MBB 222 - final material

probs more heavily questioned on this

gamma (y) subunits

are protein subunits found in some forms of Hb (in place of beta (B) subunit…)

• mother has A2B2 tetramer

• while fetus has A2Y2 tetramer

fetal a2y2 is less effective at BPG binding + CO2 linkage

• more R state with stronger average O2 affinity than mother’s A2B2

higher y = fewer sickling crises

R state

(relaxed state)

the high-affinity conformational state of hemoglobin when oxygen is bound to it.

• R state » T state O2 affinity

In this state, hemoglobin more effectively binds oxygen, facilitating oxygen transport in the body.

When O2 binds to the iron-heme groups, the subunits shift and rotate by about 15 degrees, breaking certain ionic/hydrogen bonds and transitioning the protein into the R-state

T state

(tense state)

of hemoglobin (Hb) is the deoxygenated, low-affinity conformation of the protein

• ( releases O2 ) and is stabilized by the binding of 2,3-bisphosphoglycerate (BPG) and carbon dioxide (CO2), leading to decreased oxygen affinity compared to the R state.

• When O2 binds to the T state, it pulls the iron atom into the plane of the heme, breaking inter-subunit bonds and rotating the dimers by about 15 degrees to shift into the high-affinity R state (relaxed state

Gibbs Free energy (Delta G)

BACKGROUND INFO: a system (protein molecule, or a popl’n of molecules) can change its state (chemical rx’n).

• thermodynamic state variables changes (temp, pressure, volume, + Gibbs free energy)

DEF’N :measures the energy from the reaction that is

available ("free") for performing "useful work

• system = constant pressure + temp. + closed

• -G = exergonic

• +G = endergonic

Delta G fold= -RT ln Fold

ONLY STATES BEFORE AND AFTER NOT SPECIFIC PATHWAY

exergonic

Refers to a chemical reaction that releases energy, resulting in a negative change in Gibbs free energy (ΔG < 0), which means the reaction can proceed spontaneously.

FWDS RX’N

endergonic

A type of chemical reaction that absorbs energy from its surroundings, resulting in a positive change in Gibbs free energy (ΔG > 0). This reaction requires energy input to proceed.

RVS RX’N

Negative Free gibs energy doesn’t always mean spontaneous, why?

Because spontaneity also depends on the entropy change (Delta H) of the system; if the entropy increases sufficiently, a RX’N with negative Gibbs free energy can still be non-spontaneous.

Kb and delta G bind reflect the proportion of products vs. reactants that will be found at equilibrium

Kb and delta G bind = fixed values

entropy

A measure of the disorder or randomness in a system

• conformational entropy = unfavourable (protein stability) → polypeptide chain has this **** not only source of entropy

Many diff structures count as “unfolded” D state High W and high S “disordered”

Only few structures count as “folded” N state Low W and low S, “ordered”

DeltaSfold’naught > 0 (+) favours folding

DeltaSfold’naught < 0 (-) disfavours folding

• Protein Folding: In N, a protein is highly ordered (low entropy). When it denatures or unfolds, it transitions into a random coil with many possible structural conformations, significantly increasing the system's entropy. [1]

• Hydrophobic Effect: When nonpolar molecules are placed in water, water molecules form highly ordered "cages" around them (low entropy). To minimize this restriction, hydrophobic regions cluster together, releasing the trapped water molecules back into bulk solution, which increases the total entropy and drives spontaneous protein folding.

S = R ln W, W = degeneracy

W= # of possible molecular structures w/ same energy considered the same state

Hydrophobic effect + water entropy

The hydrophobic effect refers to the tendency of nonpolar substances to aggregate in aqueous solutions, minimizing their exposure to water. This aggregation increases the entropy of water molecules, as the ordered structure around hydrophobic molecules is reduced = more disordered state.

• DeltaS(water)fold > 0 forms hydrophobic core

Hydrophobic effect (water entropy) favours …

folding

Water entropy increases from burying apolar groups in the core, but chain conformational entropy decreases by a larger amount

SO net = (-) but not by much

Attractive interactions are optimized,

including straight backbone H-bonds

of alpha-helices, and close atom packing

for London attraction

enthalpy

The total heat content of a system, reflecting the internal energy plus the product of pressure and volume.

• form non-covalent interactions - favourable

• constant pressure + temperature

• energy (Heat + work) added to system during rx’n

DeltaHfold’naught<0 folding is exothermic

DeltaGfold’naught<0 folding is exergonic

molecular interactions

force between atoms:

H-bonds, ionic, vdW interactions are all enthalpic

• Forming internal interactions

  between a.a. groups (deltaH < 0)

• breaking a.a.

  interactions with water (deltaH > 0)

NET IS <0 BECAUSE internal interactions are optimized in strength by precise atom arrangement

• straight H-bonding

• close packing London attraction

Dynamic equilibrium of unfolded + folded proteins

@ equilibrium: a protein = stably folded (N) ; protein molecule spends a tiny fraction of its time in an unfolded state (D)

atoms are constantly in motion from thermal energy; any molecule continually fluctuates in structure, randomly sampling ALL its possible conformations (some more regular, some more rare)

• protein folding usually exergonic: K fold = (- Delta G fold )

• K fold has no units

Protein stability: Enthalpy & Entropy

Protein folding (converting denatured to native state)

is characterized by multiple energetic contributions:

-enthalpy from non-covalent interactions -favourable

-conformational entropy of the chain -unfavourable

-water entropy from hydrophobic effect -favourable

Mb

Means not following chemistry rules; just utilizing concepts

• mixture of chemical species w/ diff protonation states on each ionizable group

• Fraction protonation depends on pH + pKa value of each group

• averaged pool = aggregate species (clumped tgt.)

Aggregate species rule

indicated by prime symbol ‘ (see this in delta g prime naught) for standard free Gibbs energy

naught = all chemistry, not biochemistry

EX: ATP does not exist as a single static ionic structure. Depending on the cellular pH and the concentration of magnesium ions (Mg²⁺), it exists in an equilibrium mixture of multiple forms:

• ATP⁴⁻

• HATP³⁻

• H₂ATP²⁻

• MgATP²⁻

How do we alter conditions for Fold and DeltaGfold’naught?

chemical denaturants ( urea, guanidinium) or high temperature decrease value of Kfold.

Each test tube is at equilibrium with Kfold & deltaGfoldʼ°at

fixed values ...

but these values

depend on the

test tube condition

Cm = melting concentration

-denaturant concentration where 50% of the protein is unfolded (Kfold = 1, deltaGfoldʼ° = 0)

Tm = melting temperature

-temperature where 50% of the protein is unfolded

Altering stability with a mutation

Mutation that disrupts the hydrophobic core (favoured)

(changing an apolar side chain to a polar side chain)

• lowers Cm & Tm (decreases stability)

Activity rule

Express all concentrations in molar units(M)

- then convert to chemical activity(dimensionless)

Energy converted from 1 form to another

1. energy for helices to do work unwinding DNA duplex comes from ATP fuel

2. energy for making ATP through respiration comes from food fuel (plants)

3. Energy for plants to make food fuel comes from the sun

ATP anhydride

A high-energy molecule formed by the removal of a water molecule from ATP.

ANHYDRIDE + H20 = ACID + ACID

anhydride linkage is between beta and y phosphates of ATP.

hydrolysis of anhydride = highly exergonic (-)

• structurally unfavourable → “high-energy” state

• carries PE that myosin converts into physical work

myosin

A motor protein that interacts with actin filaments to produce muscle contraction and movement in cells.

• muscle hydrolyses ATP pulls an actin cable

First Law of Thermodynamics

(Energy Conservation Law):

Different forms of energy are

interconvertible (and this conversion

neither creates or destroys the total energy) → catalytic

in a closed system. It states that the total energy of an isolated system remains constant, although it may change from one form to another, such as c KE to PE.

---

ATP anhydride:

• structurally unfavourable → “high-energy” state

• carries PE that myosin converts into physical work

non-enzymatic ATP hydrolisis releases energy as heat, not work

PE of phosphorylation functional groups

Ns = nucleoside

for an acid hydrolysis = exchanging one OH for another

highly exergonic = higher PE

PE of carbonyl functional groups

.

ATP PE energy: driving an endergonic RX’N

1. Converting an acid to an amide: moderately endergonic(+) (higher PE)

2. Converting an anhydride to an acid: highly exergonic(-)

RX’N 1 + 2 = 3 RX’N 3 (smaller PE)

3. Glu + NH3 + ATP → Gln + ADP + Pi; enzyme ( glutamine synthétase) couples the 2 partial rx’ns for a net run’ = exergonic(-)

Net deltaG depends only on starting vs. ending states

1. Glu + NH3 → Gln (+ H2O)

2. ATP + (H2O) → ADP + Pi

3. Glu + NH3 + ATP → Gln + ADP + P

How do we get to rx’n 3, what about glutamine synthetase

Condensation of Glu + NH3 (Rxn 1) followed by ATP hydrolysis (Rxn 2)

is one way to perform Rxn 3, but that's not how glutamine synthetase

does it!

Rather, glutamine synthetase does Rxn 3 using different steps:

it directly phosphorylates Glu, activating it for amination with NH3.

1. ATP → ADP

2. NH4^+ → Pi

3. RX’n 3 = RX’n (i) + RX’n (ii); H20 doesnt participate in any partial rx’ns

Energetics of familiar enzymes

Enzyme Conversion:

Protease: Amide (+ H2O) into acid with amino released

Nuclease: Ester (+ H2O) into acid with alcohol released

Myosin: Anhydride (+ H2O) into acid with acid released (ADP)

DNA polymerase or RNA polymerase: Anhydride (+ alcohol); dNTP or NTP; into ester with acid released (PPi)

all rx’ns exergonic

DNA Ligase:

Acid (+ alcohol) into ester. endergonic partial rx’n

+

ATP anhydride into acid exergonic partial rx’n

Energy coupling

With coupling by a suitable enzyme,

exergonic cleavage of a linkage with high PE (like the anhydrideof ATP)

can drive the

endergonic formation of a lower-energy linkage

(like converting an acid into an amide or ester)