Chapter 5 : Function of Globular Proteins

what are the functions of globular proteins?

1. storage of ions and molecules

2. transport of ion and molecules

3. defense against pathogens

4. muscle contractions

5. biological catalysis

ligand

a molecule that binds - typically a small molecule

binding site

region in the protein where the ligand binds

how does a ligand typically bind?

typically binds via non-covalent forces, which enables the interactions to be transient

what is kinetics of a ligand binding reversibly to a site described by?

the association rate Ka and the dissociation rate kd

• measure of protein-ligand affinity by measuring complex formation over time. Kinetics typically occur over a short time, so protein-ligand equilibrium is measured instead

what is the dissociation constant?

the inverse of the association constant

what is the relationship of the protein-ligand complex at equilibrium?

constant

what is Ka equal to?

ka/kd = equilibrium constant

what is the langmuir isotherm eqution?

[PL] = [P]t[L]t/ Kd + [L]T

what does the langmuir isotherm equation tell us?

describes a square hyperbola that is typical of saturable binding. For this gran a non-linear least-squares best fit ( non-linear regression fitting_ provides an estimate of Kd

what is [PL]/[p}t or Y/Ymax referred to ?

the fractional occupancy of the protein receptor

what are the two thermodynamic connections?

1. interaction strength

2. magnitudes

what is the lockey and key model?

proteins typically have high specificity: only certain ligands bind

how is high specificity explained….?

explained by the complementary of the bindy site and the ligand

complementary in…

• size

• shape

• charge

• hydrophobic/hydrophilic character

what is the induced fit model>

conformational changes occur upon ligand binding, this adaptation is called an induced fit. The induced fit allows for tighter binding of the ligand and canin increase the affinity of the protein for a second ligand

how is myoglobin able to function?

1. needs to store oxygen for metabolism

2. protein side-chains lack affinity for oxygen

3. some transition metals bind oxygen well but would generate free radicals in free solution

how is myoglobin prevented from creating free radicals>

• capture the oxygen molecule with a heme moiety that is sequestered inside a protein

• in mammals, myoglobin is the main oxygen storage protein

what is the heme group considered?

a strong chromophore that absorbs in ultraviolet and visible range

in uv-vis spectroscopy what color is deoxymyoglobin?

purplish color

in uv-vis spectroscopy what color is oxymyoglobin?

red

how is oxygen able to be monitored by uv-vis spectrometry?

oxygen binding alters the electronic properties of the heme, and shifts the position of the soret band to 414nm

what is the structure of hemoglobin?

tetramer of two subunits ( alpha2 and beta 2)

each subunit is tertiary structure to myoglobin

how does O2 transport occur in the body?

1. pO2 in the lugs is about 13 pka - hemoglobin binds oxygen here and transports it to the tissues

2. pO2 in tissues is about 4 pka - Hb must release oxygen

how is affinity of oxygen able to change?

• hemoglobin has multiple binding sites

• binding sites are able to interact with one another - cooperativity

positive cooperativity

first binding event, increases the affinity at remaining sites

negative cooperativity

first binding event, reduces affinity at remaining sites

how is positive cooperativity recognized?

typically by sigmoidal binding curves

what is the T state of hemoglobin?

tense state , O2 binding triggers a T-R conformation change

what is the rate state of hemoglobin?

relaxed state, this state has a higher affinity for O2 than the T state

what state is deoxyhemoglobin in?

the subunits are in the T-state

what does the conformational change between the T to R state invovle?

breaking ion pairs between Alpha 1- beta 2 interace

what is the bohr effect?

the pH difference between lungs and metabolic tissues increases the O2 transfer effciencey

which has a higher pH blood in the lungs or capillaries of metabolic tissues?

blood in the lungs

what does affinity for oxygen depend on ?

• oxygen bind well at higher pH

• oxygen is released at lower pH

what is the pH effect on O2 binding to hemoglobin ?

• a major contribution to the Bohr effect is for His HC3 of the beta subunits (other residues also involved)

• when protonated (lower pH) , the His favors the T-state

• when deprotonated , the HIS favors the R-state

what is the hemoglobin CO2 export?

• CO2 is produced by metabolism in tissues and must be exported

• some CO2 exported as dissolved bicarbonate in the blood

• some CO2 is exported in the form of a carbamate on the amino terminal residues of each of the hemoglobin polypeptide subunits

what does the formation of carbamate yield?

yields a proton which can bind to hemoglobin and promotes oxygen dissociation

• when the hemoglobin reaches the lungs, the higher pH(7.6) and high concentration of oxygen promotes O2 binding release of CO2

regulation of O2 Binding by 2,3-bisphosphoglycerate

BPG, naturally present in erythrocytes - purified Hb has substantial amounts of BPG bound, enables the sigmoidal isotherms

• BPG binds in a cavity- lined with positively charged amino acids located between the beta subunits

what state does the BPG stabilize?

T- state