Protein Structure and Function

ChE 170 Lecture 10/18/11

Protein Function is Dictated by its Structure

• Enzyme activity can depend on structural conformation

• Unique binding sites in antibodies dictate the specific ligand to which the antibody binds

Pinkas et al PLoS Biol (2007)

Active State

Herceptin and HER2

Cho, H.-S. et al. Nature (2003).

PDB ID: 1N8Z

So Why Do We Care about Protein Structure/Function?

Discussion

Importance of Understanding Protein Binding Interactions

• Antibodies and the immune response• Binding of agonistic ligands to cell surface

receptors– GPCR’s such as the AT1 receptor

• Engineering therapeutic drugs– Specificity and side effects– Affinity affects transport

• Wittrup’s model

)

)/]([

)]([

BA

Ag

KAbDR Dsurf

)/]([

)]([

Agk

KAbDR

e

Dsurf

Thurber, Schmidt, & Wittrup. Trends in Pharmacological Sciences (2007).

What Mediates Protein Structure?

• Destabilizing conditions– Heat– Chemicals (urea)– Extreme pH– High salt concentrations – Reducing agents

• Can be reversible

What Mediates Protein Structure?

• Protein folding occurs on the ribosome – Chaperones

• Further processing involves additional enzymes– Isomerase– Disulfide bond formation– Protease activation

• Protein misfolding– Ubiquitin– Can lead to disease

H3N+

COO- kf

H3N+

COO-

)ln( ff kRTG

Important Secondary Structures

• Alpha-helix– Cylindrical structure: hydrogen bonded

backbone• Residue n h-bond with n+4

• Beta-sheet– Network of hydrogen bonds: antiparallel vs.

parallel

Beta-Barrel

PDB: 1EMA

Stabilizing Forces

Covalent Bond

Disulfide Bond

Salt Bridge

Hydrogen Bond

Long-range Electrostatic

Interaction

Van der Waals Interaction

Petsko & Ringe; Protein Structure and Function; New Science Press; 2004; pg. 11

Protein Binding Interaction

Binding Affinity Dictates Half-Life

KD t1/2 Example

mM ms Non-specific

μM ms - sMultivalent cell surface; intracellular

signaling proteins

nM min-hr Antibodies

pM hr- days Growth factors/receptor

fM weeks-months Streptavidin-biotin

Protein Dissociation Example

Use Surface Plasmon Resonance to Determine Binding Affinity

Surface Plasmon Resonance

Thermodynamics of the Binding Interaction

Antibodies: Structure and Function

Antibodies: Natural Functions

• Several classes of immunoglobulins– IgG, IgA, IgM, IgD,

IgE (arranged by half-life high to low)

• IgG are the most abundant

Nester, Anderson, Roberts, and Nester; Microbiology: A Human Perspective; McGraw Hill; 2007; pg 394

Antibodies: Engineered Uses

• In vitro diagnostics– ELISA’s

• Largest class of biologic therapeutics

• Important for research in biology and medicine– Human Protein Atlas

Figure 3-1 part 1 of 3

IgGs are Composed of Two Types of Protein Chains

IgGs have Two Important Domains

Figure 3-1 part 2 of 3

Figure 3-3

Anti-parallel Beta-Sheets

Figure 3-5 part 1 of 2

Figure 3-5 part 2 of 2

The Hypervariable Regions

Figure 3-7

Figure 3-8Antibodies Bind in Different Ways

How Do We Generate Antibodies for our Own Purposes?

• Polyclonal Mixtures– Animal immunizations limited supply– Heterogeneous binding specificities

• Significant need to generate monoclonal antibodies Hybridomas!– B-cells (produce IgG) fused with myeloma

cells to produce hybrid myelomas that secrete IgG and grow continuously

Production of Monoclonal Antibodies from Hybridoma Cells

Production of Monoclonal Antibodies from Hybridoma Cells

Fluorescence-Activated Cell Sorting

(FACS)

Fluorescence-Activated Cell Sorting (FACS)

Green fluorescence

Red fluorescence

90º Light-Scatter (SSC)

Dichroic mirror

Band-pass filters

LASER Forward light-scatter (FSC)

Cell sample

Piezo

Non-target cellsTarget cells

PMT