Dilution of dipolar interactions in a spin-labeled, multimeric metalloenzyme for DEER studies

Abstract

The metallo-β-lactamases (MβLs), which require one or two Zn(II) ions in their active sites for activity, hydrolyze the amide bond in β-lactam-containing antibiotics, and render the antibiotics inactive. All known MβLs contain a mobile element near their active sites, and these mobile elements have been implicated in the catalytic mechanisms of these enzymes. However little is known about the dynamics of these elements. In this study, we prepared a site-specific, double spin-labeled analog of homotetrameric MβL L1 with spin labels at position 163 and 286 analyzed the sample with DEER (double electron electron resonance) spectroscopy. Four unique distances were observed in the DEER distance distribution, and these distances were assigned to the desired intramolecular dipolar coupling (between spin labels at positions 163 and 286 in one subunit) and to intermolecular dipolar couplings. To rid the spin-labeled analog of L1 of the intermolecular couplings, spin-labeled L1 was “diluted” by unfolding/refolding the spin-labeled enzyme in the presence of excess wild-type L1. DEER spectra of the resulting, spin-diluted enzyme revealed a single distance corresponding to the desire intramolecular dipolar coupling.

Probing substrate binding to the metal binding sites in metallo-β-lactamase L1 during catalysis

“…The steady-state kinetic constants are similar to those previously reported for wild-type L1 ( k cat = 25 ± 1 s −1 ; K m = 2.9 ± 0.1 μM). 25 Stopped-flow kinetic studies were conducted on the reaction of 40 μM ZnCo-L1 and 40 μM chromacef (Figure 2). The substrate decayed quickly over the first 100 ms of the reaction, and the concentration of the ring-opened, anionic intermediate maximized during the first 20 ms of the reaction.…”

“…The syringes, mixer, and tubing were all contained in a watertight bath that was maintained at 2 °C. 25, 33 Immediately prior to sample collection, the nozzle (and, for the shortest reaction times, the attached mixer) was removed from the bath and held 5 mm above the surface of 2-methylbutane contained in a collecting funnel and maintained at −130 °C by a surrounding bath (Update Instruments) of liquid nitrogen cooled 2-methylbutane. Samples were packed into home-designed EXAFS sample holders at −130 °C; excess 2-methylbutane was decanted, and samples were stored in liquid nitrogen until data collection.…”

Conformational dynamics of metallo-β-lactamase CcrA during catalysis investigated by using DEER spectroscopy

“…The use of two frequencies deserves comment. As pointed out earlier [42], the same information is available at both frequencies provided that the relaxation rates allow for reasonable dipolar evolution times. Q-band DEER is a much more efficient technique in terms of time and material [46, 47], and was used whenever possible.…”

“…Unbound spin label was subsequently removed by size exclusion chromatography on a Sephacryl S-200 column (1.5 cm X 40 cm of bed volume 60 ml). The efficiency of the spin labeling was estimated from the intensity of the cw-EPR signal at 25 °C, as described elsewhere [42]. …”

Investigating the position of the hairpin loop in New Delhi metallo-β-lactamase, NDM-1, during catalysis and inhibitor binding

“…[11] Deletion of the entire hairpin loop or mutations on the loop resulted in significantly lower activities of MBLs CcrA, IMP-1, and BcII. [12-15] Previous studies also suggested that this loop is dynamic during catalysis;[12, 13] however, it is not clear how the loop moves during catalysis.…”