Dataset Information

Identifying functionally important conformational changes in proteins: activation of the yeast ?-factor receptor Ste2p.

ABSTRACT: We have developed a procedure in which disulfide cross-links are used to identify regions of proteins that undergo functionally important intramolecular motion. The approach was applied to the identification of disulfide bonds that stabilize the active state of the yeast ?-mating pheromone receptor Ste2p, a member of the superfamily of G protein-coupled receptors. Cysteine residues were introduced at random positions in targeted regions of a starting allele of Ste2p that completely lacks cysteines. Libraries of mutated receptors were then screened for alleles that exhibit constitutive signaling. Two strongly activated alleles were recovered containing cysteine residues in transmembrane (TM) segments 5 and 6. Constitutive activity of these alleles was dependent on the presence of both introduced cysteines and was sensitive to reducing agent. Cross-linked peptides derived from the mutant receptors were detected by immunoblotting. Additional sites of cross-linking between TM segments 5 and 6 that did not lead to constitutive activation were also identified. These results indicate that relative motion of the TM segments 5 and 6 in the extracellular half of the membrane is sufficient to activate the receptor and that TM segment 6, but not TM segment 5, exhibits rotational mobility that is not associated with receptor activation.

SUBMITTER: Taslimi A

PROVIDER: S-EPMC3346263 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Identifying functionally important conformational changes in proteins: activation of the yeast α-factor receptor Ste2p.

Taslimi Amir A Mathew Elizabeth E Celić Andjelka A Wessel Sarah S Dumont Mark E ME

Journal of molecular biology 20120303 5

We have developed a procedure in which disulfide cross-links are used to identify regions of proteins that undergo functionally important intramolecular motion. The approach was applied to the identification of disulfide bonds that stabilize the active state of the yeast α-mating pheromone receptor Ste2p, a member of the superfamily of G protein-coupled receptors. Cysteine residues were introduced at random positions in targeted regions of a starting allele of Ste2p that completely lacks cystein ...[more]

PMID: 22387470

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Project description:BackgroundPhylogenetic profiles record the occurrence of homologs of genes across fully sequenced organisms. Proteins with similar profiles are typically components of protein complexes or metabolic pathways. Various existing methods measure similarity between two profiles and, hence, the likelihood that the two proteins co-evolve. Some methods ignore phylogenetic relationships between organisms while others account for such with metrics that explicitly model the likelihood of two proteins co-evolving on a tree. The latter methods more sensitively detect co-evolving proteins, but at a significant computational cost. Here we propose a novel heuristic to improve phylogenetic profile analysis that accounts for phylogenetic relationships between genomes in a computationally efficient fashion. We first order the genomes within profiles and then enumerate runs of consecutive matches and accurately compute the probability of observing these. We hypothesize that profiles with many runs are more likely to involve functionally related proteins than profiles in which all the matches are concentrated in one interval of the tree.ResultsWe compared our approach to various previously published methods that both ignore and incorporate the underlying phylogeny between organisms. To evaluate performance, we compare the functional similarity of rank-ordered lists of protein pairs that share similar phylogenetic profiles by assessing significance of overlap in their Gene Ontology annotations. Accounting for runs in phylogenetic profile matches improves our ability to identify functionally related pairs of proteins. Furthermore, the networks that result from our approach tend to have smaller clusters of co-evolving proteins than networks computed using previous approaches and are thus more useful for inferring functional relationships. Finally, we report that our approach is orders of magnitude more computationally efficient than full tree-based methods.ConclusionWe have developed an improved method for analyzing phylogenetic profiles. The method allows us to more accurately and efficiently infer functional relationships between proteins based on these profiles than other published approaches. As the number of fully sequenced genomes increases, it becomes more important to account for evolutionary relationships among organisms in comparative analyses. Our approach, therefore, serves as an important example of how these relationships may be accounted for in an efficient manner.

Dataset Information

Identifying functionally important conformational changes in proteins: activation of the yeast ?-factor receptor Ste2p.

Publications

Identifying functionally important conformational changes in proteins: activation of the yeast α-factor receptor Ste2p.

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