Membrane proteins

Practical within the courses 1MB202, 1BG349, 1BG351


The aim of the practical is to learn about some general principles of membrane protein structure examplified with two types of channels.

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Pdb files
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Membrane proteins belong to different structural and functional classes. You will look closer at two different types of membrane proteins, a porin and an aquaporin.

Porin structure
Gram-negative bacteria protect themselves with an outer membrane. However, the membrane has to let through nutritients and other low molecular-weight solutes that the bacterium needs. Porins are protein channels that allow diffusion of molecules up to about 0.6 kD into the periplasmic space.

Q1. How would you describe the secondary and tertiary structure of this protein?

A crystal is formed by regular packing of molecules into a lattice. The repeting unit of the crystal (called the asymmetric unit) can contain several functional or biological units. In this crystal structure, the opposite is true. The crystals form with one polypeptide chain, a monomer, in the asymmetric unit although the functional unit is larger. The biological symmetry is used in the crystal packing. Let's find out what this functional unit looks like. You can find examples of dimers, trimers, tetramers, penetamers and so on in figure 1-74, p 45 in Petsko & Ringe.

Q2. What is the functional oligomeric state of this protein (monomer/dimer/trimer/tetramer/pentamer/hexamer/heptamer)?
Q3. What thickness of the membrane would you estimate from looking at the distribution of aromatic residues?

Q4. What type of residues do you find in the region of the porin that interacts with the membrane?

Inside the barrel, the channel is partly blocked by a long loop region between strands 5 and 6. This creates a narrow region in the pore, the "eyelet", that defines up to what size molecules can pass through the pore.

Q5. What is the approximate diameter of the eyelet?
Q6. Describe the distribution of amino acids in the pores.

Q7. How do you think these residues can contribute to selecting which molecules can pass through the pore?.

Aquaporin structures
The aquaporin is a water channel present in the lense of the eye. Water transport plays a role in maintaining a transparent lense. The structure we will look at comes from the lense of cow eye and was published by Harries et al. in 2004. The aquaporin is a much more selective pore compared to the porin. Let's see if we can understand why by looking at its structure.

Q8. How would you describe the secondary and tertiary structure of this protein?
Q9. How would you describe the quartenary structure of this protein? What is the size of the functional unit (width and height)?
Q10. Describe the distribution of amino acids on the outside.
Q11. Which coordinate file represents an open state and which one represents a closed state? Choose a good orientation and display the structures as spacefilling objects to find out.

Q12. Can you see any conformational changes between the two states? Which residue, Met176 or Arg187 is most responsible for closing the pore?
Q13. Seven (or eight) water molecules are positioned in a row inside the pore. Which ones are these?
Q14. What types of residues do you find in this part of the pore?

Q15. How are the water molecules coordinated within the pore? Can you guess why metal ions cannot go through the aquaporin? Compare to what you know about the selectivity filter in the potassium channel.

Q16. In the paper describing this structure, the authors speculate that two tyrosine residues may need to move to achieve "high conductance" (high flow of water through the channel). Which are these tyrosine residues?

Q17. Two mutations associated with "human congenital cataracts" (ärftlig starr) are Glu134Gly and Thr138Arg. Try to find out how these mutations can affect the function of the aquaporin. (Hint: first calculate H-bonds, then try to make the mutations using the mutate tool of the toolbar.)

This practical was written 2007-2008 by Maria Selmer, Uppsala Universitet.