1. Protein Function –Binding
Chapter 5:
Protein Function –Binding
Protein Function Part 1: Binding. Protein Function Part 2: Enzymes is chapter 6 which will come in three parts.

2. Function of Globular Proteins: Ligand Binding
Learning Goals
1. Reversible binding of ligands is essential
Specificity of ligands and binding sites
Ligand binding is often coupled to conformational changes, sometimes quite dramatic (Induced Fit)
In multisubunit proteins, conformational changes in one subunit can affect the others (Cooperativity)
Interactions can be regulated
2. Illustrated by:
Hemoglobin, antibodies, and muscle contraction

3. Functions of Globular Proteins
Storage of ions and molecules
myoglobin, ferritin
Transport of ions and molecules
hemoglobin, serotonin transporter
Defense against pathogens
antibodies, cytokines
Muscle contraction
actin, myosin
Biological catalysis
chymotrypsin, lysozyme

4. Protein Interaction with Other Molecules
Reversible, transient process of chemical equilibrium:
A + B  AB
A molecule that binds to a protein is called a ligand
Typically a small molecule
A region in the protein where the ligand binds is called the binding site
Ligand binds via same noncovalent forces that dictate protein structure (see Chapter 4)
Allows the interactions to be transient

5. Hemoglobin and Oxygen/CO2 Binding
Hemoglobin in Erythrocytes and Myoglobin in Muscle will be our model proteins to examine ligand binding.

6. Structures of Porphyrin and Heme
FIGURE 5–1 Heme. The heme group is present in myoglobin, hemoglobin, and many other proteins, designated heme proteins. Heme consists
of a complex organic ring structure, protoporphyrin IX, with a bound iron atom in its ferrous (Fe2+) state. (a) Porphyrins, of which protoporphyrin
IX is only one example, consist of four pyrrole rings linked by methene bridges, with substitutions at one or more of the positions denoted X. (b, c) Two representations of heme (derived from PDB ID 1CCR). The iron atom of heme has six coordination bonds: four in the plane of, and bonded to, the flat porphyrin ring system, and (d) two perpendicular to it.

7. Coordination Positions – Bind with His93 = His F8
The His is the 93 aa from the N-terminal and it is in alpha helix F as the 8th amino acid of that alpha helix.

8. Myoglobin
Myoglobin is all alpha helix and random coil.

9. θ = Fraction of Protein’s Ligand Binding Sites Bound to Ligand
The Kd represents the binding constant and what concentration of ligand it takes to get effective binding.
θ = Fraction of Protein’s Ligand Binding Sites Bound to Ligand

10. Oxygen Binding to Myoglobin
Oxygen given here as partial pressure of Oxygen in kilo-Pascals. So the Kd is the partial pressure that produces 50% saturation, or P50. How is this experiment done? … what special property does myoglobin and hemoglobin have? Think…color and what that means.

11. EOC Problem 1: Relates Kd with “affinity” for ligand
What are the Kd’s ?
X has a lower Kd than the Kd of Y. It takes less concentration of A to saturate X than Y, so X has a great affinity for binding ligand A. A
EOC Problem 1: Relates Kd with “affinity” for ligand

12. How is the Binding Experiment Done?
With proteins such as Hemoglobin and Myoglobin, the absorbance spectrum changes between free and bound protein can be measured in a spectrophotometer. The spectrum of deoxy-myoglobin is different than oxy-myoglobin.
What about proteins without a chromophore? and binding colorless ligands?
Equilibrium dialysis
This is done in an Equilibrium Dialysis Chamber: two wells separated by a semipermeable membrane. The cartoon used antibodies which with exquisite specificity bind ligands. Antibodies have an overall structure that looks like a “Y” with the ligand binding at the top ends of the Y (that is it has two binding sites). The cartoonist made a mistake with the specific antibody (not on the graph but the cartoon)…what is it? (Hint the cartoonist got the top panel perfect).

13. Human serum transferrin binds iron at pH 7
Human serum transferrin binds iron at pH 7.2 with a Kd of to M
EOC Problem 5 uses simple inspection of the data to get Kd ! Make sure you get this done before Class.

14. Carbon Monoxide Binds Heme better than Oxygen Due to Steric Effect of His E7 (see next slide)

16. Comparison of Myoglobin and a Hemoglobin Polypeptide
It should be apparent that these are related and are part of a protein structure family. Hb has 4 subunits: 2 alphas and 2 betas.

17. Amino Acid Sequence of Myoglobin and Hemoglobin Polypeptides
Please note that the conserved amino acids sit amongst other that have changed (orthologs), but the structure of the protein has persistently evolved with out much change (similar 3D structure in the previous slide). The conserved aa’s have special functions which if a mutation occurred at that position, it would most likely be a nonfunctional protein and therefore eitiher a lethal or debilitating enough to not persist…passed on through the generations.
Grey Conserved, Pink Conserved in all known Hemoglobins

18. Hemoglobin Structure Showing Interchain Contact Points
See next slide for the details.

20. Contact Points in Primary Structure
The contact points look spread out, but this is a linear cartoon, it does not have 3-D structure…the dotted lines are between amino acids that are in H-bond distance from each other or less.

21. Hemoglobin has Two Different Folded States
You can see that these are different. Look at the hole of the donut. Then around to see how the conformation can move between these two states. T stands for “taut” and R for “relaxed” each state binds oxygen with different Kd’s.

22. T  R
You can see that His F8 moves straitening out the prophoryin ring.

23. Oxygen Binding Curves
EOC Problem 6 gets you further into cooperativity in oxygen binding.
Knowing this will help in Class.
The high affinity state is R and the low affinity state is T. Real functioning Hb is the middle sigmoid response. This shows that Hb goes through a conformational change based on oxygen concentration.

24. Cooperative Binding
Cooperative binding shows that when one ligand binds, it cause conformational change so the other, unoccupied, sites become better binders of the next ligand….next slide.

26. Hill Plot
We don’t need to get into the Hill plot math, but what it shows is that there are three conformational states: the low affinity state, transition to the another state, and the high affinity state as oxygen (ligand) concentration increases.

27. CO Binds Well
Increased Exposure to even Low Levels of CO results in COHb! +
The effect of exercise is trivial.

28. This is Fatal
Why does the CO binding curve go to the left more than the normal Hb (binding O2)?

29. Two Models of Sigmoid Curves
Both of these models predict the changes observed with Hb oxygen binding curve, see the next slide.
Monod-Wyman-Changeux Koshland (Induced Fit)

30. Both Models Fit the Data
The lines are the models, the actual data are the points. Regardless, Hb shows a transition from low oxygen conditions (T state) to the R state in higher oxygen conditions. Most important is that this correlates well with Hb’s ability to bind oxygen in the lungs and deliver oxygen to tissues.

31. Hb Oxygen Carrying Ability: the Bohr Effect
How does this relate to the pH’s of blood in the lungs, and tissues…particularly active muscles?

32. CO2 Transport on N-terminal Amino Groups
Hb carries some CO2 on the protein besides being soluble in blood.

33. Effect of Altitude and 2,3 bisphosphoglycerate
EOC Problem 3: examines this phenomena...be sure you know this for Class.
This is why Olympic Teams train in Colorado and other mountains sports complexes. The level of 2,3 bisphosphoglycerate increases in RBC’s and affects the delivery of oxygen to tissues, and for athletes what is most important is delivery to active muscles.

34. BPG Fits into the Hole of the Doughnut
R state without and with BPG
Positively charged R groups are in blue.

35. Normal RBC vs Sickle Cell RBC’s
The sickling of RBCs is due to a different type of Hb that when deoxygenated will form filaments which deform the RBCs, see the next slide.

36. The Difference In Shape is due to One Amino Acid Change Glu6  Val6 in Beta

37. Separation of Protein Fragments The Classic Paper Chromatography + Electrophoresis
Image from Chapter 3

38. Hb-S Polymerizes in the Deoxy form

39. Lymphocytes Have Binding Proteins
The Immune System is totally dependent on binding with ligands and larger molecules. A

40. Antibodies have at least 2 Antigen Binding Sites
IgG
Carbohydrate Bound Here
There are 5 classes of antibodies. IgG one of the most common in blood has 2 antigen binding sites. IgA (mucosal Ab) has 4, and IgM in blood has 10 antigen binding sites! Take PCB 4233 Immunology for a real exercise in protein binding.

41. IgG Protein Structure and Binding an Antigen
IgG belongs to the immunoglobin family. They bind large antigen molecules, but only bind to certain sites on the antigen…those sites are usually small being 7-15 aa’s long for protein antigens and 2-4 sugars long for polysaccharide antigens.

42. IgM Has 10 Ag Binding Sites
IgG and IgM are the major circulating antibodies

43. Cartoon of an ELISA

44. ELISA to Detect Herpes Simplex Virus in Blood Samples
Blood samples were centrifuged to remove RBC and WBC. There are 48 wells: 2 controls and the rest were different patients suspected as having a HSV infection. You can see different levels of infection…want to get into this deeper, take MCB 4503 Virology.
Positive
Negative
Controls

45. Antibodies Used with PAGE: Immunoblots
Lanes 1,23, purification of a protein kinase. The molecular weight standards are not shown, but their positions on this gel are shown by the numbers in kD (kilo Daltons). The gel was simply blotted on to a nylon membrane and then stained with an antibody that reacts with the protein kinase (the antigen). Why are these called “Westerns”?

46. Binding receptors initiates Phagocytosis.

47. Myosin

48. Myosin Aggregate

49. Actin Filament

50. Myosin Contacting an Actin Filament

51. Muscle Structure

52. Electron Microscopy of Relaxed and Contracted Muscle

53. Myosin-Actin Model

56. Things to Know and Do Before Class
Know how binding studies are done and the meaning of Kd.
Know how conformation of a protein affect ligand binding (models of myoglobin and hemoglobin)…for loading and off-loading oxygen.
How altitude affects Hb’s oxygen binding.
Why Hb-S causes red blood cells to change shape and what affect that has on Hb-S individuals.
How antibodies bind to antigens.
Be able to do EOC Problems 1,3, 5-7.