Simultaneous assignment and structure determination of a membrane protein from NMR orientational restraints

Abstract

A solid-state NMR approach for simultaneous resonance assignment and three-dimensional structure determination of a membrane protein in lipid bilayers is described. The approach is based on the scattering, hence the descriptor "shotgun," of 15 N-labeled amino acids throughout the protein sequence (and the resulting NMR spectra). The samples are obtained by protein expression in bacteria grown on media in which one type of amino acid is labeled and the others are not. Shotgun NMR short-circuits the laborious and time-consuming process of obtaining complete sequential assignments prior to the calculation of a protein structure from the NMR data by taking advantage of the orientational information inherent to the spectra of aligned proteins. As a result, it is possible to simultaneously assign resonances and measure orientational restraints for structure determination. A total of five two-dimensional 1 H/ 15 N PISEMA (polarization inversion spin exchange at the magic angle) spectra, from one uniformly and four selectively 15 N-labeled samples, were sufficient to determine the structure of the membrane-bound form of the 50-residue major pVIII coat protein of fd filamentous bacteriophage. Pisa (polarity index slat angle) wheels are an essential element in the process, which starts with the simultaneous assignment of resonances and the assembly of isolated polypeptide segments, and culminates in the complete three-dimensional structure of the protein with atomic resolution. The principles are also applicable to weakly aligned proteins studied by solution NMR spectroscopy.[The structure we determined for the membrane-bound form of the Fd bacteriophage pVIII coat protein has been deposited in the Protein Data Bank as PDB file 1MZT.]

Application of solid-state NMR restraint potentials in membrane protein modeling

“…S6 for vacuum and S7 for GBSW). The averaged tilt angles of the IP and TM helices (90.1° for IP and 22.4° for TM in vacuum, and 85.3° for IP and 22.0° for TM in GBSW) are in good agreement with those in PDB:1MZT (92.0° for IP and 20.3° for TM) [12]. Figure 2a shows both the averaged structure obtained in vacuum and PDB:1MZT.…”

“…The structure was determined by solution NMR (PDB:1FDM) [31] as well as SSNMR (PDB:1MZT) [12]. Following the structure determination protocol, we obtained 45 (vacuum) and 61 (GBSW) structures that satisfy the selection criteria (δσ ≤ 1.0 ppm and δν ≤ 1.0 kHz).…”

“…In the MC approach, since the tilt angles of the IP and TM helices are independently determined without the connecting turn (residues [19][20], the resulting structures may have geometric ambiguity. Therefore, Opella and coworkers first categorized their resulting structures into four families that have the same tilt angles but different orientations [12]. They then selected family A (as shown in Fig.…”

“…Then, we developed and optimized a protocol for SSNMR structure determination by applying the restraint potentials to several membrane proteins whose structures were previously determined using the MC approach. To illustrate the efficacy of our approach, we determined the monomer structures of the major pVIII coat protein of fd filamentous bacteriophage (fd coat) [12], the mercury ion transporter (MerF) from the bacterial mercury detoxification system [13], the TM domain of the M2 protein (M2TMP) from influenza A virus [14], and the TM domain of viral protein "u" (VpuTM) from human immunodeficiency virus 1 (HIV-1) [9]. We further utilized the monomer structures to model oligomeric states of VpuTM (tetramer and pentamer) and M2TMP (tetramer).…”

Hydration-optimized oriented phospholipid bilayer samples for solid-state NMR structural studies of membrane proteins

“…The use of lipid bilayers provides a medium that closely resembles the biological environment, and sample orientation serves as a mechanism for resonance line narrowing, and also preserves the membrane-defined protein topology. The structures of gramicidin, the M2 transmembrane segment from the acetylcholine receptor, the transmembrane segment of the M2 H + channel protein from influenza virus, and the membrane bound fd coat protein (PDB files 1MAG, 1CEK, 1MP6, 1MZT), have been determined from solid-state NMR orientational restraints using this approach [2][3][4][5].…”