1. 70% 80% 90% 100%

2. Structure-Function Relationships of Integral Membrane Proteins Hartmut “Hudel” Luecke Biochemistry, Biophysics & Computer Science Email: hudel@uci.edu http://bass.bio.uci.edu/~hudel http://bass.bio.uci.edu/~hudel

3. Two classes of integral membrane proteins

5. Porins Porins are found in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Porins control diffusion of small metabolites like sugars, ions, and amino acids across lipid bilayers.

6. Beta barrels

7. Porin trimers Jap & Walian (1996) Physiological Reviews

8. Porin cross section Jap & Walian (1996) Physiological Reviews

9. Porin folding topology Jap & Walian (1996) Physiological Reviews

10. Diacyl phospholipid in a bilayer http://blanco.biomol.uci.edu/Bilayer_Struc.html

11. Structure of the fluid dioleoylphosphatidylcholine (DOPC) bilayer The time-averaged transbilayer distributions of the quasi-molecular groups can be thought of as probability or number densities. great amount of thermal disorder revealed by the widths of the probability densities combined thermal thickness of the interfacial regions (defined by the distribution of the waters of hydration) is about equal to the 30 Å thickness of the hydrocarbon core interfaces are chemically highly heterogeneous; they are rich in possibilities for non-covalent interactions with proteins; they are also important for the folding and stability of constitutive membrane proteins because significant portions of their mass contact the interfaces. http://blanco.biomol.uci.edu/Bilayer_Struc.html 15 Å 30 Å 15 Å

12. Polarity profile of the fluid DOPC bilayer Besides being chemically heterogeneous, the interfaces are regions in which dramatic changes in polarity occur over small distances. Shown in panel B is the “polarity profile” of a DOPC bilayer derived from the structural image of panel A. It represents the average density of atomic partial charges, both + and −, calculated by weighting the absolute values of the charge densities by the number density and group volume at each position across the bilayer. http://blanco.biomol.uci.edu/Bilayer_Struc.html

13. Membrane protein biogenesis

14. Membrane protein biogenesis in eukaryotes

15. Membrane protein assembly Constitutive membrane proteins, i.e. those that are encoded in a normal cell’s genome and are responsible for vital physiological activities, are assembled by means of a complex process involving synthesis of membrane proteins by ribosomes attached transiently to a complex of proteins referred to as a translocon located in the membrane of the ER. This translocon provides a transmembrane “tunnel” into which the newly synthesized protein can be injected. After synthesis is complete, the ribosome disengages from the translocon (which enters a closed state) and the protein is released into the membrane bilayer where it assumes (in an unknown way) its final folded three-dimensional structure. http://blanco.biomol.uci.edu/Bilayer_Struc.html

16. The Translocon (Sec61) Sequential insertion of hydrophobic sequences. Hydrophobic segments insert into the membrane as they emerge from the ribosome. The orientation of the segment is opposite to the orientation of the previous segment. A segment with an N cyt -C lumen orientation is termed signal anchor (SA), and a segment with an N lumen -C cyt orientation is termed stop transfer (ST).

17. Membrane protein topology Biochemical approaches to determination of membrane protein topology. (A) Insertions. (B) Fusions. In general, membrane proteins cross the membrane in a zigzag fashion and expose their hydrophilic loops alternately in the two compartments that are separated by the membrane.

18. Probing membrane protein topology Due to the impermeability of the membrane to hydrophilic molecules, parts of a membrane protein that lie on opposite sides of the membrane are differently accessible to various agents. Easily identified target sites (TAG) are inserted in the polypeptide, and membrane-impenetrable reagents are used to determine their accessibility at one side of the membrane. Examples of target sites include N-glycosylation sites, cysteine residues, iodinatable sites, antibody epitopes, and proteolytic sites that are introduced at specific positions in the protein by site directed mutagenesis. By inserting the tag at different positions in the protein, the complete topology can be determined.

19. Probing membrane protein topology Cysteine accessibility assay. Labeling of a periplasmic (A) and a cytoplasmic (B) cysteine residue in intact cells. The cells are treated with a detectable and membrane- permeant cysteine reagent (Label) with or without pretreatment with a membrane-impermeable cysteine reagent (Block). Following the treatment, the protein is purified from the cells and assayed for labeling.

20. Probing membrane protein topology (fusion) A reporter molecule (TAG) is attached to a hydrophilic domain of a membrane protein, and the cellular location of the reporter is determined by the topogenic information in the membrane protein. The reporters are typically molecules whose properties (for example, enzymatic activity) depend on their subcellular location.

21. Protein folding funnel

22. Assembly of secondary structure elements (  helices) The last important step is to understand the energetics of the association of secondary structure elements within the membrane. Don Engelman and his colleagues have shown that transmembrane helices from bacteriorhodopsin (BR) that have been independently inserted into membranes can subsequently assemble into the native structure of BR. This indicates that the insertion steps are independent of the intra-membrane assembly process. They refer to this insertion-oligomerization process as the 'two-stage' model. Stage II of the 2-stage model "Membrane protein folding and oligomerization: the two-stage model" JL Popot and DM Engelman Biochemistry (1990) 29, 4031-7.

23. Folding pathway of porins It was shown that integral membrane proteins of the β-barrel type, for instance porins of the E. coli outer membrane, can be fully denatured in 8 M urea. Some of these proteins will spontaneously insert and refold when the urea is diluted by mixing with a large volume of urea-free buffer containing lipid vesicles in the liquid-crystalline phase (Surrey & Jähnig, 1992).

24. Gramicidin Gramicidin is a heterogeneous mixture of six antibiotic compounds divided into three categories: gramicidins A, B and C, all of which are obtained from the soil bacterial species Bacillus brevis and called collectively gramicidin D. Gramicidin D are linear pentadecapeptides, that is, they are long protein chains made up of 15 amino acids. They act by forming transmembrane channels that are permeable for cations. Gramicidins are especially effective against gram-positive bacteria but they induce hemolysis in lower concentrations than bacterial cell death thus cannot be administered internally. They are used primarily as topical antibiotics and are one of the three constituents of antibiotic Neosporin ophthalmic solution. In 1939 the American microbiologist René Dubos isolated the substance tyrothricin and later showed that it was composed of two substances, gramicidin (20%) and tyrocidine (80%). These were the first antibiotics to be manufactured commercially. Gramicidins: cation channels formyl-L-X-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-L-Trp-D-Leu-L-Y-D-Leu-L-Trp-D-Leu-L-Trp-ethanolamine where X: Val or Ile; Y: Trp (gramicidin A), Phe (gramicidin B), Tyr (gramicidin C)

25. Gramicidin A

26. Kinetics of gramicidin channel formation in lipid bilayers: transmembrane monomer association AM O'Connell, RE Koeppe 2nd, and OS Andersen Conducting gramicidin channels form predominantly by the transmembrane association of monomers, one from each side of a lipid bilayer. In single-channel experiments in planar bilayers the two gramicidin analogs, [Val1]gramicidin A (gA) and [4,4,4-F3- Val1]gramicidin A (F3gA), form dimeric channels that are structurally equivalent and have characteristically different conductances. When these gramicidins were added asymmetrically, one to each side of a preformed bilayer, the predominant channel type was the hybrid channel, formed between two chemically dissimilar monomers. These channels formed by the association of monomers residing in each half of the membrane. These results also indicate that the hydrophobic gramicidins are surprisingly membrane impermeant, a conclusion that was confirmed in experiments in which gA was added asymmetrically and symmetrically to preformed bilayers. Science (1990) 250, 1256 - 1259.

27. Gramicidin A

28. Gramicidin A dimer embedded in a bilayer

29. A lipid bilayer: The Movie This molecular dynamics simulation of a dioleoyl-phosphatidylcholine (DOPC) bilayer in excess water demonstrates the extreme thermal motion of fluid lipid bilayers. The high disorder precludes the use of structure determination by standard crystallographic methods. http://blanco.biomol.uci.edu/download/movies/dopc/DOPC_Sim_15Dec08.mov

30. Gramicidin A

31. Gramicidin A: The Movie http://bass.bio.uci.edu/~hudel/m160/gramicidin-A.mpg