Biochemistry Exam 2

myoglobin

• has 1 hemoglobin group

• all alpha helical structure

• very small

• same shape as 1 subunit of heoglobin

• tertiary structure

• present in muscles and holds oxygen and makes it available when we’re heavily respiring and need oxygen in our muscles

• sits in the muscle- doesn’t move

hemoglobin

• tetramer

• dimer of dimers- 2 alpha subunits and 2 beta subunits

• quartenary structure

• 4 hemoglobin groups

• can bind 4 oxygens- one at each hemoglobin subunit

• travels in blood- is a major component of red blood cells

• is oxygenated in the lungs (each subunit picks up an oxygen) and then moves to different places in the body that need oxygen and releases it there

hemoglobin group

• has a coordinated iron group in the center that binds to oxygen

• the iron allows for a reversible binding system

  • is a prosthetic group (non AA) that is attached to protein and allows O2 to be released

• senses the environment if it needs oxygen and responds to that

heme/hemoglobin group structure

• conjugated ring structure with an iron center

• protoporphyn ring, synthesized in mitochondria and requires components of citric acid cycle (acetyl coA + glycine)

• anabolic reaction, requires many enzymes to make large conjugated ring structure

• attached in each hemoglobin subunit

• planar rigid structure where Fe is bound

• Fe binds reversibly, attaches well but can be released when needed

oxygen attached to iron center of heme group structure

• 2 histidine residues on 2 different helices which when folded into tertiary structure are close tg in the cleft where O2 binds

• His F8 polypeptide bone (F helix) has a proximal histidine that binds to the heme group

• His E7 polypeptide backbone (E helix) has a distal histidine that H bonds with the oxygen on the heme group

  • this allows oxygen to attach, but because through H bond, is reversible

• Fe is bonded to O2 via coordination bond- coordinate covalent bond

puckered vs planar heme

• proximal histidine (His F8) gives heme group a puckered heme (still in plane but slight puckering of Fe)

• once O2 binds, there is movement of F helix and heme group becomes planar

  • planar allows for reversible oxidation of Fe from ferricyne to ferric iron (?)

deoxygenated hemoglobin state

• no oxygen bound

• in T state (tense)

• tyrosine (+) of alpha subunit H bonds to aspartic acid (-) of beta subunit

• has bigger “donut hole” in center

• not in an optimal position to H bond Asp and Asn

oxygenated hemoglobin state

• oxygen is bound

• in R state (relaxed)

• histidine flips into the donut hole in middle to stabilize this state.

• His is no longer involved in a charge interaction

• aspartic acid (+) of alpha subunit H bonds to asparigine of beta subunit

  • noncovalent bond, so keeps making and breaking the bond to switch between the two states and lock it in those positions

• not in an optimal position to H bond Tyr and Asp

oxygen as a positive allosteric effector

when O2 is bound, causes a conformational shift that allows other subunits to move in a cooperative manner

• 1) no oxygen = T state of 4 subunits

• 2) oxygen binds to 1 subunit, and the 2 subunits touching it get affected by the O2 binding and move to R state, but no O2 bound to those 2 subunits yet

  • they are primed and have higher affinity for O2 now

• 3) oxygen binds to one of those subunits, turning it into its R state

• 4) oxygen binds to the other 2 subunits = fully oxygenated state = full R state

• 5) hemoglobin exits the lungs

= sequential cooperativity

negative effectors

when hemoglobin is full of O2 and leaves the lungs and goes to tissue, it needs to lower its affinity for oxygen to release oxygen in the tissue needed

• conformation change from R state —> T state

• negative effectors bind to do this. they are found at tissue surface

• Co2, 23BP, released H+

• binds to R state, flips hemoglobin from R —> T state, induces release of O2

  • allows for switch between affinities

• the sensors that tell us to switch affinities are the same as those that tell us we need O2

sickle cell mutation + effects

Sickle cell disease results from a β6 Glu→Val mutation that creates a hydrophobic patch; when HbS is deoxygenated, hydrophobic interactions cause HbS molecules to polymerize into fibers, deforming and damaging RBCs

• clogs vessels and has lower life spans (—> sickle cell anemia)

• more details on doc

binding affinity and Kd

Kd = [P][L] / [PL]

y-axis is saturation, so look at x axis’ ligand concentration at 50% saturation

• if a lower concentration (lower Kd) is needed for the same saturation as another molecule, then it has a higher binding addinity

  • need less ligand to fill half the binding sites because of higher affinity

the bohr effect

explains how hemoglobin’s affinity for O2 changes as pH changes

As pH increases, hemoglobin’s affinity for O2 also increases.

• when a tissue is metabolically active, it produces a lot of CO2 and H+ (lower pH)

  • when these are increased (lowering pH), Hb has lower affinity for O2 —> O2 is released

• Hb getting deoxygenated: Hb moves from R —> T state

• The bohr effect releasing free H+ protonates His, giving it a positive charge. Electrostatic attraction between (+) His and (-) Asp help hold parts of the protein together, stabilizing the structure in the T state (deoxygenated state)

  • Free H+ —> protonation of His —> salt bridges form → T state stabilized → O₂ affinity decreases → O₂ is released

metabolically active tissues produce CO2 and H+ ions, which decreases the pH. In this state, Hb has a lower affinity for O2, releasing O2. In contrast, the lungs have a higher pH, which allows for the oxygenation of Hb (due to higher affinity in higher pH) = R state

lipid bilayer

• hydrophilic heads that interact with the aqueous environment

• hydrophobic tails that aggregate to the center of membrane

passive vs active transport

• passive: simple diffusion (ex small hydrophobic molecules), no E required

  • moving from high to low concentration

• active: transport of molecules (ex large, charged) through membrane with help of ATP hydrolysis, E required

  • moving from low to high concentration

channels vs carriers

channels

• passive transport channel: allows charged molecules to pass through membrane via channel and has high (linear) translocation rate (same as diffusion)

  • moving from high to low concentration

carriers

• passive transport carrier: carrier for molecules across membrane, but reaches a point where carriers are saturated bc can only carry x molecules at a time. gets saturated bc requires conformational change

  • moving from high to low concentration

• active transport carrier: driven by ATP hydrolysis to move molecules across a membrane, also reaches a point of saturation. gets saturated bc requires conformational change

  • moving from low to high concentration

K+ channels selectivity

K+ channel is a homotetramer- made up of 4 idential cubunits.

• moves K+ ions from high to low concentration (passive), but only when gate is opened

• provides access to channel but doesn’t prevent other ions (ex Na, Cl) from entering. There’s just no room for them to pass through so they generally don’t enter.

• interior of channel has amino acid groups with their C=O groups pushing out to the core

  • size of AAs resposible for how much they push C=O into channel

• Os interact with the positive K+ ions

• there is enough space between backbone and K+ that allows for K+ attraction towards top of the channel

  • there is not enough space for other ions, ex Na+ or Cl- in the channel. Too large to fit

• Narrowing on channel as order goes K+, H2O, K+, H2O etc

  • H2O provides space between the K+ ions to allow them to line up in channel but prevent repulsion

• this allows K+ molecules to be released extracellularly

primary and secondary active transport channels

• primary: antiporter that brings 2 K+ into the cell and 3 Na+ out of the cell BOTH against concentration gradient

  • builds up Na+ concentration outside cella and K+ in cell.

    • builds important gradient

  • uses ATP hydrolysis (phosphorylation of the channel) as energy

• secondary: symporter that brings 2 Na+ into the cell (down concentration gradient) and uses this gradient as energy to bring I- or anything else into the cell against its concentration gradient

Na+/K+ pump has hyperbolic curve as ion concentration increases

requires E, so is endergonic process

deltaG equation for ion movement across membrane

deltaG = RTln(C2/C1) + ZJVm

• R = 8.314 J/mol

• C1 = ion concentration at destination

• C2 = ion concentration at start

• Z - ion charge

• J = 96480 J/Vmol

• Vm = difference in electrical potential across membrane

  • if destination more positive than start; Vm = +

  • if destination more negative than start; Vm = -

  • Vm = Vm destination - Vm start

inside cell is more negative relative to outside

fluid mosaic model

• membrane in 2D fluid

• proteins and lipids diffuse laterally

• fluid membranes are unsaturated hydrocarbon tails with kinks

  • have double bonds, don’t pack tightly, room to move around

• viscous membranes are saturated hydrocarbon tails

  • no double bonds, tightly packed, don’t move around = not fluid

FRAP: fluorescence recovery after photobleaching

measures fluidity or and mobility of targets

• use a dye marked with fluorescence to dye the hydrophilic heads on membrane (red)

• photobleach some heads

  • makes the head lose its potential to glow = photobleached —> top view shows a black dot

• recovery occurs when the membrane is highly fluid and non-bleached heads move into the space where the bleached ones were, restoring fluorescence in this region

FRAP measures membrane mobility and fluidity

• membrane with more saturated fatty acids will have less mobility and lower recovery

sphingolipids

• everyone has sphingolipids (saturated fatty acid tails with sphingosine head group)

  • is it saturated??

• with Gaucher’s disease, there is an accumulation of these lipids which decreases membrane fluidity

lysosomal storage disorders decrease membrane fluidity

• apart from being a “trash can” lysosomes also break down lipids and recycle their components

• when there is a disorder such as npc mutation, lipids like sphingolipids and cholesterol accumulate abnormally disrupting membrane balance and decreasing fluidity

aquaporins

• membrane channels that form channels that allow water to passively pass through the membrane

• narrow pore size allows only water through

• NPA motif creates dipole-reorientation point, breaking H binding chain to prevent proton hopping

  • water interactions with Arg and Asn prevents proton hopping

• arginine at selectivity filter repels protons bc + repels +

rewatch lecture for this slide

movement in/out of cell due to concentration

• if inside cell is higher concentration than environment (enviro is hypotonic), water will flow into the cell and can cause cell to burst

• if inside cell is lower concentration than environment (enviro is hypertonic), water will flow out of the cell can cause cell to shrink

• isotonic is when cell and environment are at same concentration so water flow/osmosis is in both directions

membrane bound lipid transporters move lipids in cell membranes

• flippase: flips lipids from outer to inner layer, creates a gradient, so ATP dependent

• floppase: flips lipids from inner to outer layer, creates a gradient, so ATP dependent

• scramblase: flips lipids in both directions, not creating a gradient, so no ATP required