RESUMO
The solid-state NMR orientation-dependent frequencies measured for membrane proteins in macroscopically oriented lipid bilayers provide precise orientation restraints for structure determination in membranes. Here we show that this information can also be used to supplement crystallographic structural data to establish the orientation of a membrane protein in the membrane. This is achieved by incorporating a few orientation restraints, measured for the Escherichia coli outer membrane protein OmpX in magnetically oriented lipid bilayers (bicelles), in a simulated annealing calculation with the coordinates of the OmpX crystal structure. The (1)H-(15)N dipolar couplings measured for the seven Phe residues of OmpX in oriented bilayers can be assigned by back-calculation of the NMR spectrum from the crystal structure and are sufficient to establish the three-dimensional orientation of the protein in the membrane, while the (15)N chemical shifts provide a measure of cross-validation for the analysis. In C14 lipid bilayers, OmpX adopts a transmembrane orientation with a 7 degrees tilt of its beta-barrel axis relative to the membrane normal, matching the hydrophobic thickness of the barrel with that of the membrane.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Hidrolases/química , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética/métodos , Anisotropia , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Fenilalanina/química , Fosfolipídeos/química , Conformação ProteicaRESUMO
Determining the atomic resolution structures of membrane proteins is of particular interest in contemporary structural biology. Helical membrane proteins constitute one-third of the expressed proteins encoded in a genome, many drugs have membrane-bound proteins as their receptors, and mutations in membrane proteins result in human diseases. Although integral membrane proteins provide daunting technical challenges for all methods of protein structure determination, nuclear magnetic resonance (NMR) spectroscopy can be an extremely versatile and powerful method for determining their structures and characterizing their dynamics, in lipid environments that closely mimic the cell membranes. Once milligram amounts of isotopically labeled protein are expressed and purified, micelle samples can be prepared for solution NMR analysis, and lipid bilayer samples can be prepared for solid-state NMR analysis. The two approaches are complementary and can provide detailed structural and dynamic information. This paper describes the steps for membrane protein structure determination using solution and solid-state NMR. The methods for protein expression and purification, sample preparation and NMR experiments are described and illustrated with examples from the FXYD proteins, a family of regulatory subunits of the Na,K-ATPase.
Assuntos
Bicamadas Lipídicas/química , Proteínas de Membrana/química , Micelas , Ressonância Magnética Nuclear Biomolecular/métodos , Fosfoproteínas/química , Sequência de Aminoácidos , Linhagem Celular Transformada , Detergentes , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Isótopos de Nitrogênio , Fosfoproteínas/genética , Isótopos de Fósforo , Plasmídeos , Canais de Potássio/química , Canais de Potássio/genética , Estrutura Terciária de Proteína , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
The proteins PLM (phospholemman), CHIF (channel inducing factor), and Mat8 (mammary tumor protein 8 kDa) are members of the FXYD family of ion transport regulatory membrane proteins. Here we describe their cloning and expression in Escherichia coli, and their purification for NMR structural studies in lipid micelles and lipid bilayers. The molecular masses of the purified recombinant FXYD proteins, determined from SDS-PAGE and from MALDI TOF mass spectrometry, reflect monomeric species. The solution NMR and CD spectra in SDS micelles show that they adopt helical conformations. The solid-state NMR spectra in lipid bilayers give the first view of their transmembrane architecture.