BIOC 3560: Part A

stable interactions vs. transient interactions

stable:
- long lived interactions
- generally quasi-permanent interactions
- involves prosthetic groups

transient:
- short lived/temporary interactions
- these interactions are on and off (not functioning all the time)
- involves ligands

prosthetic group

- a molecule that is permanently associated with a protein and required for its function
- if the prosthetic group isn't there, the protein cannot function

ligands

- a molecule that is bound reversibly to a protein
- ligand interactions are transient

binding site

- the region of a protein surface that interacts with a ligand

a binding site must be complementary to a ligand in ....

- size
- shape
- charge
- hydrophobic/hydrophilic properties

lock and key model

- the shape of the protein is the same when the ligand is bound vs when it's not
- protein undergoes no conformational change

induced fit model

- structural adaptation between protein and ligand
- conformational change can make biding site more complementary

substrate

- ligands that are chemically altered by the protein (enzyme) they bind

Catalytic site or Active site

- the site on the enzyme where the substrate binds and chemical transformation occurs
- Binds the substrate(s) and catalyzes their chemical transformation

key concepts in protein-ligand interactions:

- reversible binding
- specificity
- conformational change
- regulation

Oxygen key properties

- poorly soluble in aqueous solutions
- inefficient diffusion through tissues

heme

- a prosthetic group
- made of protoporphyrin IX and Fe2+

protoporphyrin

- porphyrin without its metal ion

porphyrin

- four pyrrole rings connected by methine (-CH=) bridges
- can bind a metal ion in the centre
- linked into a conjugated C=C double bond system

propionate group

- heme has 2 of these groups
- heme is very hydrophobic but these groups are near the surface of the globin to interact with their surroundings while heme is buried deep within the protein

Fe3+

does not bind O2 (therefore ineffective transporter for the molecule

Fe2+

binds O2 reversibly

Coordination bonds

- 6 bonds in total
- 4 bonds to nitrogen
- 1 bond to histidine (proximal His)
- 1 bond to oxygen

myoglobin

- monomer (i.e. single polypeptide chain)
- binds and stores O2 in the muscle
- 153 residues
- binds O2 with HIGH AFFINITY
- Mb is essentially saturated
- binding curve for Mb is hyperbolic

hemoglobin

- tetramer: 2 alpha-globins and 2 beta-globins
- O2 transporter
- has LOW AFFINITY for oxygen in the TISSUES
- binding curve for Hb is sigmoidal

leghemoglobin

- found in leguminous plants
- sequesters O2, protecting O2-sensitive enzymes in N2-fixing bacteria
- also acts like an oxygen storage molecule

globins

- globular (fairly compact, almost like a sphere) alpha-helical proteins
- only 2 types of secondary structures: alpha-helices and coils

globin structure naming

- 8 alpha helices denoted A - H (N to C terminus)
- connecting loops are identified by the two helices they join
- when counting the C terminus it starts at the end of Helix H and is denoted HC1 through to the last amino group HC3

Heme binding pocket

- Formed by the E and F helices

proximal Histidine

- His residue (F8) is directly bonded to Fe2+ (5th coordination bond)
- prevents oxygen from binding to this coordination bond and therefore prevents the oxidation of Fe2+ to Fe3+

distal Histidine

- E7
- close but not bonded to heme
- distal His is positioned to interact with the O2 molecule which is bound to Fe2+ on the E7 side making up the 6th coordination bond
- distal his has a hydrogen bond between itself and oxygen

breathing

- small (<1 A) molecular motions of amino acid side chains on a nano second time scale
- the side chains are always slightly moving and shifting around allowing just enough room/time for oxygen to move into the molecule and into the heme and bind to the 6th coordination bond of Fe2+

oxy-heme

- absorbs more blue light so it appears red
- O2 binding affects electron distribution and alters the absorption of light by heme, specifically teh wavelength that light get absorbed

deoxy-heme

- absorbs more red light so it appears blue

[P]

- concentration of free protein (protein that does NOT have a ligand bound)

[L]

- ligand concentration
- can assume that [L] is always in excess

[PL]

- concentration of ligand-bound protein
- also referred to as 'complexed' protein

Kd (dissociation constant)

- equals [L] at which half of the ligand-binding sites are occupied
- measures the strength of the interaction between the ligand and the protein
- high Kd = weak binding
- low Kd = strong binding

Ka (association constant)

= 1/Kd

θ

- occupancy
- fraction of ligand-binding sites occupied
- a hyperbolic function of ligand concentration

pO2

partial pressure of O2

P50

- partial pressure of O2 at which half the ligand binding sites are occupied

Interactions that hold the subunits of Hb together:

- hydrophobic interactions
- hydrogen bonds
- ion pairs
- alpha1-beta1 / alpha2-beta2 interactions are stronger (30 residues) than alpha1-beta2 and alpha2-beta1 interactions (19 residues)

ion pairs / salt bridges

stabilize the conformation of hemoglobin

B1 subunit and His HC3

- forms salt bridges within the beta1 (B1) subunit at Asp FG1 with the alpha2 (a2) subunit at Lys C5
- Only occurs in the T state

T state

- LOW AFFINITY
- Tense state because there is a lot of salt bridges in this state
- oxygen is released in this state in the tissues
- T state = deoxy-Hb

R state

- HIGH AFFINITY
- Relaxed because there are less salt bridges in this state (there are still some but just less than the T state)
- ring has become straight (reduction in its pucker)
- R state = oxy-Hb

Cooperative binding

- O2 binding to one subunit of Hb alters the affinity for O2 in adjacent subunits
- when 1 oxygen molecule binds to Hb, it's easier for a second oxygen molecule to bind and so on
- an allosteric effect

allosteric protein

- a protein in which the binding of a ligand to one site affects the binding properties of another site

homotropic allosteric modulator

- ligand and modulator are identical
- ex. Hemoglobin (activating homotropic modulator)

heterotropic allosteric modulator

- ligand and modulator are different
- ex. ATP affects affinity of a ligand in another site
- can be activators (+) or inhibitors (-)

nH

- hill coefficient
- measures cooperativity

nH < 1

- negative cooperativity
- once one ligand binds it's harder for the next ligand to bind (rare)
- nH could be equal to n in theory but it would mean that there is perfect cooperativity
- nH is USUALLY less than n (number of binding sites)

nH > 1

positive cooperativity

nH = 1

ligand-binding is not cooperative

Concerted (MWC) model

- subunits are functionally identical
- subunits can exist in >1 conformation
- all subunits change conformation SIMULTANEOUSLY (i.e. none of the subunits are ever in different conformations)

Sequential model

- ligand binding can induce conformational change in subunits INDEPENDENTLY
- conformational change in one subunit promotes conformational change in adjacent subunit
- in this model they are still INFLUENCED to change however they do NOT have to change simultaneously

cooperative binding requires

- more than one ligand-binding site
- interactions between ligand-binding sites

anemia

- arises from the lack of Hb containing erythrocytes
- less oxygen binding capacity compared to a normal individual
- capillaries become clogged
- fewer RBC --> less Hb --> anemia

CO poisoning

- keeps Hb locked into the high affinity state so O2 is bound too tightly and will not release in peripheral tissue

Negative heterotropic allostery

- an inverse relationship between binding of O2 versus binding of H+ and/or CO2

Bohr effect

- effect of pH and CO2 on O2
- contributes to binding of O2 by Hb in lungs and release O2 from Hb in tissues

Peripheral tissues

- pH is lower (H+ is high) and [CO2] is high
- Hb binds H+ and CO2, decreasing affinity for O2

Capillaries of the lung

- CO2 is exhaled and pH rises ([H+] going down in lungs)
- Hb releases H+ and CO2 increasing affinity for O2

H+ binding

- H+ binds several side chains in Hb including HC3
- protonation of of His HC3 favours formation of the ion pair with Asp FG1 which stabilizes the T state

CO2 binding

- CO2 reacts with the alpha-amino groups at the amino-terminus (N-terminus) of each globin chain

Carbaminohemoglobin

- A product from CO2 reacting with the a-amino groups at the amino-terminus of each globin chain and contains a carbamate group

Carbamate

- group that participates in forming salt bridges that help stabilise the T-State of Hb
- This reaction creates H+ that contributes to the Bohr effect which lowers pH

2,3,-bisphosphoglycerate (BPG)

- regulates O2 binding by Hb
- BPG binds in the central cavity of Hb
- forms salt-bridges with the two beta subunits
- stabilizes the T state (BPG cannot bind in the R state)
- REDUCES AFFINITY OF HB FOR O2

HbF

- Fetal hemoglobin
- a2Y2
- Lower affinity for BPG // higher affinity for O2
- Kd for BPG higher than HbA

HbS

- Glu A3 on beta-globin is changed to valine so the tetramer has 2 fewer negative amino acids
- Valine is hydrophobic and is now near the outside of the molecule so it causes Hb to change its conformation to form aggregates
- Does not alter O2 allostery or affinity, but rather the tetramers aggregate through an abnormal hydrophobic interaction