The Molecular Architecture of Native BBSome Obtained by an Integrated Structural Approach.

The unique membrane composition of cilia is maintained by a diffusion barrier at the transition zone that is breached when the BBSome escorts signaling receptors out of cilia. Understanding how the BBSome removes proteins from cilia has been hampered by a lack of structural information. Here, we present a nearly complete Cα model of BBSome purified from cow retina.

Protein interaction perturbation profiling at amino-acid resolution.

The identification of genomic variants in healthy and diseased individuals continues to rapidly outpace our ability to functionally annotate these variants. Techniques that both systematically assay the functional consequences of nucleotide-resolution variation and can scale to hundreds of genes are urgently required. We designed a sensitive yeast two-hybrid-based 'off switch' for positive selection of interaction-disruptive variants from complex genetic libraries.

Systematic Protein-Protein Interaction Analysis Reveals Intersubcomplex Contacts in the Nuclear Pore Complex.

The nuclear pore complex (NPC) enables transport across the nuclear envelope. It is one of the largest multiprotein assemblies in the cell, built from about 30 proteins called nucleoporins (Nups), organized into distinct subcomplexes. Structure determination of the NPC is a major research goal.

Multiple protein-protein interactions converging on the Prp38 protein during activation of the human spliceosome.

Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel α-helices and lacks similarities to RNA-binding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein-protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage as Prp38.

A noncanonical PWI domain in the N-terminal helicase-associated region of the spliceosomal Brr2 protein.

The spliceosomal RNA helicase Brr2 is required for the assembly of a catalytically active spliceosome on a messenger RNA precursor. Brr2 exhibits an unusual organization with tandem helicase units, each comprising dual RecA-like domains and a Sec63 homology unit, preceded by a more than 400-residue N-terminal helicase-associated region. Whereas recent crystal structures have provided insights into the molecular architecture and regulation of the Brr2 helicase region, little is known about the structural organization and function of its N-terminal part.

Phospho-tyrosine dependent protein-protein interaction network.

Post-translational protein modifications, such as tyrosine phosphorylation, regulate protein-protein interactions (PPIs) critical for signal processing and cellular phenotypes. We extended an established yeast two-hybrid system employing human protein kinases for the analyses of phospho-tyrosine (pY)-dependent PPIs in a direct experimental, large-scale approach. We identified 292 mostly novel pY-dependent PPIs which showed high specificity with respect to kinases and interacting proteins and validated a large fraction in co-immunoprecipitation experiments from mammalian cells.

Pyrosequencing allows the detection of emergent ganciclovir resistance mutations after HCMV infection.

Prenatally acquired human cytomegalovirus (HCMV) infection is the most frequent viral infection of newborns in developed countries. Virostatic therapy is accompanied by side effects and stepwise emergence of resistant virus variants. Different genotypic approaches show limited sensitivity in detecting on-growing minor resistant virus populations.

Improved detection of mutated human cytomegalovirus UL97 by pyrosequencing.

Ganciclovir (GCV) resistance frequently occurs upon prolonged treatment of ongoing active human cytomegalovirus (HCMV) infection in individuals with immature or compromised immune functions (e.g., recipients of solid-organ and hematopoietic stem cell transplants).