BIOC 503 - Protein Function III & IV

tetramer

Hemoglobin is a ___ of two subunits (alpah2/beta2)

• each subunit is similar to myoglobin

• 2 alpha2 + 2 beta2

• 4 O2 binding sites

• 4 heme groups

subunit interactions

___ are what form the 4 binding sites for O2 in hemoglobin

• strongest ___ occur between unlike subunits

• changes as O2 binding occurs

deoxy

__-hemoglobin = T state

oxy

___-hemoglobin = R state

His146

When ___ is protonated, on the beta subunit, it forms a salt bridge/H-bond with the aspartic acid residue on that same subunit.

The C-terminal of ___ is also forming a salt bridge/H bond with the lysine residue of the neighboring alpha subunit.

flexible, R state

When the salt bridges/H bonds of the His146 residue break, it makes the beta and alpha subunits more ___ thus favoring the ___.

• make O2 binding more favorable

alpha

long range interactions are more between the 2 ___ subunits of hemoglobin.

T state

tense state of Hb

• more interactions between subunits

• more stable form of Hb

• LOWER affinity for O2

R state

relaxed state

• fewer interactions between subunits

• more flexible

• HIGHER affinity for O2

O2 binding

___ triggers a T to R state conformational change in Hb

• involves breaking ion pairs between alpha1/beta2 interface

smaller

R state makes the pore in between Hb subunits smaller, favoring interactions between them

proximal histidine

___ is the one located on the F helix of Hb and interact with iron, pulling on the helix and making it rotate slightly, causing a big conformational change into the R state.

• this conformational change is triggered by O2 binding

distal histidine

__ the one that does NOT interact with iron, but is helping O2 binding.

• kind of responsible for Hb semi-permeability to O2

• causes steric clashes when CO binds, thus lowering CO affinity wit Hb

sequential

Hb conformational changes follow the ___ model: a change in one subunit causes changes in the neighboring one.

• responsible for low to high affinity transition of Hb aka responsible for sigmoidal curve aka cooperativity

• binding curve for T state only is a low almost linear line

• binding curve for R state only is higher curve looking more like myoglobin binding curve

low

Hb has low affinity at ___ ligand concentration aka in metabolically active tissues

high

Hb has a high affinity at ___ ligand concentration aka in the lungs

66

when reaching the tissues (aka low ligand concentration), Hb affinity for O2 decreases and it releases about __% of the bound O2

7.6

pH of the lungs

higher O2 affinity

7.2

pH of metabolically active tissues

lower O2 affinity

Bohr effect

actively metabolizing tissues generate H+, lowering the pH of blood near said tissues relative to the lungs.

These H+ binds to hemoglobin and stabilizes the T state

pH difference between lungs and metabolizing tissues to protons synthesis increases efficiency of O2 transport by hemoglobin

aspartic acid

H+ released by tissues protonate His146, which form a salt bridge with neighboring ___ residue leading to T state and release of O2

CO2

• produced by metabolism in tissues as waste

• exported in the form of carbamate on amino terminal residues of each polypeptide subunit

• form additional salt bridges when binding, thus stabilizing T state

carbamate

O=C-O^-

happens when CO2 binds to amino terminal residues of polypeptide chains

• formation of ___ yields protons contributing to Bohr effect

• form additional salt bridges stabilizing T state

carbon monoxide

• fit into same binding site on Hb as O2 does

• binds heme 20 000 times better than O2 due to electron pair that can be donated

• highly toxic as it competes with O2 aka blocks functions of myoglobin, hemoglobin, and mitochondrial cytochromes

distal

While O2 binds at an angle, CO would bind perpendicularly to porphyrin ring, but this causes steric clashes with the ___ histidine and thus lower Hemoglobin affinity for CO.

linear

___ interactions between Hb and CO

2,3-biphosphoglycerate

aka BPG

• negative heterotropic Hb regulator

• produced from glycolysis intermediate

• small, negatively charged molecule binding to positively charged central cavity of Hb

• stabilized T state

• allow adaptations to altitude by slightly lowering the affinity of Hb for O2 in the lungs allowing restoration of levels of O2 released in tissues

oxygenation

binding pocket for BPG disappears upon ___ aka when Hb switches to R state

• can only bind T state

single point

Sickle cell diseases due to a ___ mutation from a glutamic acid residue to a valine residue

• from charged residue liking to be on the outside of the protein, to a hydrophobic residue needing shielding

• while shielding itself from solvent, valine causes more interactions with other hydrophobic residues, leading to sickle shape

polymerize

sickled Hb molecule can interact with each other (hydrophobic patches to hydrophobic patches) and ___ to form fibrous-like structure

cellular immune system

• targets OWN CELLS that have been infected

• clears up viruses’ particles and infecting bacteria

• macrophages, killer T cells, & inflammatory T cells

Humoral fluid immune system

• target EXTRACELLULAR pathogens

• recognize foreign proteins

• make soluble antibodies

• keeps memory of past infections

• B lymphocytes & helper T cells

antibodies

__ bind to fragments displayed on surface of invading cells

• Fc receptors

proteins that are produced by B cells that specifically bind to antigens

• mark antigen for destruction or interfere with its functions

• bind to small region (epitome) of antigen

macrophages

large phagocytes ingesting bacteria that are tagged by antibodies

antigen

substance that stimulates production of antibodies

• macromolecular in nature

• foreign to immune system

• coat proteins of bacteria and viruses

• surface carbohydrates of cells/viruses

• epitome = region of antigen bound by antibodies

• 1 antigen can have multiple epitomes

heavy chains, light chains

antibodies like immunoglobin G are made of 2 ___ and 2 ___ that arrange themself in a Y shape.

heavy, constant domain

The base of the Y-shaped antibody is only made of the two __ chains, and form a part called the ___

variable domains

the branches of the Y-shaped antibody are each made of 1 light and 1 heavy chain, and form the 2 ____

• make up the antigen finding site

disulfide bonds

antibodies exist outside of the cell in an oxidative environment, thus they are stabilized by ___

• 1 linking the 2 heavy chains

• 1 linking the left light chain to its heavy chain

• 1 linking the right light chain to its heavy chain

2

light chains have ___ regions/domain

• one constant & one variable

4

heavy chains have ___ regions/domains

indued fit

antigens bind antibodies via ____ thus causing a significant structural change to antibody

ELISA

enzyme linked immunosorbent assay

• based on antibody specificity

1. Coat surface with sample (antigens)

2. Block unoccupied sites with nonspecific proteins

3. incubate with primary antibody against specific antigen

4. incubate with secondary antibody (with an enzyme complex covalently attached to it binding color changing substrate)

5. add substrate

6. formation of colored product indicates specific antigen presence