A direct response: Exclusively heteronuclear 13C direct‐detection 2D NMR experiments are performed on 13C,15N‐enriched proteins in E. coli cells. Unfolded proteins or protein fragments provide well‐resolved carbonyl detection (see picture), whereas folded proteins or structured motifs do not give any detectable spectrum.

Carbon-13 direct-detection NMR methods have proved to be very useful for the characterization of intrinsically disordered proteins (IDPs). Here we present a suite of experiments in which amino-acid-selective editing blocks are encoded in CACON- and CANCO-type sequences to give (13) C-detected spectra containing correlations arising from a particular type or group of amino acid(s). These two general types of experiments provide the complementary intra- and inter-residue correlations necessary for sequence-specific assignment of backbone resonance frequencies. We demonstrate the capabilities of these experiments on two IDPs: fully reduced Cox17 and WIP(C) . The proposed approach constitutes an independent strategy to simplify crowded spectra as well as to perform sequence-specific assignment, thereby demonstrating its potential to study IDPs.

Gram-negative bacteria expressing class A β-lactamases pose a serious health threat due to their ability to inactivate all β-lactam antibiotics. The acyl–enzyme intermediate is a central milestone in the hydrolysis reaction catalyzed by these enzymes. However, the protonation states of the catalytic residues in this complex have never been fully analyzed experimentally due to inherent difficulties. To help unravel the ambiguity surrounding class A β-lactamase catalysis, we have used ultrahigh-resolution X-ray crystallography and the recently approved β-lactamase inhibitor avibactam to trap the acyl–enzyme complex of class A β-lactamase CTX-M-14 at varying pHs. A 0.83-Å-resolution CTX-M-14 complex structure at pH 7.9 revealed a neutral state for both Lys73 and Glu166. Furthermore, the avibactam hydroxylamine-O-sulfonate group conformation varied according to pH, and this conformational switch appeared to correspond to a change in the Lys73 protonation state at low pH. In conjunction with computational analyses, our structures suggest that Lys73 has a perturbed acid dissociation constant (pKa) compared with acyl–enzyme complexes with β-lactams, hindering its function to deprotonate Glu166 and the initiation of the deacylation reaction. Further NMR analysis demonstrated Lys73 pKato be ∼5.2 to 5.6. Together with previous ultrahigh-resolution crystal structures, these findings enable us to follow the proton transfer process of the entire acylation reaction and reveal the critical role of Lys73. They also shed light on the stability and reversibility of the avibactam carbamoyl acyl–enzyme complex, highlighting the effect of substrate functional groups in influencing the protonation states of catalytic residues and subsequently the progression of the reaction.

Intrinsically disordered proteins (IDPs) have recently attracted the attention of the scientific community challenging the well accepted structure-function paradigm. In the characterization of the dynamic features of proteins nuclear magnetic resonance spectroscopy (NMR) is a strategic tool of investigation. However the peculiar properties of IDPs, with the lack of a unique 3D structure and their high flexibility, have a strong impact on NMR observables (low chemical shift dispersion, efficient solvent exchange broadening) and thus on the quality of NMR spectra. Key aspects to be considered in the design of new NMR experiments optimized for the study of IDPs are discussed. A new experiment, based on direct detection of (13)C(α), is proposed.