Abstract As part of efforts to develop improved methods for NMR protein sample preparation and structure determination, the Northeast Structural Genomics Consortium (NESG) has implemented an NMR screening pipeline for protein target selection, construct optimization, and buffer optimization, incorporating efficient microscale NMR screening of proteins using a micro-cryoprobe. The process is feasible because the newest generation probe requires only small amounts of protein, typically 30â??200 μg in 8â??35 μl volume. Extensive automation has been made possible by the combination of database tools, mechanization of key process steps, and the use of a micro-cryoprobe that gives excellent data while requiring little optimization and manual setup. In this perspective, we describe the overall process used by the NESG for screening NMR samples as part of a sample optimization process, assessing optimal construct design and solution conditions, as well as for determining protein rotational correlation times in order to assess protein oligomerization states. Database infrastructure has been developed to allow for flexible implementation of new screening protocols and harvesting of the resulting output. The NESG micro NMR screening pipeline has also been used for detergent screening of membrane proteins. Descriptions of the individual steps in the NESG NMR sample design, production, and screening pipeline are presented in the format of a standard operating procedure.
Content Type Journal Article
Pages 11-22
DOI 10.1007/s10858-009-9386-z
Authors
Paolo Rossi, The State University of New Jersey Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers 679 Hoes Lane Piscataway NJ 08854 USA
G. V. T. Swapna, The State University of New Jersey Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers 679 Hoes Lane Piscataway NJ 08854 USA
Yuanpeng J. Huang, The State University of New Jersey Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers 679 Hoes Lane Piscataway NJ 08854 USA
James M. Aramini, The State University of New Jersey Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers 679 Hoes Lane Piscataway NJ 08854 USA
Clemens Anklin, Bruker Biospin Corporation 15 Fortune Drive Billerica MA 01821 USA
Kenith Conover, The State University of New Jersey Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers 679 Hoes Lane Piscataway NJ 08854 USA
Keith Hamilton, The State University of New Jersey Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers 679 Hoes Lane Piscataway NJ 08854 USA
Rong Xiao, The State University of New Jersey Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers 679 Hoes Lane Piscataway NJ 08854 USA
Thomas B. Acton, The State University of New Jersey Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers 679 Hoes Lane Piscataway NJ 08854 USA
Asli Ertekin, The State University of New Jersey Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers 679 Hoes Lane Piscataway NJ 08854 USA
John K. Everett, The State University of New Jersey Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers 679 Hoes Lane Piscataway NJ 08854 USA
Gaetano T. Montelione, The State University of New Jersey Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers 679 Hoes Lane Piscataway NJ 08854 USA
[Question from NMRWiki Q&A forum] 2DNOESY with N15 C13 Labelled sample or Unlabelled sample
2DNOESY with N15 C13 Labelled sample or Unlabelled sample
Dear Friends, I am in process of doing structure calculation of dimeric protein 22kD by using ARIA2.2 . Can I acquire 2DNOESY spectrum for my protein. Will it provide useful information for structure calculation? And whether I should acquire 2DNOESY with N15 C13 Labelled sample or Unlabelled sample . Which sample would be appropriate for this kind of experiment on VNMRJ 700 with biopack ?
Thanks & Regards Srinivas
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A microscale protein NMR sample screening pipeline
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