3: Protein function

How many peptides make up hemoglobin?

4 - tetramer

How many peptides make up myoglobin?

1 - monomer

What does myoglobin do?

store oxygen in muscles

release oxygen when rapidly contracting muscle need energy (ie. O₂ levels drop)

What does haemoglobin do?

transport oxygen

• responds to O2 concentration, blood pH, regulators

• take back CO2

What are myoglobin and hemoglobin called?

homologues - more specifically paralogues

What are paralogues?

• closely related

• same folds

• similar sequence identity

Describe the structure of myoglobin and haemoglobin

• each subunit of haemoglobin is similar to myoglobin

• myoglobin is a single chain that folds into a globin fold, haemoglobin has four globin folds

• heme group - contains an iron II center (allowing oxygen to bind)

Describe the structure of haemoglobin

• tetramer (four parts)

• dimer of two alpha-beta ‘protomers’

What is a protomer?

structural unit of a protein with a quaternary structure ie. aplha/beta pair in haemoglobin

What is an example of quaternary structure and describe the structure and function

• immunoglobulin Gs (IgGs)

• four subunits: 2 heavy 2 light chains

• involved in binding to antigen ligand

• variable region on each chain undergoes conformational change when bound

• 2 antigens can bind

• immunoglobulin fold

Why do antibodies have quaternary structure/multivalent?

• more efficient binding

• if one fails, still have backup

What is multivalency?

ability to bind to multiple things

Why does haemoglobin have four binding sites/quaternary structure?

• allows for more oxygen to bind

• allosteric cooperative binding

Is myoglobin more abundant in land or sea animals?

animals which dive

What colour does myoglobin give to muscles?

increased myoglobin = increased dark red muscle

ligand vs binding site

oxygen = ligand

myoglobin/protein = binding site

What is the prosthetic group?

non-protein that is bound separately to the protein ie. heme group in myoglobin

Eqm association vs. eqm dissociation constant

K_a vs K_d

is K_d or K_a preferred in biosciences?

K_d as it is useful in expressing ligand binding as it represents the conc. of free ligand at which protein is 50% saturated

high affinity vs low affinity of K_d values

high = 1 × 10^-15 M

low = 1 × 10^-2 M

What is the highest affinity? Why?

biotin

Can myoglobin transport O2?

• binds oxygen very tightly (high pressure in lungs)

• will not release it (low pressure in tissues)

Why is haemoglobin so good at what is does?

conformational state changes (tight to retain O2, loose to release)

• for effective transport, affinity must vary with pO2

R and T states

T = tense state

• more interactions, more stable

• lower affinity for O2

• stabilised by variety of salt bridge interactions

R = relaxed state

• fewer interactions, more flexible

• higher affinity for O2

How does O2 binding affect haemoglobin?

O2 binding triggers T→R conformational change

• breaking salt bridges b/w residues at alpha1-beta1 interface

T = His outside

R = His inside

What stabilises T state of haemoglobin? What destabalises it?

salt bridge between His HC3 (last residue of beta subunit) and Asp FG1

• also between Arg and Asp in alpha subunits

• and C terminus of alpha 1 and N terminus in alpha 2

destabilised by O2 binding - via flattening of heme group and breaking of salt bridges

What does HC3 and FG1 mean?

third residue, C-terminal to helix H

first residue between loop F and G helices

What is the Hill equation?

theta = fraction of binding sites occupied

L = free ligand

What is a Hill plot?

y vs. x

What does the slope of a hill plot show?

• measure of the degree of interaction (ie, the degree of 'cooperativity') between binding sites

• indicates the interaction between binding sites rather than the actual number of binding sites

What is the Hill coefficient?

the slope of the hill plot n_H

What does n_H > 1mean?

• positive cooperativity

• binding at one site increases binding at other sites

• eg. Hb and O2

What does n_H = 1mean?

• binding is not cooperative

• sites are independent

What does n_H < 1mean?

• RARE

• negative cooperativity

• binding at one site decreases binding at other sites

What is the theoretical upper limit of n_H?

n, experimentally however it is almost always lower ie. < n

What is n_H related to?

average occupancy of the binding sites not total

What are the models of cooperativity?

concerted and sequential

What is the concerted model of cooperativity?

• all or nothing

• in the absence of L, all subunits of a multimer are thought to be in the inactive T (more stable) or the active R form

• circles = T, squares = R, shading and L = ligand binding

• inactive state destabalised by L binding

• all subunits transition from T to R simultaneously

What is the sequential model of cooperativity?

• each subunit of the multimer can be in either the T or R form

• L binding procedures a change in conformation of the subunit

• a change in confirmation in one subunit induces a similar change in an adjacent subunit

• therefore binding of a second L is more likely

Are the two models of cooperativity mutually exclusive?

NO! the sequential one has concerted in it.

What are the end products of metabolism in tissues?

H+ and CO2

how much of H+ and CO2 does Hb transport?

40% of tissue H+ and up to 20% of CO2 to lungs and kidneys

True or False: H+, CO2 and O2 are all transported in the same way?

False! O2 is not transported the same way as H+ and CO2

True or False: H+, CO2 and O2 are all competing to bind to the heme group??

False!

How is H+ produced?

• metabolism directly

• when CO2 reacts with H2O to form HCO₃^-

How does pH affect O2 binding to Hb?

lower pH (higher [H⁺]) = lower affinity = help offload O2

higher pH = higher affinity = retention of O2

What are actively metabolising tissues generates?

H+ - lowering pH of blood

What happens when H+ binds to Hb?

• stabalises T state

• protonates His HC3 → forming a salt bridge with ASP FG1

• release of O2 into the tissuesf

What is the Bohr effect?

the pH difference between lungs and metabolic tissues increases efficiency of the O2 transport

How are protons (H+) transported?

• N-terminal of alpha-subunits

• His¹⁴⁶ (His HC3) of the beta subunit

• other amino acid residues

How is CO2 transported?

• 15-20% as carbamate on amino terminal residues

  • yields a proton → Bohr affect

  • forms additional salt bridges, stabilising T state

• CO2 release in lungs favours the R state

What is BPG?

2,3-bisphosphoglycerate

• derived from intermediate in glucose metabolism

• highly negative

• stabilises T state

Where does BPG bind?

on Hb at a seperate binding site to O2 but still affects O2 binding

• allosteric

• at the cavity in the middle of T state

Where is BPG found?

in RBCs

True or False: BPG is an allosteric regulator of Hb?

True! affects binding of O2 without binding to O2 site

What is the best stabiliser for T state?

BPG

What does BPG allow for?

• O2 release in the tissues

• adaptation to changes in altitude

Where is increased BPG => Hb bind O2 more weakly advantageous?

high altitude

• At sea level, about 38% of O2 in saturated Hb (in the lungs) is delivered to the tissues

• At high altitude, pO2, in lungs decreases so Hb is less saturated and O2 delivery decreases to 30%

What binds to Hb and where?

H+, BPG and CO2

all at different ALLOSTERIC sites

What do H+, BPG and CO2 do to Hb?

stabilise T state (by different mechanisms to lower affinity of Hb for O2

What do allosteric affects of H+, BPG and CO2 give rise to?

cooperativity of the binding due to changes in the conformation that are transmitted through the subunits of Hb

What can you call H+, BPG and CO2?

ligands

Is CO or CO2 more toxic? Why?

CO

• CO2 is transported in a different way than O2 (as carbonate ion)

• CO can take up O2 binding sites - heme group → and much more tightly → competitive inhibitor

Does CO stabilise Hb like O2?

Yes! stabilises R state just like O2 does