BIOL-540 M4 Protein-Ligand Interactions

2 types of interactions of proteins?

- protein acting as a reaction catalyst, or enzyme, alters the chemical configuration or composition of a bound molecule

- neither the chemical configuration nor the composition of the bound molecule is changed

protein function usually involves ____ binding to ligands.

noncovalent (weak), reversible

what allows an organism to respond rapidly and reversibly to changing conditions?

transient nature of protein-ligand interactions

binding sites are complementary to size, shape, charge, and hydrophobic/philic character of ____.

ligand

true or false: protein interactions are nonspecific.

false; specific ligand

true or false: proteins selectively bind only one or a few types of molecules.

true; specific ligand

how can some proteins bind to different ligands?

separate binding sites

how is flexibility of a protein important in protein-ligand interactions?

protein-ligand interactions are often coupled to conformational changes; a more complementary binding site allows tighter binding, induced fit

in multi-subunit proteins, a conformational change in one subunit often leads to...

conformation change in other subunits

how are protein-ligand interactions regulated?

conformational changes

oxygen can bind to a ____ group.

heme prosthetic

oxygen is ____ soluble in aqueous solutions.

poorly; therefore, its diffusion through tissues is ineffective over large distances

what caused the evolution of O2 transporters?

multicellularity; O cannot diffuse effectively by itself

____ have a strong tendency to bind oxygen.

transition metals (Fe, Cu)

how are free transition metals in reduced form harmful?

reduced metals give away electrons and O is electronegative, so harmful O species form (free radicals)

how can we prevent transition metals like Fe from reaching reduced state?

incorporate Fe into heme, a protein-bound prosthetic group that is permanently associated with protein

true or false: heme is present in all types of -globins.

true; each -globin protein has a single heme

heme consists of a complex organic ring structure called ____ that contains the bound Fe2+ atom.

protoporphyrin

6 bonds that keep Fe in place in heme?

- 4 to N atoms in the flat porphyrin ring

- 2 perpendicular to porphyrin

2 perpendicular Fe coordination bonds?

true or false: Fe3+ binds O2.

false; Fe2+ binds O2 reversibly

coordinating N bonds have electron-donating character to prevent conversion of Fe2+ to ____.

Fe3+

leghemoglobin, a plant O-binding protein, and myoglobin, an O-binding protein found in muscle tissue, are monomeric proteins. hemoglobin, which carries O in blood, is tetrameric (4 -globins). which have tertiary structure and which have quarternary structure?

all have 3º, only hemoglobin has 4º

you are studying proteins that bind to ligand L. based on Ka values, which protein has the highest affinity?

the one with the highest Ka OR lowest Kd

as Ka increases, Kd ____.

decreases (Kd = dissociation constant)

widespread protein family that function in O2 transport

globin

globins have highly-conserved __º structure of 8 ⍺-helical segments connected by bends (globin fold).

3

globins are composed of entirely ____ (2º structure?).

⍺-helical segments

histidine that binds to Fe in globins

proximal histidine (His F8)

4 types of globins in humans and other mammals?

- myoglobin (monomeric)

- hemoglobin (tetrameric)

- neuroglobin (monomeric)

- cytoglobin (monomeric)

globin that facilitates O2 diffusion in muscle tissue

myoglobin

globin that is responsible for O2 transport in the bloodstream

hemoglobin

globin that is expressed largely in neurons to protect the brain from low O2 or restricted blood supply

neuroglobin

globin that regulates levels of nitric oxide, a localized signal for muscle relaxation

cytoglobin

myoglobin consists of 153 residues + __ molecule(s) of heme.

1

equilibrium expression for reversible binding of protein (P) to ligand (L)

where ka = rate constant for forward (binding)

kd = rate constant for backward (releasing)

ratio of the rates of the forward (association) and the reverse (dissociation) reactions that form the PL complex

association constant (Ka)

as Ka increases, affinity ____.

increases (forward rxn favored)

as Ka decreases, affinity ____.

decreases (reverse rxn favored)

Ka is a measure of net ____.

binding

when [L] ____ [ligand-binding sites], the binding of the ligand by the protein does not appreciably change [L].

> (basically, assume excess L)

at equilibrium, individual proteins are constantly binding and releasing ligands; ratio of bound to free proteins (Y) is ____.

constant, where Y = fraction of ligand binding sites bound

true or false: we can measure Ymax.

true; though Ymax is never reached experimentally, we can find 1/2 Ymax point

equilibrium constant for the release of ligand; inverse of association constant

dissociation constant (Kd) = 1/Ka

as Kd decreases, ____ ligand is needed to bind.

less (small Kd = higher affinity)

when [L] = Kd,...

half of the ligand binding site are occupied

protein A has Ka = 6.0 uM-1 and protein B has Kd = 4.0 uM for binding L. which has higher affinity for L?

A

____ of oxygen is the oxygen in the air above the solution which is used because it's easier to measure (value should be the same if air and solution are given time to equilibrate).

partial pressure (pO2)

when pO2 = P50 myoglobin, what is Y?

0

____ binds free heme more than 20,000 times better than does O2.

carbon monoxide (CO)

myoglobin's distal histidine increases heme's affinity for ____.

O2 (watch video on this idk what's going on)

which interaction is more polar: Fe-O2 or Fe-CO?

Fe-O2; O attracts electrons that Fe2+ can give up to become Fe3+, C cannot (partial reduction of O2, partial oxidation of Fe)

after distal His rotates down, HB between O2 (partial -) and imidazole side chain (partial +) of His E7 (distal His)

idfk on this whole slide

red blood cells, transport O2

erythrocytes

erythrocytes are formed from precursor stem cells called ____.

hemocytoblasts

true or false: erythrocytes have organelles and a nucleus.

false; no organelles or nucleus

main function of erythrocytes?

carry oxygenated hemoglobin to tissues distal to lungs

erythrocytes are ____ of globin subunits.

tetramers

true or false: arterial blood is more O2-saturated than venous blood.

true; arteries (96% saturated) carry oxygen-rich blood from the lungs to the body's tissues, while veins (64% saturated) return oxygen-poor blood

main difference between myoglobin and hemoglobin?

myoglobin: monomeric (1 heme), only responsive to large changes in O2, primarily used for O2 storage

hemoglobin is tetrameric (4 heme), sensitive to small changes in O2, used for O2 transport

- low sequence similarity, high structural similarity

2 globin types in adult hemoglobin?

2 ⍺ chains and 2 β chains

what would happen if we used myoglobin rather than hemoglobin for transport?

its high affinity for O would prevent it from ever giving it up

how do we get hemoglobin to release O as we get further from lungs?

high affinity in lungs, lower affinity as we travel away from lungs

myoglobin and hemoglobin have low ____ similarity and high ____ similarity.

sequence, structural

black = same AA (proximal and distal AA)

blue = similar AA

white = different AA

quarternary structure of hemoglobin results from strong interactions like:

- hydrophobic effect

- hydrogen bonds

- ion pairs (salt bridges)

the ⍺1β1 and ⍺2β2 interfaces each involve more than __ residues.

30

the ⍺1β2 and ⍺2β1 interfaces each involve more than __ residues.

19