ATP Analogues for Structural Investigations: Case Studies of a DnaB Helicase and an ABC Transporter.

Nucleoside triphosphates (NTPs) are used as chemical energy source in a variety of cell systems. Structural snapshots along the NTP hydrolysis reaction coordinate are typically obtained by adding stable, nonhydrolyzable adenosine triphosphate (ATP) -analogues to the proteins, with the goal to arrest a state that mimics as closely as possible a physiologically relevant state, e.g., the pre-hydrolytic, transition and post-hydrolytic states. We here present the lessons learned on two distinct ATPases on the best use and unexpected pitfalls observed for different analogues. The proteins investigated are the bacterial DnaB helicase from Helicobacter pylori and the multidrug ATP binding cassette (ABC) transporter BmrA from Bacillus subtilis, both belonging to the same division of P-loop fold NTPases. We review the magnetic-resonance strategies which can be of use to probe the binding of the ATP-mimics, and present carbon-13, phosphorus-31, and vanadium-51 solid-state nuclear magnetic resonance (NMR) spectra of the proteins or the bound molecules to unravel conformational and dynamic changes upon binding of the ATP-mimics. Electron paramagnetic resonance (EPR), and in particular W-band electron-electron double resonance (ELDOR)-detected NMR, is of complementary use to assess binding of vanadate. We discuss which analogues best mimic the different hydrolysis states for the DnaB helicase and the ABC transporter BmrA. These might be relevant also to structural and functional studies of other NTPases

Design, Synthesis and Structure-Odor Correlation of Novel Spiro[4.5]-decan-2-ones

Abstract: Dehydration and Rupe rearrangement of 2-(3,3-dimethylcyclohexyl)hex-3-yne-2,5-diol (9) furnished as a 3% byproduct the intense vetiver-like smelling 4,7,7-trimethyl-1-methylenespiro[4.5]decan-2-one (11). Motivated by the commercial importance of vetiver oil and the lack of synthetic substitutes as well as the lack of insight into the structural requirements for vetiver odorants, an efficient synthetic route to vetiver-like smelling compounds was developed. It consists of Wittig-Horner-Emmons reaction of diverse cycloalkanones with triethyl 2-phosphonopropionate, subsequent Grignard reaction with in situ conversion to the trienolate, and classical Nazarov cyclization of the resulting dienones. This route not only leads to 11 in 61% yield in the final Nazarov cyclization, but also to 16 analogs, which provide insight into both, the Nazarov reaction and the structure-odor relationship of vetiver odorants. Other vetiver-like smelling compounds discovered include (1RS,4SR,5SR)-1,4,7,7-tetramethylspiro[4.5]decan-2-one (16), 4-methyl-1-methylenespiro[4.6]undecan-2-one (30) and 4-methyl-1-methylenespiro[4.7]dodecan-2-one (31)

Correction to: Characterization of fibril dynamics on three timescales by solid-state NMR

ISSN:0925-2738ISSN:1573-500

Paramagnetic spin labeling of a bacterial DnaB helicase for solid-state NMR

Labeling of biomolecules with a paramagnetic probe for nuclear magnetic resonance (NMR) spectroscopy enables determining long-range distance restraints, which are otherwise not accessible by classically used dipolar coupling-based NMR approaches. Distance restraints derived from paramagnetic relaxation enhancements (PREs) can facilitate the structure determination of large proteins and protein complexes. We herein present the site-directed labeling of the large oligomeric bacterial DnaB helicase from Helicobacter pylori with cysteine-reactive maleimide tags carrying either a nitroxide radical or a lanthanide ion. The success of the labeling reaction was followed by quantitative continuous-wave electron paramagnetic resonance (EPR) experiments performed on the nitroxide-labeled protein. PREs were extracted site-specifically from 2D and 3D solid-state NMR spectra. A good agreement with predicted PRE values, derived by computational modeling of nitroxide and Gd3+ tags in the low-resolution DnaB crystal structure, was found. Comparison of experimental PREs and model-predicted spin label-nucleus distances indicated that the size of the “blind sphere” around the paramagnetic center, in which NMR resonances are not detected, is slightly larger for Gd3+ (∼14 Å) than for nitroxide (∼11 Å) in 13C-detected 2D spectra of DnaB. We also present Gd3+-Gd3+ dipolar electron–electron resonance EPR experiments on DnaB supporting the conclusion that DnaB was present as a hexameric assembly.ISSN:1090-780

Characterization of fibril dynamics on three timescales by solid-state NMR

ISSN:0925-2738ISSN:1573-500

Protein sample preparation for solid-state NMR investigations

International audienc

Segmental isotope labelling and solid-state NMR of a 12 × 59 kDa motor protein: identification of structural variability

ISSN:0925-2738ISSN:1573-500

Partially-deuterated samples of HET-s(218–289) fibrils: assignment and deuterium isotope effect

International audienc

Microsecond Dynamics in Ubiquitin Probed by Solid-State N-15 NMR Spectroscopy R-1 rho Relaxation Experiments under Fast MAS (60-110 kHz)

ISSN:0947-6539ISSN:1521-376

Asparagine and Glutamine Side-Chains and Ladders in HET-s(218–289) Amyloid Fibrils Studied by Fast Magic-Angle Spinning NMR

Asparagine and glutamine side-chains can form hydrogen-bonded ladders which contribute significantly to the stability of amyloid fibrils. We show, using the example of HET-s(218–289) fibrils, that the primary amide side-chain proton resonances can be detected in cross-polarization based solid-state NMR spectra at fast magic-angle spinning (MAS). J-coupling based experiments offer the possibility to distinguish them from backbone amide groups if the spin-echo lifetimes are long enough, which turned out to be the case for the glutamine side-chains, but not for the asparagine side-chains forming asparagine ladders. We explore the sensitivity of NMR observables to asparagine ladder formation. One of the two possible asparagine ladders in HET-s(218–289), the one comprising N226 and N262, is assigned by proton-detected 3D experiments at fast MAS and significant de-shielding of one of the NH2 proton resonances indicative of hydrogen-bond formation is observed. Small rotating-frame 15N relaxation-rate constants point to rigidified asparagine side-chains in this ladder. The proton resonances are homogeneously broadened which could indicate chemical exchange, but is presently not fully understood. The second asparagine ladder (N243 and N279) in contrast remains more flexible.ISSN:2296-889