Singlet States Open the Way to Longer Time-Scales in the Measurement of Diffusion by NMR Spectroscopy

ABSTRACT: Nuclear magnetic resonance is a powerful nonintrusive technique for measuring diffusion coefficients through the use of pulsed field gradients. The main limitation to the application range of this method is imposed by the relaxation time constants of the magnetization. The recently introduced singlet-state spectroscopy affords obtaining relaxation time constants for pairs of coupled spins which can be longer by more than an order of magnitude than the spin-lattice relaxation time constants. We review in this paper the advantages that are offered by these long relaxation time constants for diffusion measurements. Using experiments that combine singlet-state and diffusion spectroscopy, slower diffusion constants can be determined. The coupling of the two methods constitutes an alternative to the use of special probes equipped with strong gradients for the study of large molecules that diffuse slowly in solution

Structural Basis of BRCC36 Function in DNA Repair and Immune Regulation

In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.ISSN:1097-2765ISSN:1097-416

Rif1 maintains telomeres and mediates DNA repair by encasing DNA ends

In yeast, Rif1 is part of the telosome, where it inhibits telomerase and checkpoint signaling at chromosome ends. In mammalian cells, Rif1 is not telomeric, but it suppresses DNA end resection at chromosomal breaks, promoting repair by nonhomologous end joining (NHEJ). Here, we describe crystal structures for the uncharacterized and conserved ∼125-kDa N-terminal domain of Rif1 from Saccharomyces cerevisiae (Rif1-NTD), revealing an α-helical fold shaped like a shepherd's crook. We identify a high-affinity DNA-binding site in the Rif1-NTD that fully encases DNA as a head-to-tail dimer. Engagement of the Rif1-NTD with telomeres proved essential for checkpoint control and telomere length regulation. Unexpectedly, Rif1-NTD also promoted NHEJ at DNA breaks in yeast, revealing a conserved role of Rif1 in DNA repair. We propose that tight associations between the Rif1-NTD and DNA gate access of processing factors to DNA ends, enabling Rif1 to mediate diverse telomere maintenance and DNA repair functions

Indirect detection of nitrogen-14 in solid-state NMR spectroscopy

This thesis describes new methods for the detection of 14N nuclei by solid-state nuclear magnetic resonance. So far, the low natural abundance (0.4 %) 15N isotope has been widely used to study nitrogen-containing samples because of its spin-1/2 nature. The limited use of the spin-1 isotope 14N (natural abundance 99.6 %) in NMR is due to its strong quadrupolar coupling constant, which leads to very broad spectra that are difficult to excite uniformly and equally difficult to detect, requiring broad receiver bandwidths. The new methods presented in this work result from two main projects covering the indirect detection of 14N via carbon and via protons. The indirect detection of 14N can be achieved by heteronuclear multiple- or single-quantum correlation (HMQC or HSQC) experiments, which rely on the transfer of coherence from a neighboring spin S = 1/2 (13C or 1H) to single- (SQ) or double-quantum (DQ) transitions of 14N nuclei. These methods allow one to observe 14N powder patterns with enhanced sensitivity. These spectra depend on the quadrupolar coupling constant CQ (typically a few MHz), the asymmetry parameter ηQ, and on the orientation of the internuclear vector rIS between the I (14N) and S (13C or 1H) nuclei with respect to the quadrupolar tensor. These parameters reveal valuable information about the structure and dynamics of nitrogen-containing solids. The indirect detection methods are discussed in detail, covering the optimization of experimental parameters; subsequently, applications to the study of amino acids at different magnetic fields are demonstrated

Singlet states open the way to longer time-scales in the measurement of diffusion by NMR spectroscopy

Nuclear magnetic resonance is a powerful nonintrusive technique for measuring diffusion coefficients through the use of pulsed field gradients. The main limitation to the application range of this method is imposed by the relaxation time constants of the magnetization. The recently introduced singlet-state spectroscopy affords obtaining relaxation time constants for pairs of coupled spins which can be longer by more than an order of magnitude than the spin-lattice relaxation time constants. We review in this paper the advantages that are offered by these long relaxation time constants for diffusion measurements. Using experiments that combine singlet-state and diffusion spectroscopy, slower diffusion constants can be determined. The coupling of the two methods constitutes an alternative to the use of special probes equipped with strong gradients for the study of large molecules that diffuse slowly in solution. © 2008 Wiley Periodicals, Inc. Concepts Magn Reson Part A 32A: 68-78, 2008

Evidence for Dynamics on a 100 ns Time Scale from Single- and Double-Quantum Nitrogen-14 NMR in Solid Peptides

The indirect detection of 14N spectra via protons in the manner of heteronuclear multiple-quantum correlation (HMQC) allows one to obtain single- (SQ) and double-quantum (DQ) 14N spectra in solids. A comparison of the SQ and DQ line widths as a function of temperature with simulations reveals motions in the tripeptide AAG with rates on the order of 107 s−1 at 49 °C

Coherence transfer between spy nuclei and nitrogen-14 in solids

Coherence transfer from ‘spy nuclei’ such as 1H or 13C (S = 1/2) was used to excite single- or double-quantum coherences of 14N nuclei (I = 1) while the S spins were aligned along the static field, in the manner of heteronuclear single-quantum correlation (HSQC) spectroscopy. For samples spinning at the magic angle, coherence transfer can be achieved through a combination of scalar couplings J(I, S) and second-order quadrupole–dipole cross-terms, also known as residual dipolar splittings (RDS). The second-order quadrupolar powder patterns in the two-dimensional spectra allow one to determine the quadrupolar parameters of 14N in amino acids

Slow Diffusion by Singlet State NMR Spectroscopy

Small diffusion coeffs. can be measured by using populations of singlet states that have a relaxation time const., Ts, which can be much longer than the longitudinal relaxation time, T1. Spatial information can be encoded with pulsed field gradients in the manner of stimulated echo sequences. Singlet states can be excited via double-quantum coherence to enhance the efficiency of phase encoding and decoding. [on SciFinder (R)