The current best membrane-protein topology-prediction methods are typically based on sequence statistics and contain hundreds of parameters that are optimized on known topologies of membrane proteins. However, because the insertion of transmembrane helices into the membrane is the outcome of molecular interactions among protein, lipids and water, it should be possible to predict topology by methods based directly on physical data, as proposed >20 years ago by Kyte and Doolittle. Here, we present two simple topology-prediction methods using a recently published experimental scale of position-specific amino acid contributions to the free energy of membrane insertion that perform on a par with the current best statistics-based topology predictors. This result suggests that prediction of membrane-protein topology and structure directly from first principles is an attainable goal, given the recently improved understanding of peptide recognition by the translocon.bioinformatics ͉ membrane insertion ͉ topology prediction ͉ translocon ͉ biological hydrophobicity scale P rediction of membrane-protein topology is a classic problem in bioinformatics (1). The very first prediction algorithms were based solely on hydrophobicity plots (2), but these early methods performed poorly in practice and were soon supplanted by machine-learning methods that extract statistical sequence preferences from databases of experimentally mapped topologies. Today, the best performing methods have been trained on extensive datasets and contain hundreds of free parameters that are optimized during the training session (3, 4). With the inclusion of information from aligned homologous sequences, one can expect modern methods to predict the correct topology for up to 80% of all multispanning membrane proteins (5, 6).Yet, from a basic science point of view, it is somewhat unsatisfying that the best methods use sequence statistics rather than physicochemical principles as the underlying basis for the prediction. After all, the cellular machineries (translocons) responsible for membrane-protein biogenesis do not have access to statistical data but rather exploit molecular interactions (lipid-protein, water-protein, and protein-protein) to ensure that membrane proteins attain their correct topology (7,8). In principle, therefore, it should be possible to match the performance of current machine-learning predictors by using methods based on the same physical properties that determine translocon-mediated membrane insertion.Despite years of biophysical studies of protein-lipid interactions (9-12), it is only recently that the first comprehensive dataset describing the insertion of transmembrane (TM) ␣-helices into the endoplasmic reticulum (ER) membrane in terms of free-energy contributions from individual amino acids in different positions along the membrane normal has been published (13). Here, we show that a simple additive free-energy model derived from these experimental data, when coupled with the ''positive-inside '' rule (14), predicts the topolog...

Many a-helical membrane proteins contain internal symmetries, indicating that they might have evolved through a gene duplication and fusion event. Here, we have characterized internal duplications among membrane proteins of known structure and in three complete genomes. We found that the majority of large transmembrane (TM) proteins contain an internal duplication. The duplications found showed a large variability both in the number of TM-segments included and in their orientation. Surprisingly, an approximately equal number of antiparallel duplications and parallel duplications were found. However, of all 11 superfamilies with an internal duplication, only for one, the AcrB Multidrug Efflux Pump, the duplicated unit could be found in its nonduplicated form. An evolutionary analysis of the AcrB homologs indicates that several independent fusions have occurred, including the fusion of the SecD and SecF proteins into the 12-TM-protein SecDF in Brucella and Staphylococcus aureus. In one additional case, the Vitamin B 12 transporter-like ABC transporters, the protein had undergone an additional fusion to form protein with 20 TM-helices in several bacterial genomes. Finally, homologs to all human membrane proteins were used to detect the presence of duplicated and nonduplicated proteins. This confirmed that only in rare cases can homologs with different duplication status be found, although internal symmetry is frequent among these proteins. One possible explanation is that it is frequent that duplication and fusion events happen simultaneously and that there is almost always a strong selective advantage for the fused form.