Chimeric β-Lactamases: Global Conservation of Parental Function and Fast Time-Scale Dynamics with Increased Slow Motions

Abstract

Enzyme engineering has been facilitated by recombination of close homologues, followed by functional screening. In one such effort, chimeras of two class-A β-lactamases – TEM-1 and PSE-4 – were created according to structure-guided protein recombination and selected for their capacity to promote bacterial proliferation in the presence of ampicillin (Voigt et al., Nat. Struct. Biol. 2002 9:553). To provide a more detailed assessment of the effects of protein recombination on the structure and function of the resulting chimeric enzymes, we characterized a series of functional TEM-1/PSE-4 chimeras possessing between 17 and 92 substitutions relative to TEM-1 β-lactamase. Circular dichroism and thermal scanning fluorimetry revealed that the chimeras were generally well folded. Despite harbouring important sequence variation relative to either of the two ‘parental’ β-lactamases, the chimeric β-lactamases displayed substrate recognition spectra and reactivity similar to their most closely-related parent. To gain further insight into the changes induced by chimerization, the chimera with 17 substitutions was investigated by NMR spin relaxation. While high order was conserved on the ps-ns timescale, a hallmark of class A β-lactamases, evidence of additional slow motions on the µs-ms timescale was extracted from model-free calculations. This is consistent with the greater number of resonances that could not be assigned in this chimera relative to the parental β-lactamases, and is consistent with this well-folded and functional chimeric β-lactamase displaying increased slow time-scale motions.

15N, 13C and 1H backbone resonance assignments of an artificially engineered TEM-1/PSE-4 class A β-lactamase chimera and its deconvoluted mutant

“…Subcloning of chimera cTEM-19m into pET-24 with the OmpA signal sequence was performed as previously described (Clouthier et al 2012). To generate the TEM-1(M68L–M69T) mutant, oligonucleotide primers were synthesized by Alpha DNA (Montréal, QC) and the QuickChange site-directed mutagenesis method developed by Stratagene (La Jolla, CA) was used with the I-Proof High Fidelity DNA polymerase (Bio-Rad, Mississauga, ON) using pET24-TEM-1 as the template (Clouthier et al 2012). The PCR product was DpnI-digested for 1 h at 37 °C to eliminate the methylated non-mutated parental DNA template and butanol-precipitated before being transformed into E. coli XL1 Blue.…”

“…One colony was selected for DNA extraction to confirm the DNA sequence of the entire coding region and for retransformation into E. coli BL21(DE3) for expression. For NMR, uniformly [ 15 N]- and [ 13 C, 15 N]-labeled cTEM-19m and TEM-1(M68L–M69T) proteins were overexpressed in modified M9 minimal medium containing 15 NH 4 Cl and 13 C-glucose and purified as previously described (Clouthier et al 2012; Gobeil et al 2014; Morin et al 2010). Samples were dialyzed against distilled, deionized water overnight at 4 °C.…”

“…Samples were dialyzed against distilled, deionized water overnight at 4 °C. For applications other than NMR, proteins were overexpressed in auto-inducing ZYP-5052 medium (Studier 2005) and purified as previously described (Clouthier et al 2012; Gobeil et al 2014). Sample purity was determined by SDS-PAGE revealed by the zinc-imidazole method.…”

“…Circular dichroism (CD) using a Jasco spectropolarimeter with a Peltier element, and thermal scanning fluorometry with Sypro Orange using a LightCycler 480 apparatus (Roche), were performed as previously described (Clouthier et al 2012). Briefly, for CD experiments, protein solutions were at a final concentration of 0.1 mg/mL (3.5 μM) in 50 mM sodium phosphate, pH 7.0, in a 2 mm path length cuvette.…”

“…Because both ‘parental’ enzymes are well characterized, the artificially-evolved chimeras resulting from their recombination provide a means to characterize the effect of multiple simultaneous substitutions on enzyme structure and activity. In an effort to better understand the high tolerance of these enzymes to mutations, we previously reported the structure, function and dynamics of a highly blended TEM-1/PSE-4 chimera, cTEM-17m (Clouthier et al 2012; Gobeil et al 2014). This chimera harbours 17 TEM-1 to PSE-4 substitutions in one of the active-site walls (residues 150–190 according to the standard Ambler numbering scheme, i.e.…”