Catherine Zwahlen

A positive feedback loop stabilizes the guanine-nucleotide exchange factor Cdc24 at sites of polarization

In Saccharomyces cerevisiae, activation of Cdc42 by its guanine‐nucleotide exchange factor Cdc24 triggers polarization of the actin cytoskeleton at bud emergence and in response to mating pheromones. The adaptor protein Bem1 localizes to sites of polarized growth where it interacts with Cdc42, Cdc24 and the PAK‐like kinase Cla4. We have isolated Bem1 mutants (Bem1‐m), which are specifically defective for binding to Cdc24. The mutations map within the conserved PB1 domain, which is necessary and sufficient to interact with the octicos peptide repeat (OPR) motif of Cdc24. Although Bem1‐m mutant proteins localize normally, bem1‐m cells are unable to maintain Cdc24 at sites of polarized growth. As a consequence, they are defective for apical bud growth and the formation of mating projections. Localization of Bem1 to the incipient bud site requires activated Cdc42, and conversely, expression of Cdc42–GTP is sufficient to accumulate Bem1 at the plasma membrane. Thus, our results suggest that Bem1 functions in a positive feedback loop: local activation of Cdc24 produces Cdc42–GTP, which recruits Bem1. In turn, Bem1 stabilizes Cdc24 at the site of polarization, leading to apical growth.

Multiple modes of peptide recognition by the PTB domain of the cell fate determinant Numb

The phosphotyrosine‐binding (PTB) domain of the cell fate determinant Numb is involved in the formation of multiple protein complexes in vivo and can bind a diverse array of peptide sequences in vitro. To investigate the structural basis for the promiscuous nature of this protein module, we have determined its solution structure by NMR in a complex with a peptide containing an NMSF sequence derived from the Numb‐associated kinase (Nak). The Nak peptide was found to adopt a significantly different structure from that of a GPpY sequence‐containing peptide previously determined. In contrast to the helical turn adopted by the GPpY peptide, the Nak peptide forms a β‐turn at the NMSF site followed by another turn near the C‐terminus. The Numb PTB domain appears to recognize peptides that differ in both primary and secondary structures by engaging various amounts of the binding surface of the protein. Our results suggest a mechanism through which a single PTB domain might interact with multiple distinct target proteins to control a complex biological process such as asymmetric cell division.

Suppression of spin diffusion in selected frequency bands of nuclear Overhauser spectra

SummaryA variant of two-dimensional nuclear Overhauser effect spectroscopy (NOESY) is described that yields information about cross-relaxation rates between pairs of spins, while the migration of magnetization through several consecutive steps (spin diffusion via neighboring spins) is largely suppressed. This can be achieved by inserting a doubly-selective inversion pulse in a conventional NOESY sequence.

Kinetic study and crystal structure of tetrakis(1,4-thioxane)palladium(II). A comparison between platinum(II) and palladium(II) reactivity

Abstract The compound [Pd(1,4-thioxane)4](BF4)2·4CH3NO2 crystallizes in the monoclinic space group P21/c with a=9.474(2), b=9.529(5), c=21.945(9) A, β=99.77(3)°, Z=2; final R=0.051 for 4325 observed reflections (Pd, C, S and O anisotropically refined). The thioxane ligands around Pd form a square-planar arrangement, with two nitromethane molecules at a distance of 3.115(2) A occupying sites perpendicular to the square-plane formed by the sulfur-bonding thioxanes giving an overall tetragonal bipyramidal coordination around Pd. Intermolecular exchange of 1,4-thioxane on [Pd(1,4-thioxane)4](BF4)2 in deuterated nitromethane and intramolecular interconversion in the bound ligand have been studied as a function of temperature and pressure by 1H NMR lineshape analysis. The first-order rate constant and activation parameters obtained for the intramolecular process, assigned to sulfur inversion, are as follows: ki298=294±26 s−1, ΔHi≠=61.9±1.9 kJ mol−1, ΔSi≠=+10.2±6.5 J K−1 mol−1, ΔVi≠=+2.7±0.2 cm3 mol−1. The intermolecular process yielded the following second-order rate constant and activation parameters: k2298=920±40 m−1 s−1, ΔH2≠=39.5±0.9 kJ mol−1, ΔS2≠=−55.6±2.8 J K−1 mol−1, ΔV2≠=−9.5±0.3 cm3 mol−1. Second-order kinetics and negative values for the entropy and volume of activation indicate an associative Ia or A exchange mechanism. The reactivity of the analogous Pt2+ compound is estimated using the proposed empirical linear correlation function ΔG≠(Pt)=−32.1+1.92ΔG≠(Pd). The calculated second-order rate constant and free energy of activation are 0.3 m−1 s−1 and 76 kJ mol−1, respectively. This relationship indicates also that the well established reactivity order Pd2+≫Pt2+ could be reversed for better π-accepting ligands than MeNC and that, in this case, five-coordinated species on Pt(II) are more stable than the Pd(II) analogue.

Chapter 1 Selective pulses in NMR

Abstract The properties of some widely used selective pulses are described, together with methods for calibration. The emphasis is on an overview of what may be expected from a given pulse shape in terms of selectivity, duration, quality of profile, sensitivity to relaxation, and range of applicability.

Towards the accurate measurement of internuclear distances in biological macromolecules by suppression of spin diffusion

The anal. of 2-dimensional NOESY spectra can be misleading if spin diffusion effects are not properly taken into account. An approach consisting in suppressing spin diffusion by selective inversion of the spins undergoing the Overhauser effect, known as QUIET-NOESY (Zwahlen, C. et al., 1994), is described. In addn., the QUIET-EXSY method (Zwahlen, C. et al., 1995), related to the suppression of spin diffusion by QUIET-NOESY, is discussed. [on SciFinder (R)]