Evaluation of the influence of anisotropic indirect nuclear spin-spin coupling tensors on effective residual dipolar couplings for model peptides

Residual dipolar couplings (RDCs) observed between nuclear spins in molecules in partially oriented media have become a valuable source of information for NMR spectroscopists seeking to structurally characterize biological macromolecules. Examination of the form of the direct (D) and indirect (J) nuclear spin-spin coupling Hamiltonians indicates that all observed RDCs contain an unknown contribution from the anisotropic part of J (J) in addition to the direct dipolar contribution, D _PQ. Here, we evaluate the influence of J on RDCs through a series of DFT calculations on model peptides. Very small corrections to one-bond RDCs measured between heavy atoms in peptides and proteins are recommended: +0.51% for N-C spin pairs, and +0.45% for C-C spin pairs. The corrections to RDCs involving at least one proton are negligible. This latter point is likely to be equally applicable to nucleic acids and oligosaccharides in addition to peptides and proteins. Finally, the orientations of the J(N, C) and J(C, C) tensors in the molecular framework are reported for glycylglycine.     

Simultaneous measurement of protein one-bond and two-bond nitrogen-carbon coupling constants using an internally referenced quantitative J-correlated [(15)N,(1)H]-TROSY-HNC experiment

A quantitative J-correlation pulse sequence is described that allows simultaneous determination of one-bond and two-bond nitrogen-carbon coupling constants for protonated or deuterated proteins. Coupling constants are calculated from volume ratios between cross peaks and reference axial peaks observed in a single 3D spectrum. Accurate backbone ¹ J _NC, ¹ J _NC, and ² J _NC coupling constants are obtained for the two [¹⁵N;¹³C]-labeled, medium-sized proteins flavodoxin and xylanase and for the [²H;¹⁵N;¹³C]-labeled, large protein DFPase. A dependence of one-bond and two-bond J _NC values on protein backbone torsion angles is readily apparent, in agreement with previously found correlations. In addition, the experiment is performed on isotropic as well as aligned protein to measure associated ¹⁵N-¹³C residual dipolar couplings.     

NMR experiments for the sign determination of homonuclear scalar and residual dipolar couplings

A modified version of the JHH-TOCSY experiment, `signed COSY', is presented that allows the determination of the sign of residual dipolar ¹H-¹H coupling constants with respect to the sign of one-bond ¹H-X coupling constants in linear three-spin systems X-¹H-¹H, where X = ¹³C or ¹⁵N. In contrast to the original JHH-TOCSY experiments, the signs of J _HH couplings may be determined for CH₂-CH₂ moieties and for uniformly ¹³C/¹⁵N-labelled samples. In addition, sensitivity is enhanced, diagonal peaks are suppressed and cross peaks are observed only between directly coupled protons, as in a COSY spectrum.     

The Specific Force of Single Intact Extensor Digitorum Longus and Soleus Mouse Muscle Fibers Declines with Aging

In the present study we measured, for the first time, the isometric specific force (SF, force normalized to cross sectional area) generated by single intact fibers from fast- (extensor digitorum longus, EDL) and slow-twitch (soleus) muscles from young adult (2–6), middle-aged (12–14) and old (20–24 month-old) mice. SF has also been measured in single intact flexor digitorum brevis fibers from young mice. Muscle fibers have been classified into fast- or slow-twitch based on the contraction kinetics. Maximum SF recorded in EDL and soleus fibers from young and middle-aged mice did not differ significantly. A significant age-dependent decline in maximum SF was recorded in EDL and soleus fibers from young or middle-aged to old mice. The SF was 377 ± 18, 417 ± 20 and 279 ± 18 kPa for EDL fibers from young, middle-aged and old mice, respectively and 397 ± 17, 405 ± 24 and 320 ± 33 kPa for soleus fibers from age-matched mice, respectively. The frequency needed to elicit maximum force in EDL and soleus fibers from middle-aged to old mice did not differ significantly. In conclusion, the specific force developed by both fast and slow-twitch single intact muscle fibers declines with aging and more significantly in the former. Received: 14 July 2000/Revised: 7 September 2000     

Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release

Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+) sparks are often observed after channel closure. CICR is a function of the net flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca(2+) channels are loosely coupled to RYR through an increase in global [Ca(2+)] due to an increase in the effective distance between L-type Ca(2+) channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.     

Solution structure of DinI provides insight into its mode of RecA inactivation

The Escherichia coli RecA protein triggers both DNA repair and mutagenesis in a process known as the SOS response. The 81-residue E. coli protein DinI inhibits activity of RecA in vivo. The solution structure of DinI has been determined by multidimensional triple resonance NMR spectroscopy, using restraints derived from two sets of residual dipolar couplings, obtained in bicelle and phage media, supplemented with J couplings and a moderate number of NOE restraints. DinI has an alpha/beta fold comprised of a three-stranded beta-sheet and two alpha-helices. The beta-sheet topology is unusual: the central strand is flanked by a parallel and an antiparallel strand and the sheet is remarkably flat. The structure of DinI shows that six negatively charged Glu and Asp residues on DinI's kinked C-terminal alpha-helix form an extended, negatively charged ridge. We propose that this ridge mimics the electrostatic character of the DNA phospodiester backbone, thereby enabling DinI to compete with single-stranded DNA for RecA binding. Biochemical data confirm that DinI is able to displace ssDNA from RecA.     

Altered G protein-coupling functions of RNA editing isoform and splicing variant serotonin2C receptors

Different isoforms of serotonin subtype 2C receptor (5-HT(2C)R) with altered G protein-coupling efficacy are generated by RNA editing, which converts genomically encoded adenosine residues into inosines. In combination, editing of five sites all located within the second intracellular loop region of 5-HT(2C)R mRNA changes the gene-encoded Ile, Asn, and Ile at positions 156, 158, and 160, respectively. We analyzed the G protein-coupling functions of previously unreported editing isoform receptors. An approximately 13-fold reduction in the agonist potency for G protein-coupling stimulation as well as a significantly reduced basal level activity was observed with the thalamus-specific isoform carrying Ile156, Gly158, and Val160 (5-HT(2C)R-IGV). In contrast, the agonist was four- to five-fold less potent with 5-HT(2C)R-MSV and -IDV, detected in the amygdala and choroid plexus, respectively, indicating a dominant role for the amino acid residue at position 158 in receptor functions. We also identified a splicing variant receptor with a truncated C terminus that displayed no ligand binding capacity or G protein-coupling activity. Examination of the alternatively spliced RNA encoding this truncated receptor suggests that editing of this variant RNA occurs after completion of splicing, resulting in complete editing at all five sites.     

Rhodopsin: A Photopigment for Phototaxis in Euglena gracilis

For over 100 years, a major focus of photobiological studies has been the unicellular flagellate, Euglena gracilis, an organism well suited for such investigations by its special complement of organelles that may be considered an ancient, yet complete “visual” system. The possible photoreceptive roles of the cytoplasmic stigma and the photoreceptor (paraflagellar swelling) of E. gracilis are still under debate, because of conflicting interpretations of the results produced so far by the different research groups working on this microorganism. This article deals with our hypothesis, first put forward in the late 1980s, that rhodopsin-like proteins are responsible for photo-detection and that the paraxial rod is involved in the control of flagellar movements. This hypothesis uses oriented dipole and electroconformational coupling mechanisms as the physical phenomena that produce signal transduction. A model for phototaxis is presented.     

A liquid crystalline medium for measuring residual dipolar couplings over a wide range of temperatures

A mixture of dilauroyl phosphatidylcholine (DLPC) and 3-(cholamidopropyl)dimethylammonio-2-hydroxyl-1-propane sulfonate (CHAPSO) in water forms disc shaped bicelles that become ordered at high magnetic fields over a wide range of temperatures. As illustrated for the FK506 binding protein (FKBP), large residual dipolar couplings can be measured for proteins dissolved in low concentrations (5% w/v) of a DLPC/CHAPSO medium at a molar ratio of 4.2:1. This system is especially useful for measuring residual dipolar couplings for molecules that are only stable at low temperatures.     

Heteronuclear correlation experiments for the determination of one-bond coupling constants

Spin-state selective experiments, HSQC-/ and CT-HMQC-/, are proposed for the simple and rapid measurement of scalar one-bond coupling constants in two-dimensional,¹ H-detected ¹⁵N-¹H or¹³ C-¹H correlation experiments based on HSQC and HMQC schemes. Pairs of subspectra are obtained, containing either the high-field or the low-field component of the doublet representing the one-bond coupling constant. The subspectral editing procedure retains the full sensitivity of HSQC and HMQC spectra recorded without heteronuclear decoupling during data acquisition, with a spectral resolution similar to that of decoupled spectra.