Lecture 5

Protein Structure and Function II

Antibodies

Proteins that recognize and bind to a particular antigen with very high specificity.

Belong to a group of serum proteins called immunoglobulins (Igs).

Each antibody has at least two identical sites that bind antigen: Antigen binding sites.

Antibody Structure Antibodies are made up

of: 2 Light Chains (identical)

~25 KDa 2 Heavy Chains (identical)

~50 KDa Each light chain bound to

heavy chain by disulfide (H-L)

Heavy chain bound to heavy chain (H-H)

CH3

CH2

Fb

Fv

Fv

FvFbFv

Hinge

Elbow

CH3

CH2

Fb

Fv

Flexibility andmotion of immunoglobulins

Hinge

CH3

CH2

CH1VH1

VLCL

First 100 a/a of amino terminal vary of both H and L chain are variable

Referred to as VL , VH, CH And CL

CDR (Complementarity Determining Regions) are what bind Ag

Remaining regions are very similar within same class

CDR Are Hypevariable

Non-covalent forces inantibody - antigen interactions

Electrostatic forces Attraction between opposite charges

Hydrogen bonds Hydrogens shared between electronegative atoms

Van der Waal’s forces Fluctuations in electron clouds around molecules

oppositely polarise neighbouring atoms

Hydrophobic forces Hydrophobic groups pack together to exclude

water (involves Van der Waal’s forces)

Five classes of antibodies:

IgG IgM IgA IgD IgE

Sequencing Of Several Immunoglobulins Revealed Five Basic Sequence Patterns ,, , , IgA, IgG, IgD, IgE and IgM The Above Classes Are Called Isotype Each class can have either or light chains Minor Differences Led To Sub-classes For IgA and IgG IgA1, IgGA2 and IgG1, IgG2, IgG3, IgG4

Sequencing Of Heavy Chains

IgG

Structure: Monomer Percentage serum antibodies: 80% Location: Blood, lymph, intestine Half-life in serum: 23 days Placental Transfer: Yes Known Functions: Enhances phagocytosis,

neutralizes toxins and viruses, protects fetus and newborn.

IgM

Structure: Pentamer Percentage serum antibodies: 5-10% Location: Blood, lymph, B cell surface (monomer) Half-life in serum: 5 days Placental Transfer: No Known Functions: First antibodies produced during

an infection.

IgA

Structure: Dimer Percentage serum antibodies: 10-15% Location: Secretions (tears, saliva, intestine, milk),

blood and lymph. Half-life in serum: 6 days Placental Transfer: No Known Functions: Localized protection of mucosal

surfaces. Provides immunity to infant digestive tract.

IgD

Structure: Monomer Percentage serum antibodies: 0.2% Location: B-cell surface, blood, and lymph Half-life in serum: 3 days Placental Transfer: No Known Functions: In serum function is

unknown. On B cell surface, initiate immune response.

IgE

Structure: Monomer Percentage serum antibodies: 0.002% Location: Bound to mast cells and basophils

throughout body. Blood. Half-life in serum: 2 days Placental Transfer: No Known Functions: Allergic reactions.

Repeating Domains of ~110 a/a Intrachain disulfide bonds within each domain

Heavy chains 1 VH and either 3 or 4 CH (CH1, CH2, CH3, CH4)

Light chains 1 VL and 1 CL

Hinge Region Rich in proline residues (flexible) Hinge found in IgG, IgA and IgD Proline residues are target for proteolytic digestion (papain and

pepsin) Rich in cysteine residues (disulfide bonds) IgM and IgE lack hinge region They instead have extra CH4 Domain

Antibody Structure

Digestion With Papain Yields 3 Fragments 2 identical Fab and 1 Fc Fab Because Fragment That is Antigen Binding Fc Because Found To Crystallize In Cold Storage

Pepsin Digestion F(ab`)2 No Fc Recovery, Digested Entirely

Mercaptoethanol Reduction (Eliminates Disulfide Bonds) And Alkylation Showed

Enzymatic Digestion Of Antibodies

Why do antibodies need an Fc region?

•Detect antigen

•Precipitate antigen

•Block the active sites of toxins or pathogen-associated

molecules

•Block interactions between host and pathogen-associated

molecules

The (Fab)2 fragment can -

•Inflammatory and effector functions associated with cells

•Inflammatory and effector functions of complement

•The trafficking of antigens into the antigen processing

pathways

but can not activate

Structure and function of the Fc region

CH3

CH2

IgA IgD IgG

CH4

CH3

CH2

IgE IgMThe hinge region is replaced by an additional Ig domain

Fc structure is common to all specificities of antibody within an ISOTYPE(although there are allotypes)

The structure acts as a receptor for complement proteins and a ligand for cellular binding sites

Immunize Animal With Antigen Multiple Clones Are Generated, Good For In Vivo For Clinical Diagnosis, Research, One Clone

That Reacts To Single Epitope Is Preferred Solution By Kohler and Milstein

Fuse A Myeloma Cell (Cancerous) With A Normal Plasma Cells

Resulting Clones Can Be Cultured Indefinitely Produces An Antibody Recognizing One Epitope

Monoclonal Antibodies

To Transport Abs Across Membranes Secretion of IgA Across Epithelium into lumen Transport of maternal Abs Across Placenta (IgG)

Many Cell Types Use FcR Ex. Mast Cells, Macrophages, Neutrophils, B, T, NK

Poly IgR Transport of IgA across epithelium

FcRN Transport of maternal IgG to fetus

Fc Receptors (FcR) Functions

IgA Antibody Transport Across Cell (Transcytosis)

Protein section review

Proteins are polymers composed of L-amino acids. Amino acids are 20 in number

Protein section review Amino acids possess two functional groups

namely carboxyl (-COOH) and amino (-NH2). In the physiological system, they exist as

zwitterions.

The structure of protein is divided into four levels of organization.

The tertiary and quaternary structures ol protein are stabilized by non-covalent bonds such as hydrogen bonds, hydrophobic interactions, ionic bonds etc.

Protein section review

The determination of primary structure of a protein: chemical and enzymatic methods are employed.

Sample question A heptapeptide (2 M, D, R, K, F, G) was isolated from the

urine of a three-toed sloth. Reaction of the heptapeptide with FDNB gave DNP-M. Limited proteolysis with carboxypeptidase indicated that M was the first amino

acid released Cyanogen bromide (CNBr) reaction with the heptapeptide released one

equivalent of free homoserine lactone Chymotrypsin digestion of the heptapeptide yielded a pentapeptide and a

dipeptide. Reaction of the pentapeptide with dansyl-Cl gave dansyl-M. Trypsin digestion yielded two M-containing tripeptides and free R. Digestion of the heptapeptide with pepsin gave a tetrapeptide (containing M, R, K

and D) and a tripeptide (M, F, and G).

DNFB & carboxypeptidase (Identify N- and C-terminal residues) CNBr acts only on methionine residues CNBr cleaves peptide chains on the carboxy side of M residues and converts them

into homoserine lactone Trypsin - cleavage on the C-side of Lys, Arg Chymotrypsin - C-side of Phe, Tyr, Trp Clostripain - like trypsin, but attacks Arg Pepsin hydrolyzes peptide chains on the N-

side of aromatic AA

Answer N-terminal AA is M    C-terminal AA is M    CNBr cleaves peptide chains on the carboxy side of M residues

and converts them into homoserine lactone) - M-?-?-?-?-?-M    Chymotrypsin (carboxy side of aromatic AA's)       - pentapeptide

- M-?-?-?-F       - dipeptide - ?-M    Trypsin digestion - since we know that M is at both the       C-

terminal and the N-terminal, this places K as the 3rd AA       residue and R as the 4th - M-?-K-R-?-?-M   

Pepsin digestion   - tetrapeptide (M,R,K & D) - sequence must be - M-D-K-R   - tripeptide (M,F & G) - sequence must be - F-G-M     

The sequence of the heptapeptide must be: M-D-K-R-F-G-M

Sample questions

The imino acid found in protein structure (a) Arginine (b) Proline (c) Histidine (d) Lysin

The bonds in protein structure that are not broken on denaturation.

(a) Hydrogen bonds (b) Peptide bonds (c) lonic bond (d) Disulfide bonds