Ganesh P. Subedi, Elijah T. Roberts, Alexander R. Davis, Paul G. Kremer, I. Jonathan Amster, Adam W. Barb
{"title":"A comprehensive assessment of selective amino acid 15N-labeling in human embryonic kidney 293 cells for NMR spectroscopy","authors":"Ganesh P. Subedi, Elijah T. Roberts, Alexander R. Davis, Paul G. Kremer, I. Jonathan Amster, Adam W. Barb","doi":"10.1007/s10858-023-00434-3","DOIUrl":null,"url":null,"abstract":"<div><p>A large proportion of human proteins contain post-translational modifications that cannot be synthesized by prokaryotes. Thus, mammalian expression systems are often employed to characterize structure/function relationships using NMR spectroscopy. Here we define the selective isotope labeling of secreted, post-translationally modified proteins using human embryonic kidney (HEK)293 cells. We determined that alpha-[<sup>15</sup>N]- atoms from 10 amino acids experience minimal metabolic scrambling (C, F, H, K, M, N, R, T, W, Y). Two more interconvert to each other (G, S). Six others experience significant scrambling (A, D, E, I, L, V). We also demonstrate that tuning culture conditions suppressed V and I scrambling. These results define expectations for <sup>15</sup>N-labeling in HEK293 cells. </p></div>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"78 2","pages":"125 - 132"},"PeriodicalIF":1.3000,"publicationDate":"2024-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Biomolecular NMR","FirstCategoryId":"99","ListUrlMain":"https://link.springer.com/article/10.1007/s10858-023-00434-3","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
A large proportion of human proteins contain post-translational modifications that cannot be synthesized by prokaryotes. Thus, mammalian expression systems are often employed to characterize structure/function relationships using NMR spectroscopy. Here we define the selective isotope labeling of secreted, post-translationally modified proteins using human embryonic kidney (HEK)293 cells. We determined that alpha-[15N]- atoms from 10 amino acids experience minimal metabolic scrambling (C, F, H, K, M, N, R, T, W, Y). Two more interconvert to each other (G, S). Six others experience significant scrambling (A, D, E, I, L, V). We also demonstrate that tuning culture conditions suppressed V and I scrambling. These results define expectations for 15N-labeling in HEK293 cells.
期刊介绍：
The Journal of Biomolecular NMR provides a forum for publishing research on technical developments and innovative applications of nuclear magnetic resonance spectroscopy for the study of structure and dynamic properties of biopolymers in solution, liquid crystals, solids and mixed environments, e.g., attached to membranes. This may include:
Three-dimensional structure determination of biological macromolecules (polypeptides/proteins, DNA, RNA, oligosaccharides) by NMR.
New NMR techniques for studies of biological macromolecules.
Novel approaches to computer-aided automated analysis of multidimensional NMR spectra.
Computational methods for the structural interpretation of NMR data, including structure refinement.
Comparisons of structures determined by NMR with those obtained by other methods, e.g. by diffraction techniques with protein single crystals.
New techniques of sample preparation for NMR experiments (biosynthetic and chemical methods for isotope labeling, preparation of nutrients for biosynthetic isotope labeling, etc.). An NMR characterization of the products must be included.
京公网安备 11010802042870号
京ICP备2023020795号-1
分享
求助内容：
应助结果提醒方式：
扫码关注我们
