Biomedical subjects

Mikaela Rapp

Publications and source records attributed to Mikaela Rapp.

5 recordsLinked to original sources

PubMed2006

Identification and evolution of dual-topology membrane proteins.

Integral membrane proteins are generally believed to have unique membrane topologies. However, it has been suggested that dual-topology proteins that adopt a mixture of two opposite orientations in the membrane may exist. Here we show that the membrane orientations of five dual-topology candidates identified in Escherichia coli are highly sensitive to changes in the distribution of positively charged residues, that genes in families containing dual-topology candidates occur in genomes either as pairs or as singletons and that gene pairs encode two oppositely oriented proteins whereas singletons encode dual-topology candidates. Our results provide strong support for the existence of dual-topology proteins and shed new light on the evolution of membrane-protein topology and structure.

Amino Acid Sequence↗

PubMed2005

Experimentally constrained topology models for 51,208 bacterial inner membrane proteins.

We have used 502 Escherichia coli inner membrane proteins with experimentally determined C-terminal locations (cytoplasmic or periplasmic) from a recently published data set, together with an additional 106 bacterial membrane proteins with known topology, as queries in BLAST searches against a data base of 658,210 bacterial open reading frames from GenBank. We find 51,208 homologs to the query sequences for which we can assign the location of the C terminus or an internal residue to the same side of the membrane as the query's C terminus. These assignments are then used as constraints for topology prediction. The 51,208 much improved topology models derived in this way cover approximately 30% of all predicted bacterial inner membrane proteins in 225 fully sequenced bacterial genomes.

Algorithms↗

PubMed2005

A scalable, GFP-based pipeline for membrane protein overexpression screening and purification.

We describe a generic, GFP-based pipeline for membrane protein overexpression and purification in Escherichia coli. We exemplify the use of the pipeline by the identification and characterization of E. coli YedZ, a new, membrane-integral flavocytochrome. The approach is scalable and suitable for high-throughput applications. The GFP-based pipeline will facilitate the characterization of the E. coli membrane proteome and serves as an important reference for the characterization of other membrane proteomes.

Escherichia coli↗

PubMed2005

Global topology analysis of the Escherichia coli inner membrane proteome.

The protein complement of cellular membranes is notoriously resistant to standard proteomic analysis and structural studies. As a result, membrane proteomes remain ill-defined. Here, we report a global topology analysis of the Escherichia coli inner membrane proteome. Using C-terminal tagging with the alkaline phosphatase and green fluorescent protein, we established the periplasmic or cytoplasmic locations of the C termini for 601 inner membrane proteins. By constraining a topology prediction algorithm with this data, we derived high-quality topology models for the 601 proteins, providing a firm foundation for future functional studies of this and other membrane proteomes. We also estimated the overexpression potential for 397 green fluorescent protein fusions; the results suggest that a large fraction of all inner membrane proteins can be produced in sufficient quantities for biochemical and structural work.

Alkaline Phosphatase↗

PubMed2004

Experimentally based topology models for E. coli inner membrane proteins.

Membrane protein topology predictions can be markedly improved by the inclusion of even very limited experimental information. We have recently introduced an approach for the production of reliable topology models based on a combination of experimental determination of the location (cytoplasmic or periplasmic) of a protein's C terminus and topology prediction. Here, we show that determination of the location of a protein's C terminus, rather than some internal loop, is the best strategy for large-scale topology mapping studies. We further report experimentally based topology models for 31 Escherichia coli inner membrane proteins, using methodology suitable for genome-scale studies.

Alkaline Phosphatase↗