proteins touching tails = hydrophobic

• Fatty acid heads are hydrophilic → proteins touching heads = hydrophilic

How can we find out the 3D (tertiary) structure of proteins?

a) X-ray crystallography ***

• proteins it works on can be crystallized

• Advantage: can provide higher resolution structures for larger proteins

• Limits: limited by need to get any given protein to form pure crystals

b) Nuclear Magnetic Resonance (NMR) spectroscopy

• Use for proteins that can’t be crystallized, provides info about dynamic changes in protein structure

• Limits: more difficult to apply as size of protein increases, less resolution than x-ray crystallography

Domains

• Protein tertiary structure = overall 3D folded shape of a protein → within that shape are domains (distinct folded regions)

• Traits of domains

  • distinct modules/structure and shape → perform specific functions

  • fold independently from each other

  • diff types/combos of modules

  • can be shuffled to create new proteins = protein evolution

    • domains can change due to mutation or environment

Protein-protein interactions

• Are interactions where proteins bind to and work with other proteins

• Happens in multiprotein complexes → where several proteins assemble together to perform a function

  • Stable interaction: proteins stay together for a long time

  • Dynamic interaction: proteins temporarily bind, then separate

• Proteins have complementary surfaces (shape, charges, hydrophobic/hydrophilic regions) so they know what to bind to

Interactomics

• The complete set of interactions between proteins in a cell/organism

• Hub proteins: a proteins interacting w/ several other proteins = important connection point in interaction network

  • Interactions can change depending on cell’s condition (presence/absence of light, nutrients or fasting, etc)

Protein folding

• Final 3D structure/protein shape depends on primary structure (amino acid sequence)

• Evidence: experiments w/ Ribonuclease

  • if you unfold a protein and then remove the chemicals that unfolded it, the protein can fold back into its original shape (Note: folded = stable structure)

  • Protein becomes unfolded and denatured w/ treatment of:

    • Urea: disrupts the weak interactions that help maintain protein’s shape by interfering w/ hydrogen bonds & hydrophobic interactions

    • Mercaptoethanol (reducing agent): in reducing environment, breaks/interferes w/ disulfide bonds (aka covalent bonds facilitating tertiary structure)

  • If both are removed, primary structure reforms and acquires tertiary structure again aka renaturation and refolding → ribonuclease is capable of self-assembly

1st and 2nd Law of Thermodynamics

• 1st Law

  • Total amount of energy is constant but can be created from one form to another

  • Amount of energy in a system can change vs. surroundings

    • System gains energy from surroundings

    • System loses energy to its surroundings

• 2nd Law

  • events in the universe go from high energy to low energy → spontaneous events (go from high energy to low energy; no energy needed)

  • energy transductions: some energy lost to surroundings

Entropy, S

• Randomness/disorder

• Increases w/ spontaneous events (high energy → low energy) & loss of energy → more randomness, doesn’t req energy

  • Spontaneous events: energetically favorable, -∆G, exergonic (releases heat)

• Decreases w/ nonspontaneous events → less randomness & more orderliness, req energy input

  • Nonspontaneous events: energetically unfavorable, +∆G, endergonic (absorbs heat)

Reversible rxns w/ ATP

• Like a rechargeable battery

• ATP hydrolysis: ATP + H₂O → ADP + Pi (battery depletion)

  • energetically favorable, -∆G, produces/releases energy that cells use to do work, spontaneous, increases entropy

• ATP synthesis: ADP + Pi → ATP + H₂O (battery charging)

  • energetically unfavorable, +∆G, nonspontaneous, supplies energy to make ATP, decreases entropy

How do cells control chemical rxns?

• In a closed system, rxns tend toward equilibrium (concentration of reactants remain unchanged but aren’t the same, rate is equal)

  • At equilibrium: high entropy (more stable, spontaneous) & low amount of energy to do work

    • ADP will be higher at equilibrium b/c is spontaneous

• But after some time, ATP amount will deplete since producing ADP is favorable → no more energy production = cell death (ADP will be higher in amount than ATP)

• Cells aren’t closed systems → aren’t at equilibrium, but at steady state where concentration of reactants & products are controlled by cell constantly using energy and replacing what it uses

• Cells at steady state

  • In dynamic nonequilibrium

  • Use energy from surroundings to make ATP → continually obtaining energy and making ATP = plenty of free energy to do work → hydrolyze/break down ATP to do work

    • has low entropy (high order)

• Ultimately, ATP hydrolysis can be coupled to energetically unfavorable rxns (nonspontaneous/endergonic rxn)

  • ATP hydrolysis supplies energy needed for nonspontaneous rxns

  • To make endergonic rxns go, cells couple endergonic rxns w/ exergonic rxns

Enzymes

• Catalyze (speed up) chemical rxns (both exergonic and endergonic), but can’t start or end rxns

  • Note: in endergonic rxns, energy still must be supplied regardless of enzyme presence → can’t make unfavorable (endergonic) rxns go w/out supplied energy

• Most are proteins (some RNA, ribozymes)

• Exhibit properties

  • 1) Req only in small amounts

  • 2) Not altered irreversibly during course of rxn, so each one can participate repeatedly in individual rxns

  • 3) Have no effect on thermodynamics of rxn

    • don’t supply energy → don’t determine whether its thermodynamically favorable or unfavorable

    • don’t determine ratio of products to reactants at equilibrium

Breaking peptide bonds

• Req energy to make peptide bonds, so when broken, energy is released = exergonic

  • Spontaneous

  • Energetically favorable

  • Increases entropy

  • Produces energy

• Process is slow b/c peptide bonds are stable → activation energy (E_A) needed to break bond → need an enzyme to speed up rxn rate b/c it reduces activation energy (aka the minimum energy needed to overcome to start a rxn)

  • How? It binds to and stabilizes transition state/enzyme substrate state which is the transition between reactants & products and where bonds are being made and broken

Enzyme-substrate complex: How does an enzyme bind a substrate and turn it into products?

• Substrates are reactants/what the enzyme acts on; don’t have to just be proteins

• Substrate binds to enzyme’s active site due to both having complementary shapes (fit together) = enzyme-substrate complex

  • held together by noncovalent bonds → weak bond so easier to release substrate, but sometimes can temporarily form covalent bond

  • some enzymes require cofactors to function (i.e. metal ions)

• Enzyme mechanisms

  • a) Aligning substrates to bring them close together and position them correctly

  • b) Changing substrate reactivity by making it more likely to react

  • c) Inducing strain: enzyme binds substrate and slightly distorts/bends it so that it’s easier to break its bonds or form new bonds = more reactive

    • Induced fit: conformation shifts so that the complementary fir between the enzyme & reactants is improved & proper reactive groups of the enzyme move into place

Enzyme rates depend on …

1. Substrate concentrations (to a point)

2. pH

3. Temperature

4. Inhibitors

Enzyme kinetics: How does changing amount of substrate affect how fast enzyme makes products?

• Velocity (v) = rate of rxn

• Rate of enzymatic rxn varies w/ concentration of substrate, [S] → relationship not linear but exponential (non-enzyme catalyzed rxn is linearly increasing)

  • Increasing substrate concentration = increase in enzyme rxn rate until it reaches V_max (where enzyme’s saturated w/ substrate) and plateaus since there’s no more enzyme available to couple w/ increasing amount of substrate

• K_m = substrate concentration when rxn’s at ½ V_max / measure of enzyme affinity for substrate

  • Lower K_m = less substrate req to meet ½ V_max, so higher affinity between enzyme & substrate

How do enzyme rates also depend on pH and temperature?

• There is an optimum pH and temp for enzymes to function best, so more or less than this pH/temp negatively impacts enzyme’s functions

• Why?

  • pH: changing pH (ion concentrations) changes bonds → enzymes loses functional structure

  • Temp: has a denaturing effect → breaks bonds and changes enzyme shape which changes structure & function (abnormal structure = abnornal function)

Inhibitors