A microscale protein NMR sample screening pipeline.
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 30-200 microg in 8-35 microl 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.
Rossi
Paolo
P
Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, 08854, USA.
Swapna
G V T
GV
Huang
Yuanpeng J
YJ
Aramini
James M
JM
Anklin
Clemens
C
Conover
Kenith
K
Hamilton
Keith
K
Xiao
Rong
R
Acton
Thomas B
TB
Ertekin
Asli
A
Everett
John K
JK
Montelione
Gaetano T
GT
D002021:Buffers; D003001:Cloning, Molecular / Q000379:methods; D030541:Databases, Genetic; D041961:Deuterium Exchange Measurement / Q000379:methods; D023281:Genomics / Q000379:methods*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization; D011506:Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics
D002021:Buffers; D011506:Proteins