Membrane protein preparation for TROSY NMR screening.
The first steps toward undertaking an NMR structural study of a new protein is very often to purify the protein and then to acquire an HSQC or TROSY NMR spectrum, the quality of which is used to assess the feasibility of an NMR-based structural determination. Relatively few integral membrane proteins (IMPs) have been subjected even to this very preliminary stage of NMR analysis. Here, NMR feasibility testing methods are outlined that are tailored for hexahistidine-tagged IMPs that have been expressed in Escherichia coli. Generally applicable protocols are presented for expression testing, purification, and NMR sample preparation. A 2D TROSY pulse sequence that has been optimized for use with IMPs is also presented.
Tian
Changlin
C
Department of Computer Science, University of South Carolina, Columbia 29208, USA.
Karra
Murthy D
MD
Ellis
Charles D
CD
Jacob
Jaison
J
Oxenoid
Kirill
K
Sönnichsen
Frank
F
Sanders
Charles R
CR
D003902:Detergents; D003903:Deuterium; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry*
D003902:Detergents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C470844:ZinT protein, E coli; D003903:Deuterium