排序方式: 共有4条查询结果,搜索用时 15 毫秒
1
1.
Libusha Kelly Ursula Pieper Narayanan Eswar Franklin A. Hays Min Li Zygy Roe-Zurz Deanna L. Kroetz Kathleen M. Giacomini Robert M. Stroud Andrej Sali 《Journal of structural and functional genomics》2009,10(4):269-280
Membrane proteins serve as cellular gatekeepers, regulators, and sensors. Prior studies have explored the functional breadth
and evolution of proteins and families of particular interest, such as the diversity of transport-associated membrane protein
families in prokaryotes and eukaryotes, the composition of integral membrane proteins, and family classification of all human
G-protein coupled receptors. However, a comprehensive analysis of the content and evolutionary associations between membrane
proteins and families in a diverse set of genomes is lacking. Here, a membrane protein annotation pipeline was developed to
define the integral membrane genome and associations between 21,379 proteins from 34 genomes; most, but not all of these proteins
belong to 598 defined families. The pipeline was used to provide target input for a structural genomics project that successfully
cloned, expressed, and purified 61 of our first 96 selected targets in yeast. Furthermore, the methodology was applied (1)
to explore the evolutionary history of the substrate-binding transmembrane domains of the human ABC transporter superfamily,
(2) to identify the multidrug resistance-associated membrane proteins in whole genomes, and (3) to identify putative new membrane
protein families. 相似文献
2.
Min Li Franklin A. Hays Zygy Roe-Zurz Libusha Kelly Chi-Min Ho Ursula Pieper Larry J.W. Miercke Kathleen M. Giacomini Andrej Sali Robert M. Stroud 《Journal of molecular biology》2009,385(3):820-4730
A medium-throughput approach is used to rapidly identify membrane proteins from a eukaryotic organism that are most amenable to expression in amounts and quality adequate to support structure determination. The goal was to expand knowledge of new membrane protein structures based on proteome-wide coverage. In the first phase, membrane proteins from the budding yeast Saccharomyces cerevisiae were selected for homologous expression in S. cerevisiae, a system that can be adapted to expression of membrane proteins from other eukaryotes. We performed medium-scale expression and solubilization tests on 351 rationally selected membrane proteins from S. cerevisiae. These targets are inclusive of all annotated and unannotated membrane protein families within the organism's membrane proteome. Two hundred seventy-two targets were expressed, and of these, 234 solubilized in the detergent n-dodecyl-β-d-maltopyranoside. Furthermore, we report the identity of a subset of targets that were purified to homogeneity to facilitate structure determinations. The extensibility of this approach is demonstrated with the expression of 10 human integral membrane proteins from the solute carrier superfamily. This discovery-oriented pipeline provides an efficient way to select proteins from particular membrane protein classes, families, or organisms that may be more suited to structure analysis than others. 相似文献
3.
Franklin A. Hays Zygy Roe-Zurz Min Li Libusha Kelly Franz Gruswitz Andrej Sali Robert M. Stroud 《Journal of structural and functional genomics》2009,10(1):9-16
Persistent hurdles impede the successful determination of high-resolution crystal structures of eukaryotic integral membrane
proteins (IMP). We designed a high-throughput structural genomics oriented pipeline that seeks to minimize effort in uncovering
high-quality, responsive non-redundant targets for crystallization. This “discovery-oriented” pipeline sidesteps two significant
bottlenecks in the IMP structure determination pipeline: expression and membrane extraction with detergent. In addition, proteins
that enter the pipeline are then rapidly vetted by their presence in the included volume on a size-exclusion column—a hallmark
of well-behaved IMP targets. A screen of 384 rationally selected eukaryotic IMPs in baker’s yeast Saccharomyces cerevisiae is outlined to demonstrate the results expected when applying this discovery-oriented pipeline to whole-organism membrane
proteomes.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Franklin A. Hays and Zygy Roe-Zurz have contributed equally to this work. 相似文献
4.
Rebba C. Boswell-Casteel Jennifer M. Johnson Kelli D. Duggan Zygy Roe-?ur? Hannah Schmitz Carter Burleson Franklin A. Hays 《The Journal of biological chemistry》2014,289(35):24440-24451
Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that transport nucleosides and, to a lesser extent, nucleobases across cell membranes. ENTs modulate efficacy for a range of human therapeutics and function in a diffusion-controlled bidirectional manner. A detailed understanding of ENT function at the molecular level has remained elusive. FUN26 (function unknown now 26) is a putative ENT homolog from S. cerevisiae that is expressed in vacuole membranes. In the present system, proteoliposome studies of purified FUN26 demonstrate robust nucleoside and nucleobase uptake into the luminal volume for a broad range of substrates. This transport activity is sensitive to nucleoside modifications in the C(2′)- and C(5′)-positions on the ribose sugar and is not stimulated by a membrane pH differential. [3H]Adenine nucleobase transport efficiency is increased ∼4-fold relative to nucleosides tested with no observed [3H]adenosine or [3H]UTP transport. FUN26 mutational studies identified residues that disrupt (G463A or G216A) or modulate (F249I or L390A) transporter function. These results demonstrate that FUN26 has a unique substrate transport profile relative to known ENT family members and that a purified ENT can be reconstituted in proteoliposomes for functional characterization in a defined system. 相似文献
1