Optimum levels of exchangeable protons in perdeuterated proteins for proton detection in MAS solid-state NMR spectroscopy
 

Optimum levels of exchangeable protons in perdeuterated proteins for proton detection in MAS solid-state NMR spectroscopy


Abstract We present a systematic study of the effect of the level of exchangeable protons on the observed amide proton linewidth obtained in perdeuterated proteins. Decreasing the amount of D2O employed in the crystallization buffer from 90 to 0%, we observe a fourfold increase in linewidth for both 1H and 15N resonances. At the same time, we find a gradual increase in the signal-to-noise ratio (SNR) for 1Hâ??15N correlations in dipolar coupling based experiments for H2O concentrations of up to 40%. Beyond 40%, a significant reduction in SNR is observed. Scalar-coupling based 1Hâ??15N correlation experiments yield a nearly constant SNR for samples prepared with â?¤30% H2O. Samples in which more H2O is employed for crystallization show a significantly reduced NMR intensity. Calculation of the SNR by taking into account the reduction in 1H T 1 in samples containing more protons (SNR per unit time), yields a maximum SNR for samples crystallized using 30 and 40% H2O for scalar and dipolar coupling based experiments, respectively. A sensitivity gain of 3.8 is obtained by increasing the H2O concentration from 10 to 40% in the CP based experiment, whereas the linewidth only becomes 1.5 times broader. In general, we find that CP is more favorable compared to INEPT based transfer when the number of possible 1H,1H interactions increases. At low levels of deuteration (â?¥60% H2O in the crystallization buffer), resonances from rigid residues are broadened beyond detection. All experiments are carried out at MAS frequency of 24 kHz employing perdeuterated samples of the chicken α-spectrin SH3 domain.

• Content Type Journal Article
• Pages 67-73
• DOI 10.1007/s10858-009-9369-0
• Authors
  • Ã?mit Akbey, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Robert-Rössle-Str. 10 13125 Berlin Germany
  • Sascha Lange, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Robert-Rössle-Str. 10 13125 Berlin Germany
  • W. Trent Franks, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Robert-Rössle-Str. 10 13125 Berlin Germany
  • Rasmus Linser, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Robert-Rössle-Str. 10 13125 Berlin Germany
  • Kristina Rehbein, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Robert-Rössle-Str. 10 13125 Berlin Germany
  • Anne Diehl, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Robert-Rössle-Str. 10 13125 Berlin Germany
  • Barth-Jan van Rossum, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Robert-Rössle-Str. 10 13125 Berlin Germany
  • Bernd Reif, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Robert-Rössle-Str. 10 13125 Berlin Germany
  • Hartmut Oschkinat, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Robert-Rössle-Str. 10 13125 Berlin Germany

• • Journal Journal of Biomolecular NMR
  • Online ISSN 1573-5001
  • Print ISSN 0925-2738
  • Journal Volume Volume 46
  • Journal Issue Volume 46, Number 1

Source: Journal of Biomolecular NMR