Comprehensive evaluation of solution nuclear magnetic resonance spectroscopy sample preparation for helical integral membrane proteins.
The preparation of high quality samples is a critical challenge for the structural characterization of helical integral membrane proteins. Solving the structures of this diverse class of proteins by solution nuclear magnetic resonance spectroscopy (NMR) requires that well-resolved 2D 1H/15N chemical shift correlation spectra be obtained. Acquiring these spectra demands the production of samples with high levels of purity and excellent homogeneity throughout the sample. In addition, high yields of isotopically enriched protein and efficient purification protocols are required. We describe two robust sample preparation methods for preparing high quality, homogeneous samples of helical integral membrane proteins. These sample preparation protocols have been combined with screens for detergents and sample conditions leading to the efficient production of samples suitable for solution NMR spectroscopy. We have examined 18 helical integral membrane proteins, ranging in size from approximately 9 kDa to 29 kDa with 1-4 transmembrane helices, originating from a number of bacterial and viral genomes. 2D 1H/15N chemical shift correlation spectra acquired for each protein demonstrate well-resolved resonances, and >90% detection of the predicted resonances. These results indicate that with proper sample preparation, high quality solution NMR spectra of helical integral membrane proteins can be obtained greatly enhancing the probability for structural characterization of these important proteins.
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Richard C
RC
Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306-4390, USA.
Moore
Jacob D
JD
Nguyen
Hau B
HB
Sharma
Mukesh
M
Chase
Rose
R
Gao
Fei Philip
FP
Mobley
Charles K
CK
Sanders
Charles R
CR
Ma
Liping
L
Sönnichsen
Frank D
FD
Lee
Sangwon
S
Howell
Stanley C
SC
Opella
Stanley J
SJ
Cross
Timothy A
TA
D002247:Carbon Isotopes / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D005069:Evaluation Studies as Topic; D008565:Membrane Proteins / Q000737:chemistry*; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D017433:Protein Structure, Secondary
D002247:Carbon Isotopes; D003902:Detergents; D008565:Membrane Proteins; D009587:Nitrogen Isotopes