Methodology for measuring conformation of solvent-disrupted protein subunits using T-WAVE ion mobility MS: an investigation into eukaryotic initiation factors.
The methodology developed in the research presented herein makes use of chaotropic solvents to gently dissociate subunits from an intact macromolecular complex and subsequently allows for the measurement of collision cross section (CCS) for both the recombinant (R-eIF3k) and solvent dissociated form of the subunit (S-eIF3k). In this particular case, the k subunit from the eukaryotic initiation factor 3 (eIF3) was investigated in detail. Experimental and theoretical CCS values show both the recombinant and solvent disrupted forms of the protein to be essentially the same. The ultimate goal of the project is to structurally characterize all the binding partners of eIF3, determine which subunits interact directly, and investigate how subunits may change conformation when they form complexes with other proteins. Research presented herein is the first report showing retention of solution conformation of a protein as evidenced by CCS measurements of both recombinant and solvent disrupted versions of the same protein.
Leary
Julie A
JA
Department of Molecular and Cellular Biology, University of California at Davis, Davis, California 95616, USA. jaleary@ucdavis.edu
Schenauer
Matthew R
MR
Stefanescu
Raluca
R
Andaya
Armann
A
Ruotolo
Brandon T
BT
Robinson
Carol V
CV
Thalassinos
Konstantinos
K
Scrivens
James H
JH
Sokabe
Masaaki
M
Hershey
John W B
JW
D003198:Computer Simulation; D013058:Mass Spectrometry / Q000379:methods*; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D008956:Models, Chemical; D011487:Protein Conformation; D011489:Protein Denaturation; D021122:Protein Subunits; D012997:Solvents / Q000737:chemistry*
C468124:EIF3K protein, human; D008869:Microtubule-Associated Proteins; D021122:Protein Subunits; D012997:Solvents