Module 3

Myoglobin vs Hemoglobin

Myoglobin: monomer, singular polypeptide creating single domain

• stores oxygen

Hemoglobin: tetramer with quart structure - similar fold but 4 subunits

• transports oxygen

Heme group coordinates the oxygen that binds

Other ways to describe hemoglobin

Immunoglubulin Gs (antibody)

Tetramer bc composed of 4 chains

• but symmetry indicates that its a dimer of light chain - heavy chain pairs

Different representations of antibodies (Immunoglubulin G)

4 domains in left corner 4 ovals in schematic (two yellows and 2 blues)

4 domains hanging at bottome (all blues) form the constant region

right in bottom is a variable version of Immunoglubulin G fold

• similar to left original one but with some adjustments and additions

Why is hemoglobin multimeric? - why does it have multiple domains/binding. sites

Binding properties comparison

Myoglobin Function

Release of O2 is when oxygen conc in tissue drops

Mb and diving mammals

Proteins and ligand explanation with Mb

O2 is the ligand

Mb and oxygen binding

prosthetic: bc its seperate to the protein

Heme prosthetic group

1 ligand above and below heme ring

• on other sides is the his residue and then O2

Equilibrium Association and Dissociation Constants

Why Kd is preferred over Ka

How Kd looks on the graph + important eq to remember

x-axis: theta = shows how many binding sites occupied

• hyperbolic in shape

• kd is also indicated on the graph - it is the y-axis value when 50% of binding sites are occupied

What Kd value tells us about how tight interactions are

low affinity: weak binding

high affinity: tight binding receptor

avidin-biotin is so tight that its practically irreversible

Biotin

vitamin that can only be provided by the diet

• it binds to protein avidin in raw egg white - eggs’ defence mechanism against bacteria

  • avidin in egg white will extract the biotin out of cell and kill them

Mb and oxygen binding curve

y-axis: conc

x-axis: saturation of binding sites

Could myoglobin transport O2?

Myoglobin binds O2 very tightly and O2

• Myoglobin would pick up O2 very tightly in the lungs and become almost saturated but when moved to the tissues, it will deliver very little

List of traits that would be necessary for an oxygen transporting molecule on the side

Affinity varying with pO2 (hemoglobin)

How it can change it’s affinity to binding O2 like this is bc it changes between conformations that have a high affinity (at lung partial pressure) and then low affinity at tissues partial pressure

Mechanism that helps switch bw high affinity and low affinity states based on O2 conc

R and T States of Hemoglobin

O2 binding in one subunit can trigger the conformational change in both that subunit and others

R and T States crystal structure

salt bridge interactions stabilise the T state

• O2 binding destabilises these salt bridge interactions and allows transition to R state

Important specific subunit interactions

Salt bridge bw positive charge on histadine HC3 side chain and Aspartic acid FG1 carboxylate group

Lys C5 and H HC3 also have salt bridge interactions

Examples of subunit interactions

if oxygen binds to any one of the heme groups, it destabilise and changes to R group

Conformation change triggered y O2 bonding

O2 pulls Iron 2 through the heme ring to other side and flattens the heme ring which stabilises the transition to the R state

Ka changes for cooperative binding - Hill Equation

Ka is the reciprocal of Kd

Hill equation breakdown

What the slope of Hill plot gives us

Hill Plot cooperativity interpretation for hemoglobin

Upper limit of Hill plot - max

What are the two models of cooperativity

concerted or sequential

Concerted model

ALL OR NONE

circles represent subunits in the low affinity T states and squares represent subunits in high affinity R states

• shaded L means there’s a ligand bound to that subunit

In the absence of L, all subunits are in the inactive T or active R form but bc T form is more stable → more would be in that state

• successive binding of ligand to subunits increases the likelihood of that transition from T state to R state

Sequential model

The highlighted in yellow shows the elements of concerted model - physical reality is probably better represented by a composite model somewhere in the middle

Hb also transports H+ and CO2 differently

pH effect on O2 binding to hemoglobin

right shift indicates weaker binding and left shift indicates tighter binding

Effect of H+ on binding of O2 to Hb (lower pH)

Lower pH = lower affinity of Hb for O2 = T state stabilised

Mechanism by which Hemoglobin picks up protons

His HC3 forms that salt bridge with Asp FG1

Effect of H+ on bindings curve mechanism

Hemoglobin and CO2 export

The negative carbonate group also helps form additional salt bridges stabilising the T state but when CO2 released - these salt bridges are disrupted and R state is favoured

Overview of BPG

2,3-BPG binding function - T state

How BPG effects binding of O2 to Hb

BPG binding to deoxyHb

BPG is binding into the cavity of T state of hemoglobin

• it interacts with positively charged residues (Lys)

  • Bohr effect and precense of BPG stabilise T state