5- Protein Structure 2

5- Protein Structure 2 - 580.221-Lecture 5 Fall 2008...

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580.221-Lecture 5 Fall 2008 1 Protein Structure 2 – Myoglobin vs Hemoglobin Eileen Haase Fall 2007 Lecture # 5 1 Levels of protein structure 2 Motifs, Domains Specificity versus Affinity Affinity : Between Receptor and 1 Ligand Does the receptor “like” the ligand? How much time does it want to spend with this ligand? Specificity : Between Receptor and > 2 different Ligands (but only one ligand at a time) Which does the receptor like better, Ligand A or Ligand B? 3 Quantifying Affinity–1 Receptor, 1 Ligand Largest Kd – weakest bond [R], [L], >> [RL] 4 Smallest Kd – tightest bond [RL] >> [R], [L]    L K L d Fractional Occupancy,  when a receptor can only bind one molecule of ligand - univalent 5 Fractional Occupancy can also be thought of as “saturation”. When =1, the receptors are 100% occupied, fully saturated, and cannot hold anymore ligand Multivalent binding If we know the affinity of one site, how to predict affinity of 2 sites? Antigen – can bind more than one antibody independently Antibody can bind antigen with both arms Hemoglobin–can bind more than one O 2 cooperatively (not independently) 6
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580.221-Lecture 5 Fall 2008 2 Antibodies have two binding sites 7 Antigen binds different antibodies Antigen binds multiple antibodies Can treat antibody with papain to break 3 parts: Fab (2) and Fc 8 How do you calculate Kd for an antibody that can independently bind 2 molecules of antigen? Antibody simultaneously binding 2 epitopes RL R + L Kd 1 = 1 mM R 2 L 2 R 2 + L 2 Kd 2 = ?? Note: 2 identical receptors, R, on the same antibody and 2 identical molecules of ligand 9 antibody, and 2 identical molecules of ligand (epitope), L G 1 o = -RT ln Kd 1 G 2 o = -RT ln Kd 2 since the free energies are additive: G 2 o = 2 G 1 o (since 2 reactions are taking place: R 2 L 2 R 2 L + L and R 2 L 2 R 2 + L 2 ) Simultaneous Binding G 2 o = 2 G 1 o (energy to bind 2 molecules to 2 receptors is 2 x the energy to bind one molecule) -RT ln K d2 = -2RT ln K d1 or ln K d2 = 2 ln K d1 10 Hence K d2 = (K d1 ) 2 If K d1 = 1mM, then K d2 = 1 M The multivalent effect (avidity) is very useful to make weak binding appear strong. What happens if the binding of one site does affect the binding of another site another site? In hemoglobin, the Kd changes for each of the four binding sites–the binding of oxygen to an additional site is not independent 11 Diffusion of O 2 across pulmonary capillary 12
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580.221-Lecture 5 Fall 2008 3 How do you predict binding with more than one site, with more than one ligand? If you know the affinity of one site, how do you predict the affinity of another site? 13 Fractional Saturation for one ligand/receptor–Univalent binding L 14    L K d What about multiple binding sites?
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5- Protein Structure 2 - 580.221-Lecture 5 Fall 2008...

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