|Alpha helical proteins:
|Beta barrel proteins:
Experiment types used to determine topologies
The riporter enzyme was fusioned or inserted to a given point of
the transmembrane protein investigated. The activity of the riporter
enzyme in this chimera shows the localisation of fusion point.
Fusion with alkaline phosphatase. Alkaline phosphatase is a
periplasmic bacterial enzyme. It is active only in the periplasmic
Sandwich fusion with alkaline phosphatase. Alkaline phosphatase
is inserted into a given point of the transmembrane protein
investigated. In this case the transmembrane protein is not
truncated. Alkaline phosphatase is active only the periplasmic
space of bacterii.
Fusion with beta-galactosidase. Beta-galactosidase is a bacterial
enzyme, which is active only in the cytoplasm.
Fusion with alkaline-phosphatase and beta-galactosidase. Because
these enzymes are active on the opposide side of the bacterial
inner membrane, only one of them can be active in a given
Fusion with beta-lactamase. Beta-lactamase is a bacterial enzyme,
which protects cells against lysis by beta-lactam antibiotics such
as ampicillin. It is active only in the periplasmic space.
Fusion with biotin acceptor domain. Membrane topology studies based on
the BAD make use of the compartment-specific in vivo biotinylation
of the domain or the high sensitivity to detect the biotinylated
proteins in combination with proteolysis.
Prolactin fusion. The construct can be digested with PNGase
if the fusion position is in the cytoplasm. If digestion
occured, the fragment(s) can be detected by gel electroforesis.
Green fluorescent protein fusion. It is possible to fuse GFP
to the C-terminal of the investigated protein, or fusing the
investigated protein to GFP (prot-GFP or GFP-prot).
Fusion with invertase plus histidinol dehydrogenase (invHIS4C),
which is active only at the cytoplasmic site.
The system utilizes complementation between separable domains of ubiquitin:
the N terminus of ubiquitin (Nub, amino acids 1-34) and the C terminus of
ubiquitin (Cub, amino acids 35-76), which is followed by a reporter protein
(Rep). Wild-type Nub (NubI, with I being isoleucine at position 13)
spontaneously assembles with Cub-Rep, resulting in proteolytic cleavage at
the C terminus of Cub by a ubiquitin-specific protease(s) and subsequent
release of the reporter fragment. However a mutant of Nub (NubG) in which
Ile-13 is changed to Gly-13 is unable to assemble with Cub-Rep unless two
proteins X and Y that interact with each other are fused to the Cub-R and
to the NubG so that this interaction can force the reassociation of the two
halves of ubiquitin. As a result, the interaction between two proteins X
and Y can be monitored by the cleavage and subsequent activation of the
Fusion with Suc2p invertase. Suc2p becomes rapidly modified by
asparagine-linked glycosylation at multiple sites upon
translocation to the lumen of the ER, resulting in a 20-26-kDa
increase in molecular mass.
Post translational modification: glycolysation or phosphorylation.
Transmembrane proteins are glycolysated in the endoplasmic reticulum
at specific sequence motifs, if the motif is outside. Inserting
or deleting glycolysation specific sequence motif, and investigating
the molecule mass of the modified protein can help to localise the
place of the insertion point.
Wild-type N-glycolysation site can be proven to be glycosylated,
usually with inhibition of glycolysation and molecular weight
shift, or with mutation elimiminating the glycolisation site.
Glycosylation of the domain indicates a luminal position of the
fusion point while no glycosylation indicates a cytoplasmic location.
C-Mannosylation is a unique form of protein glycosylations,
involving the C-glycosidic attachment of a mannosyl
residue to the indole moiety of Trp.
Large scale analysis and mass spectrometry.
Ubiquitination can be occur on NH2 group at the intracellular side of the protein.
Membrane proteins, like all proteins, contain cleavage sites
for various proteolytic enzymes. Externally added proteolytic
enzymes cannot cross the membrane, and therefore cytoplasmic
sites are protected against cleavage by the membrane upon
exposure of bacterial right-side-out membranes or spheroplasts
whereas these sites are not protected against cleavage in
Digestion with a proteinase enzyme (proper name is given in
the value tag) and determining the molecular weight shift.
Proper name of the protease is given in the Value field of the
database entry. The abbreviations used are: ProtK, Proteinase K;
ProtKEP, Proteinase K digestion followed by epitop detection;
Tryp, Trypsin; ChyT, Chymotrypsin; V8, Endopeptidase Glu-C (V8
peptidase); ArgE, Arginin-C endopeptidase; AmpK,
Aminoipeptidase K; AmpM, Aminopeptidase M; Amp, Aminopeptidase;
Subs, Substilisine; CarbA, Carboxipeptidase A; CarbY,
Carboxypeptidase Y; Kall, Kallikrein; ThLy, Thermolysin;
GlnC, Endopeptidase Gln-C; Papa, Papain; LysC, Lys-C endopetidase;
Peps, Pepsin; Clos, Clostripain; Lys-X, Endopeptidase Lys-X;
Arg-X, Endopeptidase Arg-X.
Signal peptidase enzyme on a N-terminal labelled protein can be
used to localise the signal cleveage site.
Labeling membrane embedded parts of a protein with
([125I]TID) followed by proteolysis. The peptides
attached to the membrane remained labeled and can be identified.
Localisation of inserted epitope using specific antibody.
Proper name of the protease is given in the Value field of the
database entry. The abbreviations used are: FMDV, Foot and mouth
disease virus; FETfl, Functional Epitope Tagging of Flag; FETcm,
Functional Epitope Tagging of c-myc; HAEI, Hemagglutinin; ColA,
Colicin A; PP, 6-4 Photoproduct.
Inserting artificial epitope for immunolocalisation.
Using monoclonal antibody against the protein's endogen epitope.
The cysteine residue is a relatively hydrophobic, nonbulky
residue, and its introduction at most positions in a membrane
protein is likely to be tolerated. This feature and the ease
of specific chemical modification with sulfhydryl reagents are
the basis of several methods aiming at topological and
structure-function-related information on membrane proteins.
In cysteine scanning mutagenesis, a series of single cysteine
mutants created and the reactivity of the single cysteine
mutant to various sulfhydryl reagents is assessed under different conditions.
Chemical modification of lysin....
Fluorescence quenching using PM-labelled (N-(1-pyrenyl)-maleimide)
single-Cys mutants, water-soluble (e.g. acrylamide) and lipid-soluble
(e.g. 5-doxylstearic acid, 12-doxylstearic acid) quenchers.
Extracting topology data directly from the 3D structure of the protein.
3D Structure from PDB processed by the TMDET algorithm. Side definitions
are defined using original publications.
Sequence motif, which always is specific to one side of the membrane.
The Value field contains the name of the motif (currently only Walker
A or B, ABC Signature, ATP-Binding, Transit and OGlyc).
Deletion or insertion some residues or segments and investigating
the properties of the protein. The name of the method is on the
Value field. It can be:
Trypsin Cleavage Site Insertion: 31 aa long insertion
containing a lot of trypsin cleavage sites. (PMID:10498725).
Factor Xa protease cleavage site insertion.
Deletion are in permissive site, i.e. properties (function,
structure) are not affected by the deletion, indicating surface
Deleting transmembrane region or regions.
The potential transmembrane segments are inserted to fusion
vectors to find signal anchor and/or stop transfer sequences.
Glycosylation altered by upstream transmembrane region deletion
PhoA fusion activity altered by upstream transmembrane