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Structure-Function Relationships of
Integral Membrane Proteins
Hartmut “Hudel” Luecke
Biochemistry, Biophysics &
Computer Science
Email: [email protected]
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
membranepermeant 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)
Stage II of the 2-stage model
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.
"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).
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 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.
Gramicidin A
Gramicidin A
Kinetics of gramicidin channel formation
transmembrane monomer association
in
lipid
bilayers:
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-F3Val1]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