Most essential proteins perform their function as part of a protein complex. Many such complexes are large, multi-subunit entities whose structures are well beyond the capabilities of traditional methods of structural determination. This TR&D builds upon previous pioneering achievements by the YRC in developing novel techniques to determine the structure of proteins and protein complexes. We will develop an arsenal of new techniques that will complement current methods of structural determination, empowering the scientific community to address structural questions that were previously out of reach.
In Specific Aim 1, we will develop our already highly successful protein cross-linking/mass spectrometry (XL-MS) technology to: (1) Facilitate its adoption throughout the scientific community through development of a quality control toolkit; (2) Increase its sensitivity through improved fragmentation of cross-linked peptides; (3) Incorporate quantitative capabilities allowing XL-MS to be used to study dynamic populations of protein complex conformations and; (4) Develop a comprehensive set of computational tools to identify cross-linked peptides and statistically validate those identifications.
In Specific Aim 2, we will develop a complementary method, molecular painting, which will allow the determination of surface-surface interactions in protein complexes ? even in vivo where current techniques such as HD exchange cannot be applied. In our third and final specific aim we will use co-evolution data to model protein structures and interfaces based on covariation of pairs of residues. We will develop technology to apply deep mutational scanning data (a technology developed in the YRC) to model protein interfaces if sufficient sequences are not available for co-evolution methods to be used. We will integrate these predictions with data generated by cross-linking and molecular painting. Through these aims we will drive the field of higher order protein structure determination into the future.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Biotechnology Resource Grants (P41)
Project #
2P41GM103533-21
Application #
9208073
Study Section
Special Emphasis Panel (ZRG1)
Project Start
1997-09-30
Project End
2022-03-31
Budget Start
2017-01-01
Budget End
2017-12-31
Support Year
21
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Washington
Department
Biochemistry
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Searle, Brian C; Pino, Lindsay K; Egertson, Jarrett D et al. (2018) Chromatogram libraries improve peptide detection and quantification by data independent acquisition mass spectrometry. Nat Commun 9:5128
Payea, Matthew J; Sloma, Michael F; Kon, Yoshiko et al. (2018) Widespread temperature sensitivity and tRNA decay due to mutations in a yeast tRNA. RNA 24:410-422
Seixas, Adriana; Alzugaray, MarĂ­a Fernanda; Tirloni, Lucas et al. (2018) Expression profile of Rhipicephalus microplus vitellogenin receptor during oogenesis. Ticks Tick Borne Dis 9:72-81
Luan, Qing; Zelter, Alex; MacCoss, Michael J et al. (2018) Identification of Wiskott-Aldrich syndrome protein (WASP) binding sites on the branched actin filament nucleator Arp2/3 complex. Proc Natl Acad Sci U S A 115:E1409-E1418
Smukowski Heil, Caiti; Burton, Joshua N; Liachko, Ivan et al. (2018) Identification of a novel interspecific hybrid yeast from a metagenomic spontaneously inoculated beer sample using Hi-C. Yeast 35:71-84
Ziegler, Yvonne S; Moresco, James J; Tu, Patricia G et al. (2018) Proteomic analysis identifies highly expressed plasma membrane proteins for detection and therapeutic targeting of specific breast cancer subtypes. Clin Proteomics 15:30
Nishimura, Noriyuki; Tsuchiya, Wataru; Moresco, James J et al. (2018) Control of seed dormancy and germination by DOG1-AHG1 PP2C phosphatase complex via binding to heme. Nat Commun 9:2132
McClatchy, Daniel B; Ma, Yuanhui; Liem, David A et al. (2018) Quantitative temporal analysis of protein dynamics in cardiac remodeling. J Mol Cell Cardiol 121:163-172
Starita, Lea M; Islam, Muhtadi M; Banerjee, Tapahsama et al. (2018) A Multiplex Homology-Directed DNA Repair Assay Reveals the Impact of More Than 1,000 BRCA1 Missense Substitution Variants on Protein Function. Am J Hum Genet 103:498-508
Wang, Zheng; Wu, Catherine; Aslanian, Aaron et al. (2018) Defective RNA polymerase III is negatively regulated by the SUMO-Ubiquitin-Cdc48 pathway. Elife 7:

Showing the most recent 10 out of 372 publications