Zhichun Liang

Multifrequency Electron Spin Resonance Study of the Dynamics of Spin Labeled T4 Lysozyme

An extensive set of electron spin resonance spectra was obtained over a wide range of frequencies (9, 95, 170, and 240 GHz) and temperatures (2 to 32 degrees C) to explore the dynamic modes of nitroxide-labeled T4 lysozyme in solution. A commonly used nitroxide side chain (R1), or a methylated analogue with hindered internal motion (R2), was substituted for the native side chain at solvent-exposed helical sites, 72 or 131. The spectra at all four frequencies were simultaneously fit with the slowly relaxing local structure (SRLS) model. Good fits were achieved at all the temperatures. Two principle dynamic modes are included in the SRLS model, the global tumbling of the protein and the internal motion consisting of backbone fluctuations and side chain isomerizations. Three distinct spectral components were required for R1 and two for R2 to account for the spectra at all temperatures. One is a highly ordered and slow motional component, which is observed in the spectra of both R1 and R2; it may correspond to conformers stabilized by interaction with the protein surface. The fraction of this component decreases with increasing temperature and is more populated in the R2 spectra, possibly arising from stronger interaction of the nitroxide ring with the protein surface due to the additional methyl group. The other two components of R1 and the second component of R2 are characterized by fast anisotropic diffusion and relatively low ordering, most likely corresponding to conformers having little or no interactions with nearby residues. Ficoll of different concentrations was added to increase the solution viscosity, thereby slowing down the global tumbling of the protein. A significant effect of Ficoll on the internal motion of an immobilized component was apparent in R2 but not in R1. The ability of such multifrequency studies to separate the effects of faster internal modes of motion from slower overall motions is clearly demonstrated, and its utility in future studies is considered.

Protein Dynamics in the Solid State from 2H NMR Line Shape Analysis: A Consistent Perspective

Deuterium line shape analysis of CD3 groups has emerged as a particularly useful tool for studying microsecond-millisecond protein motions in the solid state. The models devised so far consist of several independently conceived simple jump-type motions. They are comprised of physical quantities encoded in their simplest form; improvements are only possible by adding yet another simple motion, thereby changing the model. The various treatments developed are case-specific; hence comparison among the different systems is not possible. Here we develop a new methodology for (2)H NMR line shape analysis free of these limitations. It is based on the microscopic-order-macroscopic-disorder (MOMD) approach. In MOMD motions are described by diffusion tensors, spatial restrictions by potentials/ordering tensors, and geometric features by relative tensor orientations. Jump-type motions are recovered in the limit of large orientational potentials. Model improvement is accomplished by monitoring the magnitude, symmetry, and orientation of the various tensors. The generality of MOMD makes possible comparison among different scenarios. CD3 line shapes from the Chicken Villin Headpiece Subdomain and the Streptomyces Subtilisin Inhibitor are used as experimental examples. All of these spectra are reproduced by using rhombic local potentials constrained for simplicity to be given by the L = 2 spherical harmonics, and by axial diffusion tensors. Potential strength and rhombicity are found to be ca. 2-3 k(B)T. The diffusion tensor is tilted at 120° from the C-CD3 axis. The perpendicular (parallel) correlation times for local motion are 0.1-1.0 ms (3.3-30 μs). Activation energies in the 1.1-8.0 kcal/mol range are estimated. Future prospects include extension to the (2)H relaxation limit, application to the (15)N and (13)C NMR nuclei, and accounting for collective motions and anisotropic media.

Protein Dynamics in the Solid State from 2H NMR Line Shape Analysis. II. MOMD Applied to C–D and C–CD3 Probes

Deuterium line shape analysis from mobile C–D and C–CD3 groups has emerged as a particularly useful tool for studying dynamics in the solid state. The theoretical models devised so far consist typically of sets of independent dynamic modes. Each such mode is simple and usually case-specific. In this scenario, model improvement entails adding yet another mode (thereby changing the overall model), comparison of different cases is difficult, and ambiguity is unavoidable. We recently developed the microscopic order macroscopic disorder (MOMD) approach as a single-mode alternative. In MOMD, the local spatial restrictions are expressed by an anisotropic potential, the local motion by a diffusion tensor, and the local molecular geometry by relative (magnetic and model-related) tensor orientations, all of adjustable symmetry. This approach provides a consistent method of analysis, thus resolving the issues above. In this study, we apply MOMD to PS-adsorbed LKα14 peptide and dimethylammonium tetraphenylborate (C–CD3 and N–CD3 dynamics, respectively), as well as HhaI methyltransferase target DNA and phase III of benzene-6-hexanoate (C–D dynamics). The success with fitting these four disparate cases, as well as the two cases in the previous report, demonstrates the generality of this MOMD-based approach. In this study, C–D and C–CD3 are both found to execute axial diffusion (rates R⊥ and R∥) in the presence of a rhombic potential given by the L = 2 spherical harmonics (coefficients c02 and c22). R⊥ (R∥) is in the 102–103 (104–105) s–1 range, and c02 and c22 are on the order of 2–3 kBT. Specific parameter values are determined for each mobile site. The diffusion and quadrupolar tensors are tilted at either 120° (consistent with trans–gauche isomerization) or nearly 110.5° (consistent with methyl exchange). Future prospects include extension of the MOMD formalism to include MAS, and application to 15N and 13C nuclei.

Conformational Response of Influenza A M2 Transmembrane Domain to Amantadine Drug Binding at Low pH (pH 5.5)

The M2 protein from influenza A plays important roles in its viral cycle. It contains a single transmembrane helix, which oligomerizes into a homotetrameric proton channel that conducts in the low-pH environment of the host-cell endosome and Golgi apparatus, leading to virion uncoating at an early stage of infection. We studied conformational rearrangements that occur in the M2 core transmembrane domain residing on the lipid bilayer, flanked by juxtamembrane residues (M2TMD21−49 fragment), upon its interaction with amantadine drug at pH 5.5 when M2 is conductive. We also tested the role of specific mutation and lipid chain length. Electron spin resonance (ESR) spectroscopy and electron microscopy were applied to M2TMD21−49, labeled at the residue L46C with either nitroxide spin-label or Nanogold® reagent, respectively. Electron microscopy confirmed that M2TMD21−49 reconstituted into DOPC/POPS at 1:10,000 peptide-to-lipid molar ratio (P/L) either with or without amantadine, is an admixture of monomers, dimers, and tetramers, confirming our model based on a dimer intermediate in the assembly of M2TMD21−49. As reported by double electron-electron resonance (DEER), in DOPC/POPS membranes amantadine shifts oligomer equilibrium to favor tetramers, as evidenced by an increase in DEER modulation depth for P/Ls ranging from 1:18,000 to 1:160. Furthermore, amantadine binding shortens the inter-spin distances (for nitroxide labels) by 5–8 Å, indicating drug induced channel closure on the C-terminal side. No such effect was observed for the thinner membrane of DLPC/DLPS, emphasizing the role of bilayer thickness. The analysis of continuous wave (cw) ESR spectra of spin-labeled L46C residue provides additional support to a more compact helix bundle in amantadine-bound M2TMD 21−49 through increased motional ordering. In contrast to wild-type M2TMD21−49, the amantadine-bound form does not exhibit noticeable conformational changes in the case of G34A mutation found in certain drug-resistant influenza strains. Thus, the inhibited M2TMD21−49 channel is a stable tetramer with a closed C-terminal exit pore. This work is aimed at contributing to the development of structure-based anti-influenza pharmaceuticals.