1. AH Biology: cells and proteins- PPT 5
Reversible Binding of Phosphate and Control of Conformation

2. What are we learning about?
Phosphate groups Kinases Phosphatases The effect of phosphate groups on conformation of proteins ATP Phosphorylation of myosin and its interaction with actin ATPases

3. Kinase
Kinases are often responsible for the phosphorylation of other proteins using ATP.
The addition or removal of phosphate from particular R groups can be used to cause reversible conformational changes in proteins.
This is a covalent modification of the protein.
This is a common form of post-translational modification.
In this way the activity of many cellular proteins, such as enzymes and receptors, are regulated.

4. Kinase A phosphate group is highly charged.
This alters the position of the
charged bonding in the three
dimensional structure of the
protein. This causes a
Conformational (shape) change in the protein.

5. Kinases and the cell cycle
Cyclins are proteins involved in regulating the cell cycle. Cyclins build up during the phases of the cell cycle. They help move the cell into mitosis through the activation of different Cdks (cyclin dependent kinases) Each Cdk phosporylates different target proteins in the cell. There are 4 classes of cyclin-Cdk each involved in a different phase: G1-Cdk, G1/S-Cdk, S-Cdk and M-Cdk + mitosis promoting factor (MPF)

6. Cyclins in the cell cycle

7. Phosphatase
Phosphatase catalyses dephosphorylation of other proteins by the hydrolysis of phosphate from the protein molecule. This changes the conformation of the protein as a result of charge interactions of the R groups in the protein. The cell cycle is finally pushed into the M phase by the phosphatase Cdc25. This removes an inhibitory phosphate from Mitosis Promoting Factors (MPF), activating mitosis.

8. Phosphatase and glycogen metabolism
Gluconeogenesis:
Glucose-6-phosphatase is an important enzyme involved in the dephosphorylation of glucose-6-phosphate produced from the metabolism of glycogen.
This generates glucose which is then available for excretion from the cell or directly for respiration.

9. Regeneration of ATP
ATP is regenerated in respiration. Most respiration takes place in the mitochondria via oxidative phosphorylation. This creates a proton gradient that is used to drive the membrane-bound enzyme ATP synthase and thus produces ATP.

10. ATP and the Sodium potassium pump
Some proteins (ATPases) use ATP for their phosphorylation.
The pump creates an electrochemical gradient across the cell membrane that can be used to provide energy for other processes, such as the transport of glucose.
The binding of ATP releases phosphate, which provides energy for the reaction and binds to the protein channel.
The binding of the phosphate changes the conformation of the protein so that it releases three Na+ to the extracellular space.
At the same time the affinity for K+ increases and as it does phosphate is released, changing the conformation again back to its original state and releasing two K+ to the intracellular space.
The affinity for Na+ is again increased in this state.
This creates a net gain of sodium ions in the extracellular space and potassium ions in the intracellular space.
This produces an electrochemical gradient and thus an energy source that can be used to power other reactions in the cell when coupled with a separate ion channel.
The glucose co-transporter is an example of this.

11. Signal transduction
Extracellular hydrophilic signalling molecules are involved in the activation of extracellular receptor proteins that then interact with intracellular proteins through a series of kinases and phosphatases. This cascade of phosphorylation and dephosphorylation quickly activates intracellular events. Insulin and the blood sugar level are controlled in this way, as is cell death (apoptosis).

12. Skeletal/striated muscle and contraction using ATP
Striated muscle (skeletal muscle) is composed of muscle fibres, which are long multinucleated cells.
The following details are for not required for this course, but may be of interest to candidates:
Bone
Perimysium (connective tissue sheath surrounding a fascile)
Blood vessel
Muscle fibre
Fascile (a bundle of muscle fibres)
Endomysium (the connective tissue sheath around a muscle fibre)
Epimysium (the sheath of connective tissue surrounding a muscle)
Tendon

13. Transmission electron microscope image: human striated muscle
Each muscle fibre contains longitudinal sections of parallel myofibrils.
Each myofibril has a striated (striped) appearance and is subdivided into sarcomeres.

14. Sarcomere myosin actin
The sarcomere is a functional unit of muscle capable of contraction and is composed of the protein filaments actin and myosin, which overlap:
I band: actin only
A band: actin and myosin overlap
H band: myosin only.
Z lines indicate the extent of one sarcomere.

15. Sarcomere
A muscle contracts as the actin (thin filaments) and myosin (thick filaments) slide past each other.
The distance between the Z lines decreases during muscle contraction and as a result the muscle shortens.
This can be seen in the muscle as the A bands remain the same length but the I band and H zone get shorter during the contraction.

16. Muscle contraction via ATPase
Myosin has heads that act as cross bridges as they bind to actin at specific binding sites and allow the muscle to contract. This involves the binding of ADP and Pi on the myosin to make the initial movement of actin. As ADP and Pi are released the myosin head moves along the actin. ATP then binds to the myosin allowing the myosin to move along the actin fibre again. ATP is converted to ADP and Pi releasing energy which the myosin uses.
Once the myosin binding site on the actin is free the myosin head can rebind. At this stage ADP and Pi are still attached to the myosin head from the previous contraction.
The rebinding releases the ADP and a phosphate ion, dragging the myosin head along the actin filament.
ATP then binds to the myosin head.
When ATP binds to myosin, the myosin head detaches from actin, swings forwards and rebinds to a new myosin binding site along the length of the actin.
ATP is converted to ADP and Pi, and the energy from this conversion is stored in the myosin head to power the transition.
This whole process repeats until the contraction ends, Ca2+ concentration decreases and the protein tropomyosin again blocks the myosin binding sites on the actin.

17. What Were we learning about?
Phosphate groups Kinases Phosphatases The effect of phosphate groups on conformation of proteins ATP Phosphorylation of myosin and its interaction with actin ATPases