protein structure and function che 170 lecture 10/18/11
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Protein Structure and Function
ChE 170 Lecture 10/18/11
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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
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Herceptin and HER2
Cho, H.-S. et al. Nature (2003).
PDB ID: 1N8Z
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So Why Do We Care about Protein Structure/Function?
Discussion
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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
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Thurber, Schmidt, & Wittrup. Trends in Pharmacological Sciences (2007).
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What Mediates Protein Structure?
• Destabilizing conditions– Heat– Chemicals (urea)– Extreme pH– High salt concentrations – Reducing agents
• Can be reversible
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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
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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
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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
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Protein Binding Interaction
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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
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Protein Dissociation Example
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Use Surface Plasmon Resonance to Determine Binding Affinity
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Surface Plasmon Resonance
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Thermodynamics of the Binding Interaction
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Antibodies: Structure and Function
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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
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Antibodies: Engineered Uses
• In vitro diagnostics– ELISA’s
• Largest class of biologic therapeutics
• Important for research in biology and medicine– Human Protein Atlas
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Figure 3-1 part 1 of 3
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IgGs are Composed of Two Types of Protein Chains
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IgGs have Two Important Domains
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Figure 3-1 part 2 of 3
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Figure 3-3
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Anti-parallel Beta-Sheets
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Figure 3-5 part 1 of 2
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Figure 3-5 part 2 of 2
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The Hypervariable Regions
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Figure 3-7
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Figure 3-8Antibodies Bind in Different Ways
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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
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Production of Monoclonal Antibodies from Hybridoma Cells
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Production of Monoclonal Antibodies from Hybridoma Cells
Fluorescence-Activated Cell Sorting
(FACS)
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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