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Structure-Function Relationships of Integral Membrane Proteins

Hartmut “Hudel” Luecke

Biochemistry, Biophysics &

Computer Science

Email: hudel@uci.edu http://bass.bio.uci.edu/~hudel

Two classes of integral membrane proteins

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.

Beta barrels

Porin trimers

Jap & Walian (1996) Physiological Reviews

Porin cross section

Jap & Walian (1996) Physiological Reviews

Porin folding topology

Jap & Walian (1996) Physiological Reviews

Membrane protein biogenesis

Membrane protein biogenesis in eukaryotes

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

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 Ncyt-Clumen orientation is termed signal anchor (SA), and a segment with an Nlumen-Ccyt orientation is termed stop transfer (ST).

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.

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.

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.

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.

Protein folding funnel

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.

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).

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)

Gramicidin A

Gramicidin A

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.

Gramicidin A

Gramicidin A

Gramicidin A dimer embedded in a bilayer

Gramicidin A

Gramicidin A: The Movie

http://bass.bio.uci.edu/~hudel/m160/gramicidin-A.mpg