Structural biology of transmembrane domains: efficient production and characterization of transmembrane peptides by NMR.
Structural characterization of transmembrane peptides (TMPs) is justified because transmembrane domains of membrane proteins appear to often function independently of the rest of the protein. However, the challenge in obtaining milligrams of isotopically labeled TMPs to study these highly hydrophobic peptides by nuclear magnetic resonance (NMR) is significant. In the present work, a protocol is developed to produce, isotopically label, and purify TMPs in high yield as well as to initially characterize the TMPs with CD and both solution and solid-state NMR. Six TMPs from three integral membrane proteins, CorA, M2, and KdpF, were studied. CorA and KdpF are from Mycobacterium tuberculosis, while M2 is from influenza A virus. Several milligrams of each of these TMPs ranging from 25 to 89 residues were obtained per liter of M9 culture. The initial structural characterization results showed that these peptides were well folded in both detergent micelles and lipid bilayer preparations. The high yield, the simplicity of purification, and the convenient protocol represents a suitable approach for NMR studies and a starting point for characterizing the transmembrane domains of membrane proteins.
Hu
Jian
J
Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, USA.
Qin
Huajun
H
Li
Conggang
C
Sharma
Mukesh
M
Cross
Timothy A
TA
Gao
Fei Philip
FP
D000595:Amino Acid Sequence; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008823:Micelles; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism
D008051:Lipid Bilayers; D008565:Membrane Proteins; D008823:Micelles; D010455:Peptides; D011993:Recombinant Fusion Proteins