Biomedical subjects

M Hohwy

Publications and source records attributed to M Hohwy.

6 recordsLinked to original sources

PubMed2001

Targeting of an abundant cytosolic form of the protein import receptor at Toc159 to the outer chloroplast membrane.

Chloroplast biogenesis requires the large-scale import of cytosolically synthesized precursor proteins. A trimeric translocon (Toc complex) containing two homologous GTP-binding proteins (atToc33 and atToc159) and a channel protein (atToc75) facilitates protein translocation across the outer envelope membrane. The mechanisms governing function and assembly of the Toc complex are not yet understood. This study demonstrates that atToc159 and its pea orthologue exist in an abundant, previously unrecognized soluble form, and partition between cytosol-containing soluble fractions and the chloroplast outer membrane. We show that soluble atToc159 binds directly to the cytosolic domain of atToc33 in a homotypic interaction, contributing to the integration of atToc159 into the chloroplast outer membrane. The data suggest that the function of the Toc complex involves switching of atToc159 between a soluble and an integral membrane form.

Arabidopsis↗

PubMed2001

1H-1H MAS correlation spectroscopy and distance measurements in a deuterated peptide.

In this Communication, we demonstrate the use of deuteration together with back substitution of exchangeable protons as a means of attenuating the strong 1H-1H couplings that broaden 1H magic angle spinning (MAS) spectra of solids. The approach facilitates 15N-1H correlation experiments as well as experiments for the measurement of 1H-1H distances. The distance measurement relies on the excellent resolution in the 1H MAS spectrum and homonuclear double quantum recoupling techniques. The 1H-1H dipolar recoupling can be analyzed in an analytical fashion by fitting the data to a 2- or 3-spin system. The experiments are performed on a sample of the dipeptide N-Ac-Val-Leu-OH, which was synthesized from uniformly [2H, 15N] labeled materials and back-exchanged in H2O.

Deuterium↗

PubMed2001

Conformation of alamethicin in oriented phospholipid bilayers determined by (15)N solid-state nuclear magnetic resonance.

The conformation of the 20-residue antibiotic ionophore alamethicin in macroscopically oriented phospholipid bilayers has been studied using (15)N solid-state nuclear magnetic resonance (NMR) spectroscopy in combination with molecular modeling and molecular dynamics simulations. Differently (15)N-labeled variants of alamethicin and an analog with three of the alpha-amino-isobutyric acid residues replaced by alanines have been investigated to establish experimental structural constraints and determine the orientation of alamethicin in hydrated phospholipid (dimyristoylphosphatidylcholine) bilayers and to investigate the potential for a major kink in the region of the central Pro(14) residue. From the anisotropic (15)N chemical shifts and (1)H-(15)N dipolar couplings determined for alamethicin with (15)N-labeling on the Ala(6), Val(9), and Val(15) residues and incorporated into phospholipid bilayer with a peptide:lipid molar ratio of 1:8, we deduce that alamethicin has a largely linear alpha-helical structure spanning the membrane with the molecular axis tilted by 10-20 degrees relative to the bilayer normal. In particular, we find compatibility with a straight alpha-helix tilted by 17 degrees and a slightly kinked molecular dynamics structure tilted by 11 degrees relative to the bilayer normal. In contrast, the structural constraints derived by solid-state NMR appear not to be compatible with any of several model structures crossing the membrane with vanishing tilt angle or the earlier reported x-ray diffraction structure (Fox and Richards, Nature. 300:325-330, 1982). The solid-state NMR-compatible structures may support the formation of a left-handed and parallel multimeric ion channel.

Alamethicin↗

PubMed2000

NH-NH vector correlation in peptides by solid-state NMR.

We present a novel solid-state magic angle-spinning NMR method for measuring the NH(i)-NH(i+1) projection angle θ(i,i+1) in peptides. The experiment is applicable to uniformly (15)N-labeled peptides and is demonstrated on the chemotactic tripeptide N-formyl-l-Met-l-Leu-l-Phe. The projection angle θ(i,i+1) is directly related to the peptide backbone torsion angles φ(i) and psi(i). The method utilizes the T-MREV recoupling scheme to restore (15)N-(1)H interactions, and proton-mediated spin diffusion to establish (15)N-(15)N correlations. T-MREV has recently been shown to increase the dynamic range of the (15)N-(1)H recoupling by gamma-encoding, and permits an accurate determination of the recoupled NH dipolar interaction. The results are interpreted in a quasi-analytical fashion that permits efficient extraction of the structural parameters.

Magnetic Resonance Spectroscopy↗

PubMed1999

Efficient spectral simulations in NMR of rotating solids. The gamma-COMPUTE algorithm.

We explore the time-translational relation between one of the powder angles (gamma) and the sample rotation angle (omegart) in NMR spectroscopy of rotating solids. Averaging over the gamma powder angle is shown to be generally equivalent to a cross correlation of two periodic functions. This leads to a fundamental relation concerning the phases of NMR spectra of rotating solids as well as improved strategies for efficient simulation of experimental spectra. Using these results in combination with the frequency-domain simulation procedure COMPUTE (M. Edén et al., J. Magn. Reson. A 120, 56 (1996)), it proves possible to reduce the computation time for spectral simulations by typically a factor 10-30 relative to the state-of-the-art calculations using the original COMPUTE algorithm. The advantage and the general applicability of the new simulation procedure, referred to as gamma-COMPUTE, are demonstrated by simulation of single- and multiple-pulse MAS NMR spectra of 31P-31P and 1H-1H spin pairs influenced by anisotropic chemical shielding and homonuclear dipolar interactions.

Algorithms↗

PubMed1998

1H chemical shielding anisotropies from polycrystalline powders using MSHOT-3 based CRAMPS

It is demonstrated that combined rotation and multiple-pulse spectroscopy (CRAMPS) based on MSHOT-3 homonuclear multiple-pulse decoupling represents a powerful method for determination of 1H chemical shielding anisotropies from polycrystalline powders. By virtue of high-order dipolar decoupling, large spectral width, resonance offset stability, and the absence of artifacts fromtilted-axis precession, MSHOT-3-based CRAMPS enables straightforward sampling of high-quality spectra. Comparison with explicit calculations, taking the effect of the multiple-pulse sequence into account, shows that the spectra may be simulated and iteratively fitted using standard software for the calculation of magic-angle spinning spectra influenced by chemical shielding anisotropy with the shielding interaction reduced by the scaling factor of the MSHOT-3 decoupling sequence. The method is demonstrated by experimental determination of 1H chemical shielding anisotropies for adipic acid, Ca(OH)2, malonic acid, and KHSO4. The data are compared with those determined previously from single-crystal NMR studies. Copyright 1998 Academic Press.

Journal Article↗