Protein Structure and Function ChE 170 Lecture 10/18/11.
-
Upload
silvester-stanley -
Category
Documents
-
view
228 -
download
11
Transcript of Protein Structure and Function ChE 170 Lecture 10/18/11.
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