Interpreting Effects of Allosteric Agents

pO2

FractionalSaturation

100

50

Shift to R state Shift to

T state

CO2

BPGH+

OH-O2

Release O2

Bind O2

-Hb 10

80

95

4 RULES Governing Allosteric Proteins

RULE: Allostery is a property of proteins that containmultiple subunits or a single subunit with multiple binding sites.

RULE: Allosteric proteins must be able to bind more than one ligand.

RULE: A ligand has the capacity to induce a change in the shape of an allosteric protein.

RULE: By changing the protein’s shape, a ligand can facilitate (positive) or deter (negative) the binding of additional ligands to the protein.

MODELS OF ALLOSTERY

CONCERTED: (ALL OR NONE)

Subunits exist in two conformational states

All subunits have the same conformation; no hybrids

Ligand binds to any subunit in either conformation

See Strategies p.120

Conservation of symmetry is the driving force

The two states are in equilibrium

Problems with Concerted Model

• Symmetry is not preserved in most oligomeric proteins

• Some allosteric oligomeric proteins do not have identical subunits

• Cannot explain in a two state model how negative and positive cooperativity occur in the same protein

Sequential Model

S S S SS S S

S

S S

S

S

S

S

Ligand binding induces change in subunit

Change is progressive

Symmetry is not preserved

Mechanical coupling between subunits may be weak

Sickle Cell Anemia(a genetic disease)

One Amino acid in a protein can make

a difference between life and

death

Is it a membrane protein?

Is it a cytosolic protein?

Distortion

Is it something other than a protein?

Hb (alpha) N- Val-Leu-Ser-Pro-Ala-Asp-Lys-Thr……..

Hb (beta) N- Val-His-Leu-Thr-Pro- -Glu-Lys…….GluVal

Glu6 Val6 (each beta chain)

Glu GAA or GAG

Val GUA or GUG

A U replaces an A in the codon (a pyrimidine/purine exchange)

Transposition

Sickle Cell

Val

Polymerized deoxy Hb Aggregates Insoluble fibers

Distorts the overall shape of hemoglobin

Distorts the overall shape of an erythrocyte

Distorted erythrocytes cannot pass through capillaries

AntibodiesAncient Greeks:

Thucydides (460-400 B.C.) Speaking of the plague of Athens,

Yet, it was in those that had recovered from thedisease that the sick and dying found most compassion. These knew what it was from experience,and had now no fear for themselves; for the sameman was never attacked twice-never at least fatally.

Early Theories

Pauling 1940: The “Instructive” Theory

Antigens act like templates that direct the folding of a nascent antibody chain

MacFarlane Burnet: The Selective or clonal theory

The combining site on an antibody moleculeis completely determined before it encountersan antigen

leukocytes macrophages

lymphocytes

Bone Marrow

T-lymphocytes (T-cells)B-lymphocytes

Antibodies

Cytokines

Cytotoxic T cells (TC cells)

Helper T cells(TH cells)

Precursor

Humoral

Cell-Mediated

Essential Features• Each antibody-producing B-cell makes a single

kind of antibody…no antigen is needed.

• Specificity is determined by amino acid sequence…each cell’s DNA is distinctive.

• Immature cells that make antibodies are destroyed early in life…self tolerance

• Mature cells make and display antibodies on their surface

• Interaction with antigen triggers cell to divide, making large amounts of a particular antibody that persists until antigen is gone

Antibodies are large complex molecules

Composition:

2 heavy, 2 light chains

Hinge region

Disulfide bonds

Antigen-binding site

Constant Variable

5 Classes of Antibodies (Immunoglobulins, Ig)

• IgA…external secretions, tears,saliva, bronchial and intestinal mucous

• IgG…principal antibody in the serum, originally called gamma globulin

• IgD…least understood

• IgE…no clear function, has receptor on mast cells and stimulates production of histamine and is linked to allergic responses

• IgM…first class to appear, highest combining sites, effective against bacteria

Mechanism• Preexisting B cell synthesizes IgM first

• IgG, IgA, IgD, IgE of same specificity are made later

• Light chain is unchanged during switch

• Variable region of heavy chain is unchanged during switch

• Only the Constant region of heavy chain changes…(class switching or CH switching)

• In the mouse, switching uses the appropriate Constant region gene, [C, C, C, C, C] for the antibody class

Major histocompatibility complex (MHC)

Function: In conjunction with T-cell surveillance, MHC is a series of polymorphic proteins designed to display digested peptide fragments on the surface of the cell. The basis of cell-mediated immunity

Interacts with TC cells to initiate infected cell destruction

Interacts with TH cells to alert system of infection

MHC-1

MHC-2

On all cell surfaces

On surface of phagocytic cells (macrophages, etc.)

Displays fragments from internal protein digests

Displays digested fragments from external protein sources

Cell-Mediated Immunity

Important!

Internal proteins digested by proteases cannot bind to MHC-2

External proteins that enter the cell by an endosomal pathway cannot bind to MHC-1.

MHC-1 interaction is a signal to TC cells to destroy the infected cell

MHC-2 interaction is a signal to TH cells that the system is under attack by a virus, bacteria, etc. and to send for help in the form of antibody-producing cells