Biomedical subjects
Publications and source records attributed to Beat H Meier.
The application of a spin-state-selective coherence transfer experiment (INADEQUATE-SSS) to solid-state NMR spectroscopy is described. Two-dimensional (13)C double-quantum/single-quantum spectra without J splittings in both dimensions lead to enhanced spectral resolution. The method is demonstrated to significantly improve the spectral resolution of the crowded C'-C(alpha) region of two proteins.
We report the observation of undetected (until now) residues of the prion protein fragment HET-s(218-289) which give rise to well-resolved (13)C, (15)N, and (1)H NMR resonances under high-resolution magic-angle spinning (HRMAS) conditions. The observed signals belong to large polymeric units as shown by measuring the lateral diffusion constants. The amino acids identified in the spectra are compatible with their localization in the segments of the protein which could not be detected in earlier solid-state NMR experiments. The observed chemical shifts indicate that these residues are in a random-coil conformation. Complementary experiments which detect only dynamic or static residues, respectively, strongly suggest that they belong to different parts of the same molecule.
Magnetic-resonance force microscopy is combined with cross-polarization and spin-decoupling NMR techniques to obtain double-resonance NMR signals of micrometer-scaled objects. The effective one-dimensional spatial resolution obtained in our experiments performed on a KPF6 single crystal sample is approximately 0.5 microm. The spectral linewidth of 900 Hz is sample limited. The described double-resonance techniques can introduce new chemical specificity to the magnetic-force sensor.
The morphogenic opiate pentapeptide leucine-enkephalin (lenk) in a hydrated dimyristoylphosphatidylcholine (DMPC) bilayer is studied using NMR spectroscopy and molecular dynamics simulation. Contrary to the frequent assumption that the peptide attains a single fixed conformation in the presence of membranes, we find that the lenk molecule is flexible, switching between specific bent conformations. The constraints to the orientation of the aromatic rings that are identified by the NMR experiment are found by the MD simulation to be related to the depth of the peptide in the bilayer. The motion of the N-H vectors of the peptide bonds with respect to the magnetic field direction as observed by MD largely explain the magnitude of the observed residual dipolar coupling (RDC), which are much reduced over the static (15)N-(1)H coupling. The measured RDCs are nevertheless significantly larger than the predicted ones, possibly due the absence of long-time motions in the simulations. The conformational behavior of lenk at the DMPC surface is compared to that in the aqueous solution, both in the neutral and in the zwitterionic forms.
The partial 15N and 13C solid-state NMR resonance assignment of the HET-s prion protein fragment 218-289 in its amyloid form is presented. It is based on experiments measured at MAS frequencies in the range of 20-40 kHz using exclusively adiabatic polarization-transfer schemes. The resonance assignment within each residue is based on two-dimensional 13C-13C correlation spectra utilizing the DREAM mixing scheme. The sequential linking of the assigned residues used a set of two- and three-dimensional 15N-13C correlation experiments. Almost all cross peaks visible in the spectra are assigned, but only resonances from 43 of the 78 amino-acid residues could be detected. The missing residues are thought to be highly disordered and/or highly dynamic giving rise to broad resonance lines that escaped detection in the experiments applied. The line widths of the observed resonances are narrow and comparable to line widths observed in micro-crystalline samples. The 43 assigned residues are located in two fragments of about 20 residues.
We describe the simplification of 13C-13C correlation spectra obtained from a microcrystalline protein sample expressed on a growth medium of 10% fully 13C labeled glucose diluted in 90% natural abundance glucose as compared to a fully labeled sample. Such a labeling scheme facilitates the backbone and side-chain resonance assignment of Phe, Tyr, His, Asp, Asn, Ile, Lys and Pro and yields an unambiguous stereospecific assignment of the valine Cgamma1, Cgamma2 13C resonances and of Leucine Cdelta2.
Silk from the wild silkworm Samia cynthia ricini with a molecular mass of about 300kDa consists of alternating repeats of nano-crystalline poly-(Ala) and non-crystalline glycine-rich domains. The backbone torsion angles between pairs of these two amino acids is determined by DOQSY solid-state NMR spectroscopy: the alanine-rich domains are predominantly in a beta-sheet conformation, whereas the glycine-rich domains are found to be partially in an extended beta-sheet conformation and partially in an approximately 3(1)-helical conformation. In the cast film from liquid silk significantly different secondary structures were found: the alanine-rich domains are alpha-helical conformation, whereas the results for glycine-labelled sample are explained by a random-coil state. A detailed error analysis of the technique is presented.
This paper centers on a theoretical study of amplitude-modulated heteronuclear decoupling in solid-state NMR under magic-angle spinning (MAS). A spin system with a single isolated rare spin coupled to a large number of abundant spins is used in the analysis. The phase-alternating decoupling scheme (XiX decoupling) is analyzed using bimodal Floquet theory and the operator-based perturbation method developed by van Vleck. An effective Hamiltonian correct to second order is calculated for the spin system under XiX decoupling. The results of these calculations indicate that under XiX decoupling the main contribution to the residual line width comes from a cross-term between the heteronuclear and the homonuclear dipolar couplings. This is in contrast to continuous-wave decoupling, where the residual line width is dominated by the cross-term between the heteronuclear dipolar coupling and the chemical-shielding tensor of the irradiated spin. For high-power decoupling the method results in very good decoupling provided that certain unfavorable recoupling conditions, imposed by specific ratios of the amplitude modulation frequency and the MAS frequency, are avoided. For low-power decoupling, the method leads to acceptable decoupling when the pulse length corresponds to an integer multiple of a 2pi rotation and the rf-field amplitude is less than a quarter of the MAS frequency. The performance of the XiX scheme is analyzed over a range of values of the rf power, and numerical results that agree well with the most recent experimental observations are presented.
The application of two or more different time-dependent coherent perturbations with, in general, incommensurable frequencies occurs quite commonly in NMR experiments. Here we develop a unified description of the entire class of experiments using bimodal Floquet theory and van Vleck-Primas perturbation theory. This treatment leads to a time-independent effective Hamiltonian in Hilbert space and can be looked at as a generalization of average Hamiltonian theory to several incommensurate time dependencies. As a prototype experiment we treat the application of continuous-wave (cw) radio-frequency irradiation in combination with magic-angle sample spinning. Practically relevant examples of this type of experiments are heteronuclear spin decoupling and recoupling experiments using cw irradiation, e.g., rotary-resonance recoupling. Perturbations up to the third order must be taken into account to explain all experimentally observed resonance conditions.
Prions are believed to be infectious, self-propagating polymers of otherwise soluble, host-encoded proteins. This concept is now strongly supported by the recent findings that amyloid fibrils of recombinant prion proteins from yeast, Podospora anserina and mammals can induce prion phenotypes in the corresponding hosts. However, the structural basis of prion infectivity remains largely elusive because acquisition of atomic resolution structural properties of amyloid fibrils represents a largely unsolved technical challenge. HET-s, the prion protein of P. anserina, contains a carboxy-terminal prion domain comprising residues 218-289. Amyloid fibrils of HET-s(218-289) are necessary and sufficient for the induction and propagation of prion infectivity. Here, we have used fluorescence studies, quenched hydrogen exchange NMR and solid-state NMR to determine the sequence-specific positions of amyloid fibril secondary structure elements of HET-s(218-289). This approach revealed four beta-strands constituted by two pseudo-repeat sequences, each forming a beta-strand-turn-beta-strand motif. By using a structure-based mutagenesis approach, we show that this conformation is the functional and infectious entity of the HET-s prion. These results correlate distinct structural elements with prion infectivity.
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An adiabatic double-quantum polarization-transfer experiment is described. It can be characterized as an adiabatic variant of the POST-C7 experiment. A continuous variation of the phase increment between pulses leads to the introduction of a fictitious Zeeman field that allows for an adiabatic passage through the recoupling condition. This results in a chemical-shift-offset-compensated adiabatic experiment, which leads to an efficient and broadbanded polarization transfer or to a double-quantum excitation. Similar variations of other C- or R-type experiments can be envisioned.
We demonstrate that two-dimensional solid-state NMR chemical-shift correlation spectra can be recorded under low-power conditions. Except for the cross-polarization period, no rf-field amplitudes above 40 kHz are used. Such experiments require the use of fast (>50 kHz) magic-angle spinning (MAS). A comparison with the high-power version of the experiment shows no general line broadening but some changes in the polarization-transfer dynamics.
Protein deposition as amyloid fibrils underlies many debilitating human disorders. The complexity and size of disease-related polypeptides, however, often hinders a detailed rational approach to study effects that contribute to the process of amyloid formation. We report here a simplified peptide sequence successfully designed de novo to fold into a coiled-coil conformation under ambient conditions but to transform into amyloid fibrils at elevated temperatures. We have determined the crystal structure of the coiled-coil form and propose a detailed molecular model for the peptide in its fibrillar state. The relative stabilities of the two structural forms and the kinetics of their interconversion were found to be highly sensitive to small sequence changes. The results reveal the importance of specific packing interactions on the kinetics of amyloid formation and show the potential of this exceptionally favorable system for probing details of the molecular origins of amyloid disease.
Structural information can be extracted from one-bond residual dipolar couplings (RDC) measured in NMR spectra of systems in field-ordered media. RDC can be on the order of J-couplings if the anisotropy of alignment is approximately 10(-2), 10-fold stronger than that typically used for structural studies of water-soluble proteins. In such systems the performance of (1)H--> (15)N polarization transfer methods of the INEPT type is not satisfactory. In this study we show the effectiveness of adiabatic-passage cross-polarization (APCP) in transferring the (1)H--> (15)N polarization in the bicelle-associated peptide Leucine Enkephalin (Lenk). APCP is efficient both in static samples and in samples spun at the magic angle (MAS) or any other angle of the spinning axis to the magnetic field (variable-angle spinning, VAS). The anisotropic spectrum of an aligned static sample and the isotropic spectrum of the sample under MAS provide a set of possible values for the (1)H-(15)N RDC of phospholipid-associated Lenk. The unambiguous determination of the (1)H-(15)N RDC was accomplished by means of VAS experiments.
It is demonstrated that internuclear distances can be evaluated from rotational-resonance (RR) experiments in uniformly (13)C-labelled compounds. The errors in the obtained distances are less than 10% without the need to know any parameters of the spin system except the isotropic chemical shifts of all spins. We describe the multi-spin system with a simple fictitious spin-1/2 model. The influence of the couplings to the passive spins (J and dipolar coupling) is described by an empirical constant offset from the rotational-resonance condition. Using simulated data for a three-spin system, we show that the two-spin model describes the rotational-resonance transfer curves well as long as none of the passive spins is close to a rotational-resonance condition with one of the active spins. The usability of the two-spin model is demonstrated experimentally using a sample of acetylcholine perchlorate with labelling schemes of various levels of complexity. Doubly-, triply-, and fully labelled compounds lead to strongly varying RR polarization-transfer curves but the evaluated distances using the two-spin model are identical within the expected error limits and coincide with the distance from the X-ray structure. Rotational-resonance distance measurements in fully labelled compounds allow, in particular, the measurement of weak couplings in the presence of strong couplings.
Proteins are not always available in amounts desirable for solid-state magic-angle spinning (MAS) nuclear-magnetic resonance (NMR) spectroscopy. To maximize the signal-to-noise ratio achievable with small samples, the filling factor must be optimized by using small-diameter MAS rotors. These rotors have the added benefit of allowing higher radio frequency field amplitudes during polarization transfer steps and during decoupling periods as well as allowing higher spinning frequencies. We demonstrate the advantages of relatively fast MAS (30 kHz using a 2.5 mm rotor) compared to MAS at 12 kHz for the 10.4 kDa model protein Crh with 93 residues and show that the signal-to-noise ratio in two-dimensional correlation spectra can be significantly improved by taking advantage of optimized pulse sequences available with rapid MAS.
Rotational-resonance magic-angle spinning NMR experiments are frequently used to measure dipolar couplings and to determine internuclear distances. So far most measurements were performed on samples containing isolated spin pairs. Thus, extensive structure elucidation, for example in biomolecules, requires the preparation of a whole set of doubly labeled samples. Here, we describe the analysis of the rotational-resonance polarization-exchange curves obtained from a single, uniformly labeled sample. It is shown experimentally that, at a magnetic field of 14.09 T, the rotational-resonance conditions in uniformly (13)C-labeled threonine are sufficiently narrow to permit the measurement of five distances between the four carbon spins with an accuracy of better than 10%. The polarization-exchange curves are analyzed using a modified two-spin model consisting of the two active spins. The modified model includes an additional offset in the final polarization, which comes from the coupling to the additional, passive, spins. The validity of this approach is experimentally verified for uniformly (13)C-labeled threonine. The broader applicability of such a model is demonstrated by numerical simulations which quantify the errors as a function of the most relevant parameters in the spin system.