Matthew T. Eddy

Intermolecular Structure Determination of Amyloid Fibrils with Magic-Angle Spinning and Dynamic Nuclear Polarization NMR

We describe magic-angle spinning NMR experiments designed to elucidate the interstrand architecture of amyloid fibrils. Three methods are introduced for this purpose, two being based on the analysis of long-range (13)C-(13)C correlation spectra and the third based on the identification of intermolecular interactions in (13)C-(15)N spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (PI3-SH3 or PI3K-SH3), that efficient (13)C-(13)C correlation spectra display a resonance degeneracy that establishes a parallel, in-register alignment of the proteins in the amyloid fibrils. In addition, this degeneracy can be circumvented to yield direct intermolecular constraints. The (13)C-(13)C experiments are corroborated by (15)N-(13)C correlation spectra obtained from a mixed [(15)N,(12)C]/[(14)N,(13)C] sample which directly quantify interstrand distances. Furthermore, when the spectra are recorded with signal enhancement provided by dynamic nuclear polarization (DNP) at 100 K, we demonstrate a dramatic increase (from 23 to 52) in the number of intermolecular (15)N-(13)C constraints detectable in the spectra. The increase in the information content is due to the enhanced signal intensities and to the fact that dynamic processes, leading to spectral intensity losses, are quenched at low temperatures. Thus, acquisition of low temperature spectra addresses a problem that is frequently encountered in MAS spectra of proteins. In total, the experiments provide 111 intermolecular (13)C-(13)C and (15)N-(13)C constraints that establish that the PI3-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.

Radio frequency-driven recoupling at high magic-angle spinning frequencies: Homonuclear recoupling sans heteronuclear decoupling

We describe solid-state NMR homonuclear recoupling experiments at high magic-angle spinning (MAS) frequencies using the radio frequency-driven recoupling (RFDR) scheme. The effect of heteronuclear decoupling interference during RFDR recoupling at high spinning frequencies is investigated experimentally and via numerical simulations, resulting in the identification of optimal decoupling conditions. The effects of MAS frequency, RF field amplitude, bandwidth, and chemical shift offsets are examined. Most significantly, it is shown that broadband homonuclear correlation spectra can be efficiently obtained using RFDR without decoupling during the mixing period in fully protonated samples, thus considerably reducing the rf power requirements for acquisition of (13)C-(13)C correlation spectra. The utility of RFDR sans decoupling is demonstrated with broadband correlation spectra of a peptide and a model protein at high MAS frequencies and high magnetic field.

Magic Angle Spinning NMR Investigation of Influenza A M218−60: Support for an Allosteric Mechanism of Inhibition

The tetrameric M2 proton channel from influenza A virus conducts protons at low pH and is inhibited by aminoadamantyl drugs such as amantadine and rimantadine (Rmt). We report magic angle spinning NMR spectra of POPC and DPhPC membrane-embedded M2(18-60), both apo and in the presence of Rmt. Similar line widths in the spectra of apo and bound M2 indicate that Rmt does not have a significant impact on the dynamics or conformational heterogeneity of this construct. Substantial chemical shift changes for many residues in the transmembrane region support an allosteric mechanism of inhibition. An Rmt titration supports a binding stoichiometry of >1 Rmt molecule per channel and shows that nonspecific binding or changes in membrane composition are unlikely sources of the chemical shift changes. In addition, doubling of spectral lines in all of the observed samples provides evidence that the channel assembles with twofold symmetry.

Structure and mechanism of the influenza A M218–60 dimer of dimers

We report a magic angle spinning (MAS) NMR structure of the drug-resistant S31N mutation of M218-60 from Influenza A. The protein was dispersed in diphytanoyl-sn-glycero-3-phosphocholine lipid bilayers, and the spectra and an extensive set of constraints indicate that M218-60 consists of a dimer of dimers. In particular, ∼280 structural constraints were obtained using dipole recoupling experiments that yielded well-resolved (13)C-(15)N, (13)C-(13)C, and (1)H-(15)N 2D, 3D, and 4D MAS spectra, all of which show cross-peak doubling. Interhelical distances were measured using mixed (15)N/(13)C labeling and with deuterated protein, MAS at ωr/2π = 60 kHz, ω0H/2π = 1000 MHz, and (1)H detection of methyl-methyl contacts. The experiments reveal a compact structure consisting of a tetramer composed of four transmembrane helices, in which two opposing helices are displaced and rotated in the direction of the membrane normal relative to a four-fold symmetric arrangement, yielding a two-fold symmetric structure. Side chain conformations of the important gating and pH-sensing residues W41 and H37 are found to differ markedly from four-fold symmetry. The rmsd of the structure is 0.7 Å for backbone heavy atoms and 1.1 Å for all heavy atoms. This two-fold symmetric structure is different from all of the previous structures of M2, many of which were determined in detergent and/or with shorter constructs that are not fully active. The structure has implications for the mechanism of H(+) transport since the distance between His and Trp residues on different helices is found to be short. The structure also exhibits two-fold symmetry in the vicinity of the binding site of adamantyl inhibitors, and steric constraints may explain the mechanism of the drug-resistant S31N mutation.

Proton assisted recoupling at high spinning frequencies.

We demonstrate the successful application of (13)C-(13)C proton assisted recoupling (PAR) on [U-(13)C,(15)N] N-f-MLF-OH and [U-(13)C,(15)N] protein GB1 at high magic angle spinning (MAS) frequencies (omega(r)/2pi = 65 kHz). Specifically, by combining PAR mixing with low power heteronuclear decoupling (omega(1H)/2pi approximately 16 kHz) and high spinning frequencies, we obtain high resolution 2D spectra displaying long-range (13)C-(13)C contacts from which distance estimates can be extracted. These experiments therefore demonstrate the possibility of performing high resolution structural studies in the limit of high spinning frequency and low power (1)H decoupling, a regime which optimizes the resolution of protein samples and preserves their integrity.

Lipid Dynamics and Protein-Lipid Interactions in 2D Crystals Formed with the β-barrel Integral Membrane Protein VDAC1

We employ a combination of (13)C/(15)N magic angle spinning (MAS) NMR and (2)H NMR to study the structural and functional consequences of different membrane environments on VDAC1 and, conversely, the effect of VDAC1 on the structure of the lipid bilayer. MAS spectra reveal a well-structured VDAC1 in 2D crystals of dimyristoylphosphatidylcholine (DMPC) and diphytanoylphosphatidylcholine (DPhPC), and their temperature dependence suggests that the VDAC structure does not change conformation above and below the lipid phase transition temperature. The same data show that the N-terminus remains structured at both low and high temperatures. Importantly, functional studies based on electrophysiological measurements on these same samples show fully functional channels, even without the presence of Triton X-100 that has been found necessary for in vitro-refolded channels. (2)H solid-state NMR and differential scanning calorimetry were used to investigate the dynamics and phase behavior of the lipids within the VDAC1 2D crystals. (2)H NMR spectra indicate that the presence of protein in DMPC results in a broad lipid phase transition that is shifted from 19 to ~27 °C and show the existence of different lipid populations, consistent with the presence of both annular and bulk lipids in the functionally and structurally homogeneous samples.

Secondary Structure in the Core of Amyloid Fibrils Formed from Human β2m and its Truncated Variant ΔN6

Amyloid fibrils formed from initially soluble proteins with diverse sequences are associated with an array of human diseases. In the human disorder, dialysis-related amyloidosis (DRA), fibrils contain two major constituents, full-length human β2-microglobulin (hβ2m) and a truncation variant, ΔN6 which lacks the N-terminal six amino acids. These fibrils are assembled from initially natively folded proteins with an all antiparallel β-stranded structure. Here, backbone conformations of wild-type hβ2m and ΔN6 in their amyloid forms have been determined using a combination of dilute isotopic labeling strategies and multidimensional magic angle spinning (MAS) NMR techniques at high magnetic fields, providing valuable structural information at the atomic-level about the fibril architecture. The secondary structures of both fibril types, determined by the assignment of ∼80% of the backbone resonances of these 100- and 94-residue proteins, respectively, reveal substantial backbone rearrangement compared with the location of β-strands in their native immunoglobulin folds. The identification of seven β-strands in hβ2m fibrils indicates that approximately 70 residues are in a β-strand conformation in the fibril core. By contrast, nine β-strands comprise the fibrils formed from ΔN6, indicating a more extensive core. The precise location and length of β-strands in the two fibril forms also differ. The results indicate fibrils of ΔN6 and hβ2m have an extensive core architecture involving the majority of residues in the polypeptide sequence. The common elements of the backbone structure of the two proteins likely facilitates their ability to copolymerize during amyloid fibril assembly.

Heteronuclear proton assisted recoupling

We describe a theoretical framework for understanding the heteronuclear version of the third spin assisted recoupling polarization transfer mechanism and demonstrate its potential for detecting long-distance intramolecular and intermolecular (15)N-(13)C contacts in biomolecular systems. The pulse sequence, proton assisted insensitive nuclei cross polarization (PAIN-CP) relies on a cross term between (1)H-(15)N and (1)H-(13)C dipolar couplings to mediate zero- and∕or double-quantum (15)N-(13)C recoupling. In particular, using average Hamiltonian theory we derive effective Hamiltonians for PAIN-CP and show that the transfer is mediated by trilinear terms of the form N(±)C(∓)H(z) (ZQ) or N(±)C(±)H(z) (DQ) depending on the rf field strengths employed. We use analytical and numerical simulations to explain the structure of the PAIN-CP optimization maps and to delineate the appropriate matching conditions. We also detail the dependence of the PAIN-CP polarization transfer with respect to local molecular geometry and explain the observed reduction in dipolar truncation. In addition, we demonstrate the utility of PAIN-CP in structural studies with (15)N-(13)C spectra of two uniformly (13)C,(15)N labeled model microcrystalline proteins-GB1, a 56 amino acid peptide, and Crh, a 85 amino acid domain swapped dimer (MW=2×10.4 kDa). The spectra acquired at high magic angle spinning frequencies (ω(r)∕2π>20 kHz) and magnetic fields (ω(0H)∕2π=700-900 MHz) using moderate rf fields, yield multiple long-distance intramonomer and intermonomer (15)N-(13)C contacts. We use these distance restraints, in combination with the available x-ray structure as a homology model, to perform a calculation of the monomer subunit of the Crh protein.

Rapid three-dimensional MAS NMR spectroscopy at critical sensitivity

Solid-state magic angle spinning nuclear magnetic resonance (MAS NMR) is maturing rapidly. Progress is highlighted by recent examples that demonstrate its capability to yield site-specific assignments and atomic resolution structural information on fibrillar,[1–3] membrane-associated,[4–6] and non-crystalline proteins.[7–9] Furthermore, applications to systems of ever-increasing molecular size are limited only by the signal-to-noise ratio (S/N) in the multidimensional spectra required for adequate resolution,, rather than by more fundamental limitations from spin relaxation. Therefore, the most pressing need in MAS NMR is arguably for more efficient data acquisition methods.

15N-15N Proton Assisted Recoupling in Magic Angle Spinning NMR

We describe a new magic angle spinning (MAS) NMR experiment for obtaining (15)N-(15)N correlation spectra. The approach yields direct information about the secondary and tertiary structure of proteins, including identification of alpha-helical stretches and interstrand connectivity in antiparallel beta-sheets, which are of major interest for structural studies of membrane proteins and amyloid fibrils. The method, (15)N-(15)N proton assisted recoupling (PAR), relies on a second-order mechanism, third spin assisted recoupling (TSAR), used previously in the context of (15)N-(13)C and (13)C-(13)C polarization transfer schemes. In comparison to (15)N-(15)N proton-driven spin diffusion experiments, the PAR technique accelerates polarization transfer between (15)Ns by a factor of approximately 10(2)-10(3) and is furthermore applicable over the entire range of currently available MAS frequencies (10-70 kHz).