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.

Order in disorder: The characterization of intrinsically disordered proteins by NMR spectroscopy is a necessity on the one hand and a continuous challenge on the other. We propose two experiments that provide diagnostic parameters to monitor the degree of unfolding of a polypeptide. The test was performed on the yeast Cox17 protein, known to gain its function through maturation from an intrinsically disordered state (see figure).

A number of foldamer backbones have been described as useful mimics of protein secondary structure elements, enabling for example the design of synthetic oligomers with the ability to engage specific protein surfaces. Synthetic folded backbones can also be used to create artificial proteins in which a folded peptide segment (e.g. α-helix, a loop) is replaced by its unnatural counterpart, with the expectation that the resulting molecule would maintain its ability to fold while manifesting new exploitable features. The similarities in screw sense, pitch and polarity between α-helices and oligourea 2.5-helices suggest that a tertiary structure could be retained when swapping the two backbones in a protein sequence. In the present work we move a step towards the creation of such composite proteins by replacing the 10-residue long original α-helical segment in the Cys2His2 zinc finger 3 of transcription factor Egr1 (also known as Zif268) by an oligourea sequence bearing two appropriately spaced imidazole side chains for zinc coordination. We show by spectroscopic techniques and mass spectrometry analysis under native conditions that the ability of the peptide/oligourea hybrid to coordinate zinc ion is not affected by the foldamer replacement. Moreover, detailed NMR analysis provides evidence that the engineered zinc finger motif adopts a folded structure in which the native beta-sheet arrangement of the peptide region and global arrangement of DNA binding side chains are preserved. Titration in the presence of Egr1 target DNA sequence supports binding to GC bases as reported for the wild type zinc finger.

The inside cover picture shows how “spin gymnastics” can be used to “avoid the crowd”. A major problem with the NMR spectra of intrinsically disordered proteins is the chemical shift dispersion arising from their high flexibility and lack of unique 3D structure. On , I. C. Felli, R. Pierattelli, et al. present a strategy for drastically reducing crosspeak overlap by conducting amino‐acid‐selective 13C NMR experiments (CAS‐NMR).

Franck-Condon factors (vibrational transition probabilities) and r-centroids have been evaluated by a numerical integration procedure for the bands of A 0 +g − X 1 Σ + , B 0 +g − X 1 Σ + systems of AuH and A 0 + − X 1 Σ + system of AuD molecules using a suitable potential. The physical and astrophysical significances of our evaluated Franck-Condon factors and r-centroids have also been discussed, which would be helpful in the prediction of the presence of AuH and AuD molecules in various celestial bodies.