Study of the Hydrophobic Cavity of β‐Cryptogein through Laser‐Polarized Xenon NMR Spectroscopy

Abstract

The interaction of xenon with beta-cryptogein, a basic 10 kDa protein belonging to the elicitin family, has been studied by using dissolved thermal and laser-polarized gas in liquid-state NMR. 13C and 1H chemical-shift-mapping experiments were unfruitful, the proton lines only experienced a slight narrowing but no significant frequency variation when the xenon concentration was increased. Nevertheless magnetization transfer from hyperpolarized xenon to protons of the protein demonstrates an undoubted interaction and enables localization of the noble-gas-binding site. Due to the proton-proton cross-relaxation efficiency, however, this experiment is subjected to important spin-diffusion. An automatic procedure that takes spin-diffusion into account when assigning the protons that interact with xenon is then used. The binding site, as defined by 30 Xe--H interactions, is situated in the inner core of the protein. The protons that interact with xenon border the channel by which sterols are known to enter into the cavity. These results support the idea that xenon is a good probe for hydrophobic protein regions.

Dynamics of Xenon Binding Inside the Hydrophobic Cavity of Pseudo-Wild-type Bacteriophage T4 Lysozyme Explored through Xenon-Based NMR Spectroscopy

“…Moreover the shorter xenon T 1 at low field induces an efficient xenon relaxation during the shaking of the NMR tube in the fringe field of the NMR magnet, shaking needed to renew dissolved laser-polarized xenon before starting any SPINOE experiment. As a consequence and as noted, the absolute xenon magnetization cannot be as large in the case of T4 lysozyme as values obtained for tobacco LTP1 13 or β-cryptogein …”

“…As a consequence and as noted, the absolute xenon magnetization cannot be as large in the case of T4 lysozyme as values obtained for tobacco LTP1 13 or β-cryptogein. 43 From a biophysical point of view, this much longer correlation time reveals that the mobility of xenon inside the cavity of T4 lysozyme is much more restricted than in wheat LTP1, which is expected as the latter has a much larger cavity volume (>300 Å 3 ). On the basis of the X-ray structure of the complex (PDB code: 1C6T, ref 19), this tendency is expected since the cavity of the WT* T4 lysozyme is slightly enlarged during xenon binding.…”

“…Since laserpolarized xenon allows accurate NMR measurements, the xenon resonance line width has never been exploited, although different behaviors exist. For instance, in the case of LTP1 proteins 8,13 or β cryptogein 43 no significant variation of the xenon resonance line width is observed as a function of the bound xenon fraction. On the other hand, in the case of maltose-binding protein, a significant broadening of the xenon resonance is observed when the protein concentration increases.…”

“…From a structural point of view each approach has its own advantages and drawbacks. For example SPINOE techniques were able to locate xenon inside β-cryptogein 43 for which X-ray crystallography under xenon pressure was unsuccessful. 49 In contrast, the SPINOE spectra obtained on WT* T4 lysozyme exhibit poor signal-to-noise ratios, and in this case X-ray analysis of the crystallized protein in the presence of pressurized xenon was better suited for determining the xenon binding site.…”

Sensitivity enhancement in solution NMR: Emerging ideas and new frontiers

“…The hydrophobic cavity of the 10 kDa protein β-cryptogein was studied by SPINOE at 1.6 mM in the presence of 5.2 mM ~15% hyperpolarized xenon. A small but clearly distinguishable increase in 1 H resonance intensity (by ~1%) was observed in the inner core of the protein [127]. Additional details about SPINOE can be found elsewhere [122].…”