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

Abstract

Wild-type bacteriophage T4 lysozyme contains a hydrophobic cavity with binding properties that have been extensively studied by X-ray crystallography and NMR. In the present study, the monitoring of 1H chemical shift variations under xenon pressure enables the determination of the noble gas binding constant (K = 60.2 M(-1)). Although the interaction site is highly localized, dipolar cross-relaxation effects between laser-polarized xenon and nearby protons (SPINOE) are rather poor. This is explained by the high value of the xenon-proton dipolar correlation time (0.8 ns), much longer than the previously reported values for xenon in medium-size proteins. This indicates that xenon is highly localized within the protein cavity, as confirmed by the large chemical shift difference between free and bound xenon. The exploitation of the xenon line width variation vs xenon pressure and protein concentration allows the extraction of the exchange correlation time between free and bound xenon. Comparison to the exchange experienced by protein protons indicates that the exchange between the open and closed conformations of T4 lysozyme is not required for xenon binding.

Use of experimental crystallographic phases to examine the hydration of polar and nonpolar cavities in T4 lysozyme

“…The L99A cavity in T4 lysozyme is, in many respects, the prototypical nonpolar cavity in proteins. It was one of the first such cavities to be engineered (16) and has been the subject of thermodynamic, structural (16), ligand-binding (17)(18)(19), NMR (20)(21)(22), pressure-dependent, and theoretical studies (23,24). With a volume of Ϸ120 Å 3 , it is larger than one typically obtains from a ''large-to-small'' substitution (e.g., Leu 3 Ala or Phe 3 Ala) in the core of a protein (16,25).…”

“…It might be noted that, under pressure, this cavity will bind xenon, which has a volume of 45.3 Å 3 (19). There is no evidence that a major change in conformation is required to allow the ligand to reach the binding site (22). Halle and coworkers (30)(31)(32) have also shown that exchange of water at internal sites in proteins is rapid.…”

Sensitivity enhancement in solution NMR: Emerging ideas and new frontiers

“…Hence, this effect enhances NMR sensitivity for the detection of protons in solution [122, 125]. SPINOE was used primarily to study organic molecules containing xenon-accessible cavities such as α-cyclodextrin and cryptophane-A [123] as well as in the context of proteins larger than 100 amino acids [126]. 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.…”

Observation of Noise‐Triggered Chaotic Emissions in an NMR‐Maser

“…[3,4] These solutions have had appeal through the emergence of new application fields, [5][6][7] but their direct extension to all liquid-state NMR techniques might be less straightforward than anticipated. Indeed, whatever the magnetic field, the nuclear magnetization of a solution containing 50 mmol L À1 of 129 Xe polarized at 30 % is identical to that of bulk water in a 11.7 T magnet at room temperature.…”