Aims

• To reinforce knowledge about the structure of helical membrane proteins.

Objectives

After this practical you will:

• be aware of the structures of some transmembrane proteins.

Exercises

1. Protein Data Bank entry 1H68 contains the structure of sensory rhodopsin II from Natronobacterium pharaonis. Look at the distribution of hydrophobic and hydrophilic residues in this structure.

2. Protein Data Bank entry 1BL8 is a potassium channel protein from Streptomyces lividans.

   1. Locate the potassium selectivity filter. The residues involved are listed in UniProt entry KCSA_STRCO. Which protein atoms make contact with potassium ions?

   2. The four Tyr residues from the selectivity filter interact with eight Trp side chains from the pore helices to form a massive sheet of twelve aromatic residues. Locate these residues in the structure. Notice the hydrogen bonding between the Tyr hydroxyls and Trp nitrogens.

3. The HOLE program can be used to analyse and visualise the pores of membrane proteins such as ion channels and aquaporins. The method used by HOLE is described in:

   • Smart OS, Goodfellow JM, Wallace BA. The pore dimensions of gramicidin A. Biophys J. 1993 Dec;65(6):2455-60. (PubMed)

   Read the description of this method.

   1. Describe the method used by the HOLE program in your own words.

   2. The HOLE program requires that the user provides an initial point within the channel and a vector in the approximate direction of the channel. Discuss whether it would be possible for a program to identify these automatically, so that the user doesn't need to provide these.

   3. Compare the method used in HOLE with the method for identifying channels that is described in section 2(d) of:

      • Tilton RF Jr, Singh UC, Weiner SJ, Connolly ML, Kuntz ID Jr, Kollman PA, Max N, Case DA. Computational studies of the interaction of myoglobin and xenon. J Mol Biol. 1986 Nov 20;192(2):443-56. (PubMed)

4. Protein Data Bank entry 1ZLL contains an ensemble of 20 NMR-derived structure of human phospholamban.

   Simmerman et al. (1996) have identified residues whose mutation to Ala or Phe affected Phospholamban's ability to form a pentamer. Locate these residues on the three-dimensional structure of phospholamban.

   • Simmerman HK, Kobayashi YM, Autry JM, Jones LR. A leucine zipper stabilizes the pentameric membrane domain of phospholamban and forms a coiled-coil pore structure. J Biol Chem. 1996 Mar 8;271(10):5941-6. (PubMed)