Protein Function

Ligand

any molecule that is specific and reversible in binding to a protein at a binding site

Ligands can be

proteins, small molecules, ions, peptides, nucleic acids

Ligand binding alters

the proteins conformation or activity

Binding site

region on a protein that specifically binds to a ligand through non-covalent interactions

Binding sites may also be referred to as

active sites in enzymes and orthosteric sites for receptors

Lock and Key model

protein-ligand model where the protein’s binding site is preformed and rigid and perfectly complements the ligand with no conformational change

Why is the lock and key model important

historically significant but often taken over by the induced fit and conformational selection models

Induced fit model

model where the protein undergoes a conformational change when binding to the ligand, optimises complementary bonds between the binding site and the ligand

What happens in the induced fit model?

the ligand induces a shape change in the protein to improve the fit

Cofactor

non-protein molecule/ion required for protein function- can be coenzymes or metal ions or proesthetic groups or cosubstrates

Coenzymes

an organic cofactor often derived from a vitamin and participating in enzyme-catalyzed reactions often carrying chemical groups or electrons

Examples of cofactors

NAD+, FAD, CoA and thyamine pyrophosphate

Every catalytic cycle, cofactors

bind and dissociate

Metal ion

an inorganic cofactor

Prosthetic group

type of coenzyme that is tightly or covalently bound and not released during catalysis and often important in protein structure and function

Examples of prosthetic groups

heme in myoglobin/hemoglobin, FAD in succinate dehydrogenase, and pyridoxal phosphate in transaminases

Cosubstrates

type of coenzyme that are transiently bound to proteins and bind and dissociate each catalytic cycle

Heme

iron-containing porphrin group

Where is heme found

myoglobin, hemoglobin and cytochromes

Structure of heme

protoporphryin IX ring with a central Fe2+ ion that coordinates 4 N atoms of the ring into equilateral positions and one histidine axially with O2 binding the 6th axial site

Fetal hemoglobin

found in the fetus and contains two α subunits and two y ubunits

What do the y subunits in fetal hemoglobin change?

they bind 2,3 BPG less tightly than adult hemoglobin so that the fetal hemoglobin has higher affinity to O2 and allowing O2 transfer from mother to child through placenta

Myoglobin

monomeric globular oxygen-storage protein found in muscle tissue contains a single heme group

Myoglobin displays a

simple hyperbolic O2- binding curve and high O2 affinity

Myoglobin functions to

store O2 in the muscles and allow O2 diffusion into the muscle cells

Hemoglobin

tetrameric O2-transport protein in Red Blood Cells composed of 2 ɑ subunits and two ß subunits each of which contain a heme group

Hemoglobin displays

cooperative O2 binding or sigmoidal binding curve from allosteric communication between subunits allowing efficient O2 unloading and loading

Dissociation constant (KD)

equilibrium constant for dissociation of protein-ligand complexes (PL→P+L)

KD=

[P][L]/[PL]

Lower KD indicates

tighter binding (higher affinity)

KD refers to the

ligand concentration at which half of the binding sites are occupied (M mM etc.)

P50

partial pressure of O2 at which hemoglobin/myoglobin is 50% saturated

P50 is analogous to

KD for O2 binding proteins

Lower P50 means

higher O2 affinity

P50 is higher in

hemoglobin than in myoglobin

What does P50 difference between hemoglobin and myoglobin indicate?

reflects hemoglobin’s role as a transporter rather than just storage

Allostery

regulatory mechanism where binding of a molecule at one site of the protein induces a conformation change that alters the activity or affinity at a distant site of the protein

effector/modulator

molecule binding at an allosteric site

allosteric site

site that when bound induces protein affinity or activity

Allosteric regulation can be either

activating or inhibitory or homotropic or heterotropic

Homotropic

same molecule as the substrate

Heterosteric

different molecule than the substrate

Positive cooperativity

type of homotropic allostery where the binding of one ligand increases affinity for subsequent ligand molecules at other binding sites

Positive cooperativity leads to what type of curve?

sigmoidal or kinetic curve

Examples of positive cooperativity

hemoglobin where the binding of one subunit stabilizes the R state and promotes O2 binding to the other subunits

Negative cooperativity

a type of homotropic allostery in which binding of on ligand molecule decreases affinity for subsequent molecules to bind

Negative cooperativity produces a

binding curve less steep than a hyperbola

Negative cooperativity is less

common that positive and produces a large range of responses

T-state

“tense” low-affinity conformation of hemoglobin in the absence of O2 and has lower affinity for O2

T-state is stabilized by

a network of inner-subunit salt bridges and ion pairs with C-terminus residues

R-state

“relaxed” high affinity conformation of hemoglobin common when O2 is bound to the protein

R-state transfer from the T-state involves

breaking of ion pairs and quaternary structural change

Hill plot

linearised graphical representation of cooperative binding data

Hill plots plot

log(θ/1-θ) vs. log[L] or log(pO2)

A slope of 1 indicates

no cooperativity

What does the slope linear region of the Hill plot mean?

it is the degree of cooperativity

A slope of >1

positive cooperativity

A slope of <1

negative cooperativity

Hill coefficient (nH)

The slope of the Hill plot and quantitative measure of cooperativity out of 4

nH + 1

non-cooperative/hyperbolic

nH for hemoglobin

~2.8-3.0 so strong but not infinite cooperativity

nH > 1

positive cooperativity

nH < 1

negative cooperativity

Immunoglobin G

most abundant antibody isotype in the human serum

Immunoglobin G looks like a

Y-shaped glycoprotein made of two heavy chains and two light chains held together by disulfide bonds

Where are the antigen binding Fab domains?

at the tips of the Y

Where is the constant Fc?

in the base

Immunoglobin G function

humoral immune response neutralizing pathogens and tagging them for destruction

Antigen

any molecule (although typically a protein, polysaccharide, or nucleic acid from a pathogen or foreign substance) that is recognized and bound by an antibody or B/T cell receptor and triggering an immune response

Epitope

the specific region on an antigen that is recognized and directly contacted by the variable domains of an antibody

Epitopes can be

linear (contiguous residues), or conformational (non-contiguous residues brought together by protein folding

Polyclonal antibodies

mixture of antibodies produced by many different B-cell clones, each recognizinf a different epitope on the same antigen

Polyclonal antibodies result from

immunizing an animal with an antigen

Polyclonal antibodies compared to monoclonal antibodies

recognize multiple antigens simultaneously but are less specific

Monoclonal antibodies

identical antibody molecules produced by many different B-cell clones all recognizing the same epitope with the same affinity

Monoclonal antibodies are used in

research (ELISA, immunoprecipitation, Western blots) and therapeutic agents (autoimmune disease treatment etc.)

BPG

2,3 Bisphosphoglycerate, highly anionic metabolite very common in RBCs

BPG is found where in RBCs?

the central cavity of the deoxyhemoglobin T-state and stabilizes the T-state to lower O2 binding affinity

Levels of BPG increase at

high altitude, shifting O2 binding curve to the right and facilitating O2 delivery to tissues