TOPDB Topology Data Bank of Transmembrane Proteins
Topology, Structure and Prediction.
Database revision: (4190 entries, 75211 topology data) Quick search:    

Database status

Database revision:
Revision date: 01/01/70
Entries: 4190
Topology data: 75211
Alpha helical proteins: 4067
Beta barrel proteins: 123
PubMed links: 4270
PDB links: 12816
UniProt links: 4190
Visitors: 601740

Experiment types used to determine topologies

Fusion

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.
PhoA Fusion with alkaline phosphatase. Alkaline phosphatase is a periplasmic bacterial enzyme. It is active only in the periplasmic space.
PhoAS 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.
LacZ Fusion with beta-galactosidase. Beta-galactosidase is a bacterial enzyme, which is active only in the cytoplasm.
PhoALacZ 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 construct.
BlaM 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.
BAD 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.
PL 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.
GFP 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).
HIS Fusion with invertase plus histidinol dehydrogenase (invHIS4C), which is active only at the cytoplasmic site.
SplitUbiquitin 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 reporter genes.
Suc2 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.
Other Other fusions.

PostTransMod

Post translational modification: glycolysation or phosphorylation.
NGlyc 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.
Cman C-Mannosylation is a unique form of protein glycosylations, involving the C-glycosidic attachment of a mannosyl residue to the indole moiety of Trp.
Phosphorylation Large scale analysis and mass spectrometry.
Ubiquitination Ubiquitination can be occur on NH2 group at the intracellular side of the protein.

Protease

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 inside-out membranes.
Partial Proteolysis 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 Signal peptidase enzyme on a N-terminal labelled protein can be used to localise the signal cleveage site.
TID Labeling membrane embedded parts of a protein with 3-(trifluoromethyl)-3-(m-[125]iodophenyl)diazirine ([125I]TID) followed by proteolysis. The peptides attached to the membrane remained labeled and can be identified.

Immunolocalisation

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.
Epitope Insertion Inserting artificial epitope for immunolocalisation.
Endogen Epitope Using monoclonal antibody against the protein's endogen epitope.

Chemical_modification

Cys 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.
Lys Chemical modification of lysin....
Quenching 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.

Structure

Extracting topology data directly from the 3D structure of the protein.
PDBTM 3D Structure from PDB processed by the TMDET algorithm. Side definitions are defined using original publications.

Other

Revertants Revertants
SeqMotif 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).
Tailoring 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:
TCSI
Trypsin Cleavage Site Insertion: 31 aa long insertion containing a lot of trypsin cleavage sites. (PMID:10498725).
fXa
Factor Xa protease cleavage site insertion.
DPerm
Deletion are in permissive site, i.e. properties (function, structure) are not affected by the deletion, indicating surface loops.
DTM
Deleting transmembrane region or regions.
M0M1
The potential transmembrane segments are inserted to fusion vectors to find signal anchor and/or stop transfer sequences.
DTMNGlyc
Glycosylation altered by upstream transmembrane region deletion
DTMPhoA
PhoA fusion activity altered by upstream transmembrane region deletion
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