Published: August 01, 2011 r2011 American Chemical Society 10758 dx.doi.org/10.1021/jp205002n | J. Phys. Chem. B 2011, 115, 10758–10767 ARTICLE pubs.acs.org/JPCB Conformational Disorder of Membrane Peptides Investigated from Solid-State NMR Line Widths and Line Shapes Yongchao Su and Mei Hong* Department of Chemistry, Iowa State University, Ames, Iowa 50011, United States b S Supporting Information ’ INTRODUCTION Solid-state NMR (SSNMR) spectroscopy has become a power- ful probe of the molecular structure and dynamics of insoluble biological macromolecules such as membrane proteins, 1,2 amyloid fibrils, 3À5 and cell walls. 6,7 A persistent challenge in structure determination of these biological solids is spectral resolution. While biomolecules in solution have narrow line widths due to their fast isotropic tumbling, solid systems, without such motion, exhibit broad NMR line widths due to the presence of orienta- tion-dependent nuclear spin interactions such as chemical shift anisotropy (CSA) and dipolar coupling. Magic angle spin- ning (MAS) 8 and hetero- and homonuclear dipolar decoupling techniques 9À14 eliminate most of this orientational broadening. Nevertheless, residual dipolar couplings between protons and heteronuclei due to imperfect decoupling still contribute sizable line widths, and dipolar couplings between isotopically enriched 13 C spins broaden lines when MAS rates are insufficient. In addi- tion, 13 CÀ 13 C J-couplings are difficult to remove in directly detected 13 C spectra and thus contribute a fixed amount of linewidth. These coherent line broadening mechanisms are ameliorated by the use of higher magnetic fields, 15 faster MAS, 14,16 and reduction of the proton density by perdeuteration of proteins followed by H/D exchange. 17À19 In contrast to the coherent linewidth contributions, an inco- herent line broadening mechanism is transverse T 2 relaxation, which results from random fluctuations of local magnetic fields induced by molecular motion. 20,21 When motional rates are comparable to the strength of the dipolar decoupling field strengths or MAS rate, extreme line broadening occurs that abolishes the spectral intensity altogether. This intermediate time scale motional broadening has been studied in both small molecules 22,23 and membrane peptides. 24À26 The third line broadening mechanism, which cannot be removed by radio frequency pulses or higher magnetic fields, is conformational disorder. Conformational distribution gives rise to multiple isotropic chemical shifts for each chemically unique nuclear spin in the same manner that chemically inequivalent spins cause different isotropic shifts. This inhomogeneous line broad- ening causes characteristic 2D correlation line shapes 27,28 and can give very broad peaks, even if the homogeneous line widths due to residual couplings and T 2 relaxation are small. The large differ- ence between inhomogeneous and homogeneous line widths has Received: May 29, 2011 Revised: July 29, 2011 ABSTRACT: A challenge in the application of solid-state NMR spectroscopy to membrane peptides and proteins is the relatively broad line widths compared to those for solution NMR spectra. To under- stand the linewidth contributions to membrane protein NMR spectra, we have measured the inhomogeneous and homogeneous line widths of several well-studied membrane peptides under immobilized conditions. 13 C T 2 relaxation times of uniformly 13 C-labeled residues show that the homogeneous line widths of the peptides are comparable to those of crystalline model compounds under identical 1 H decoupling and magic angle spinning conditions, indicating that the homogeneous line widths are determined by conformation-independent factors, including residual dipolar coupling, J-coupling, and intrinsic T 2 relaxation. However, the membrane peptides exhibit larger apparent line widths than the crystalline compounds, indicating conformational disorder. A cationic cell-penetrating peptide, the human immunodeficiency virus TAT, exhibits the largest apparent line widths, which are about five-fold larger than the homogeneous line widths, while the transmembrane helix of the influenza M2 peptide and the β-hairpin antimicrobial peptide PG-1 show moderately larger apparent line widths than the crystalline compounds. These results are consistent with the random coil nature of the TAT peptide, which contrasts with the intramolecularly hydrogen bonded M2 and PG-1. Cross peak line shapes of 2D double-quantum correlation spectra show that the conformational disorder can occur at the residue level and can result from three origins, lipidÀpeptide interaction, intrinsic conformational disorder encoded in the amino acid sequence, and side-chain rotameric averaging. A particularly important lipidÀpeptide interaction for cationic membrane peptides is guanidiniumÀphosphate ion pair interaction. Thus, NMR line widths and line shapes are useful for understanding the conformational disorder of membrane peptides and proteins.