ProteinsProteins

Protein FunctionProtein Function CatalysisCatalysis StructureStructure MovementMovement DefenseDefense RegulationRegulation TransportTransport AntibodiesAntibodies

Monomers—Monomers—Amino AcidsAmino Acids

R-groupsR-groups HydrophilicHydrophilic HydrophobicHydrophobic Uncharged Uncharged ChargedCharged LargeLarge SmallSmall

Confer unique Confer unique chemical chemical properties on properties on each aaeach aa

PROTEIN LEVELS OF PROTEIN LEVELS OF STRUCTURESTRUCTURE

PRIMARY STRUCTUREPRIMARY STRUCTURE

Is a unique characteristic of every Is a unique characteristic of every proteinprotein

Is encoded by the nucleotide Is encoded by the nucleotide sequence of DNAsequence of DNA

Is thus a form of genetic informationIs thus a form of genetic information Is read from the amino terminus to Is read from the amino terminus to

the carboxyl terminusthe carboxyl terminus

Nature of Protein SequencesNature of Protein Sequences

Sequences and composition reflect Sequences and composition reflect the function of the protein:the function of the protein: Membrane proteins have more Membrane proteins have more

hydrophobic residues.hydrophobic residues. Homologous proteins from different Homologous proteins from different

organisms have similar sequences.organisms have similar sequences. e.g., cytochrome c is highly conservede.g., cytochrome c is highly conserved

Cytochrome cCytochrome c

SECONDARY STRUCTURE I: THE -HELIX

HelixHelix

If N-terminus is at bottom, then all peptide N-H bonds point “down” and all peptide C=O bonds point “up”.

N-H of residue n is H-bonded to C=O of residue n+4.

Secondary Structure II: The -Strand

approx.3.4 A

Several -strands assemble into a

-sheet (a tertiary structural element)

TERTIARY STRUCTURETERTIARY STRUCTURE

3-D structure. 3-D structure. Form follows function!!Form follows function!! Native vs denaturedNative vs denatured

Determinants of tertiary structureDeterminants of tertiary structure Amino acid sequenceAmino acid sequence Environment in which the protein Environment in which the protein

residesresides

Stabilizing InteractionsStabilizing Interactions

Hydrogen Bonds Electrostatic interactions (“salt-

bridges” or ion pairs) van der Waals interactions (dipole-

dipole and dispersion) Hydrophobic interactions Disulfide bridges

Protein DenaturationProtein Denaturation

•Denaturants--Anything that can Denaturants--Anything that can disrupt stabilizing interactionsdisrupt stabilizing interactions

• HeatHeat• SaltsSalts• pHpH• Organic solventsOrganic solvents

Quaternary StructureQuaternary Structure

ANTIBODIESANTIBODIES Extremely specificExtremely specific Definitions:Definitions:

AntigenAntigen Epitope (antigenic determinant)Epitope (antigenic determinant) HaptenHapten

FLUORESCEIN – a hapten

SSSS

Light Chain

Light ChainSS

SS

Antibody StructureAntibody Structure

Constant Constant

Constant Constant

VV

V

V

Antigen binding site

Antigen binding

site

Heavy Chains

Antibody StructureAntibody Structure

Antibody StructureAntibody Structure

Recognition and BindingRecognition and Binding

The N-terminal region of antibody light chains and heavy chains form the antigen binding site

The variability in amino acid sequence provides the structural basis for the diversity of antigen-binding sites

Antigen BindingAntigen Binding

Variable

Light

Variable

Heavy

Antigen 1Antigen 3

Antigen 2

Polyclonal vs Monoclonal Polyclonal vs Monoclonal AbsAbs

101077-10-1099 genetically distinct genetically distinct lymphocytes, each producing a lymphocytes, each producing a single type of Ab.single type of Ab.

Polyclonal—normal immune Polyclonal—normal immune response. Several Abs, recognition of response. Several Abs, recognition of various epitopes with varying various epitopes with varying affinities.affinities.

Monoclonal Monoclonal

Monoclonal Ab ProductionMonoclonal Ab Production Given:Given:

Normal cells—MortalNormal cells—Mortal Transformed cells—Immortal Transformed cells—Immortal Two Pathways of DNA SynthesisTwo Pathways of DNA Synthesis

MajorMajor Salvage—Requires HGPRTSalvage—Requires HGPRT

8-azaguanine—HGPRT poison. 8-azaguanine—HGPRT poison. Aminopterin---Interferes w/ major Aminopterin---Interferes w/ major

pathwaypathway PEG---promotes cell fusionPEG---promotes cell fusion

HAT SelectionHAT Selection1)1) Select HGPRTSelect HGPRT- - mutant mutant

myeloma by treatment myeloma by treatment with 8-azaguaninewith 8-azaguanine

2)2) Fuse HGPRTFuse HGPRT- - mutant mutant myeloma with normal cells myeloma with normal cells using PEGusing PEG

3)3) Select with aminopterinSelect with aminopterin1)1) Normal?Normal?

2)2) Myeloma?Myeloma?

3)3) Hybridoma?Hybridoma?

4)4) Screen for desired Screen for desired monoclonal. monoclonal.

MAbs in the LabMAbs in the Lab Macs extremely useful in molecular Macs extremely useful in molecular

biology and medicinebiology and medicine ApplicationsApplications

Affinity columnsAffinity columns Western blotsWestern blots ELISA (Enzyme Linked ImmunoSorbent ELISA (Enzyme Linked ImmunoSorbent

Assay)Assay)

Back

The Future?The Future?

Single Chain Antibodies Single Chain Antibodies Catalytic antibodiesCatalytic antibodies Bifunctional antibodiesBifunctional antibodies Etc. Etc.

VL

VH

N

C

VL

VH

VL

VH

Features of a single-chain antibody (sFv).

LinkerConsists of the variable light (VL) chain of an antibody joined via a linker to the variable heavy (VH) domain.

The linker typically consists of a flexible/soluble peptide (for example, [GGGGS]6)

The sFv maintains the antigen binding specificity (but not always the affinity) of the parent antibody.

CH2

CH2

CH3

CH3CH1

CH1

CL

CL

Back