This work introduces an integrated microfluidic device for measuring rapid H/D exchange (HDX) in proteins. By monitoring backbone amide HDX on the millisecond to low second time scale, we are able to characterize conformational dynamics in weakly structured regions, such as loops and molten globule-like domains that are inaccessible in conventional HDX experiments. The device accommodates the entire MS-based HDX workflow on a single chip with residence times sufficiently small (ca. 8 s) that back-exchange is negligible (≤5%), even without cooling. Components include an adjustable position capillary mixer providing a variable-time labeling pulse, a static mixer for HDX quenching, a proteolytic microreactor for rapid protein digestion, and on-chip electrospray ionization (ESI). In the present work, we characterize device performance using three model systems, each illustrating a different application of 'time-resolved' HDX. Ubiquitin is used to illustrate a crude, high throughput structural analysis based on a single subsecond HDX time-point. In experiments using cytochrome c, we distinguish dynamic behavior in loops, establishing a link between flexibility and interactions with the heme prosthetic group. Finally, we localize an unusually high 'burst-phase' of HDX in the large tetrameric enzyme DAHP synthase to a 'molten globule-like' region surrounding the active site.

In this work, we introduce an integrated, electrospray mass spectrometry-coupled microfluidic chip that supports the complete workflow for 'bottom up' hydrogen/deuterium exchange (HDX) pulse labelling experiments. HDX pulse labelling is used to measure structural changes in proteins that occur after the initiation of a reaction, most commonly folding. In the present case, we demonstrate the device on the β-lactamase enzyme TEM-1, identifying active site changes that occur upon acylation by a covalent inhibitor and subtle changes in conformational dynamics that occur away from the active site over a period of several second after the inhibitor is bound. Our results demonstrate the power of microfluidics-enabled sub-second HDX pulse labelling as a tool for studying allostery and show some intriguing correlations with mutagenesis studies.

Carbapenem-hydrolyzing class D ␤-lactamases (CHDLs) are a subgroup of class D ␤-lactamases, which are enzymes that hydrolyze ␤-lactams. They have attracted interest due to the emergence of multidrug-resistant Acinetobacter baumannii, which is not responsive to treatment with carbapenems, the usual antibiotics of choice for this bacterium. Unlike other class D ␤-lactamases, these enzymes efficiently hydrolyze carbapenem antibiotics. To explore the structural requirements for the catalysis of carbapenems by these enzymes, we determined the crystal structure of the OXA-58 CHDL of A. baumannii following acylation of its active-site serine by a 6␣-hydroxymethyl penicillin derivative that is a structural mimetic for a carbapenem. In addition, several point mutation variants of the active site of OXA-58, as identified by the crystal structure analysis, were characterized kinetically. These combined studies confirm the mechanistic relevance of a hydrophobic bridge formed over the active site. This structural feature is suggested to stabilize the hydrolysis-productive acyl-enzyme species formed from the carbapenem substrates of this enzyme. Furthermore, our structural studies provide strong evidence that the hydroxyethyl group of carbapenems samples different orientations in the active sites of CHDLs, and the optimum orientation for catalysis depends on the topology of the active site allowing proper closure of the active site. We propose that CHDLs use the plasticity of the active site to drive the mechanism of carbapenem hydrolysis toward efficiency.

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Carbapenems such as imipenem and meropenem are β‐lactam antibiotics with a wide spectrum of activity, initially designed to overcome β‐lactam resistance due to their stability toward most β‐lactamases. However, as a result of selective pressure these carbapenems have succumbed to the hydrolytic power of β‐lactamases. These enzymes hydrolyze the β‐lactam bond of these antibiotics. They are classified in four classes based on their primary sequences, classes A, B, C and D. Classes A, C and D are serine‐active site β‐lactamases and class B β‐lactamases are metallo‐enzymes. The progressively increasing ability of all four classes of β‐lactamases to hydrolyze carbapenems has been documented. Among them, Carbapenem‐hydrolyzing Class D β‐lactamases (CHDLs) are directly associated with outbreaks of carbapenem‐resistant Acinetobacter baumannii around the world and have emerged as a major problem in treatment of multidrug resistant A. baumannii, for which carbapenems have been the drug of choice. We have undertaken a structural study using H/D exchange and a kinetic study using a number of OXA‐58 mutants to delineate the mechanism of hydrolysis of imipenem by OXA‐58. Our studies show that OXA‐58 employs enzyme dynamics to gain specificity toward imipenem hydrolysis.

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