Conformational Landscape of α-Halopropiophenones Determined by nJC-H NMR Reveals Unexpected Patterns and Geometric Constraints.

The conformational features of α-halopropiophenones were investigated to understand the influence of α-halogens on conformation through hyperconjugative interactions, electrostatics, and steric factors. Using NMR, C-H scalar coupling constants were measured in different solvents, revealing a pattern in the conformational equilibria, which we validated by computational means. This behavior arises largely from hyperconjugative effects with the exception of the fluoro-derivatives, which are also influenced by steric and electrostatic interactions. In all cases, the contribution to hyperconjugation of the α-halo ketones is driven by the oxygen lone pair (rather than the C-X bond), which donates electron density to the adjacent C-C bonds. Additionally, C-Cα bond rotation generates distortions in the side chain, responsible for destabilization, thus affecting system conjugation. These structural features identified for the α-halo ketones are also reflected in their reactivity, which is distinct from that expected for nucleophilic addition.

Insights into the Allosteric Response to Acidity by the Helicobacter pylori NikR Transcription Factor.

Helicobacter pylori NikR (HpNikR) is a nickel-responsive transcription factor that regulates genes involved in nickel homeostasis, which is essential for the survival of this pathogen within the acidic human stomach. HpNikR also responds to drops in pH and regulates genes controlling acid acclimation of the bacteria, independently of nickel. We previously showed that nickel binding biases the conformational ensemble of HpNikR to the more DNA-binding competent states via an allosteric network of residues encompassing the nickel binding sites and the interface between the metal- and DNA-binding domains. Here, we examine how acidity promotes this response using 19F-NMR, mutagenesis, and DNA-binding studies. 19F-NMR revealed that a drop in pH from 7.6 to 6.0 does little to shift the conformational ensemble of HpNikR to the DNA binding-compatible cis conformer. Nevertheless, DNA-binding affinities of apo-HpNikR at pH 6.0 and Ni(II)-HpNikR at pH 7.6 are comparable for the ureA promoter. Histidine residues of the nickel binding sites were shown to be important for pH-dependent DNA binding and thus likely impart positive charge to the protein, initiating long-range electrostatic interactions with DNA that induce DNA complexation. The results point to a different DNA-binding mechanism in response to acidity compared to the conformational selection mechanism in response to nickel and overall provide new insights into the influence of pH on HpNikR activity, which contributes to H. pylori viability.

Advances in the study of GPCRs by 19F NMR.

Crystallography and cryo-electron microscopy have advanced atomic resolution perspectives of inactive and active states of G protein-coupled receptors (GPCRs), alone and in complex with G proteins or arrestin. 19F NMR can play a role in ascertaining activation mechanisms and understanding the complete energy landscape associated with signal transduction. Fluorinated reporters are introduced biosynthetically via fluorinated amino acid analogs or chemically, via thiol-specific fluorinated reporters. The chemical shift sensitivity of these reporters makes it possible to discern details of conformational ensembles. In addition to spectroscopic details, paramagnetic species can be incorporated through orthogonal techniques to obtain distance information on fluorinated reporters, while T2-and T1-based relaxation experiments provide details on exchange kinetics in addition to fluctuations within a given state.

Allosteric regulation of the nickel-responsive NikR transcription factor from Helicobacter pylori.

Nickel is essential for the survival of the pathogenic bacteria Helicobacter pylori in the fluctuating pH of the human stomach. Due to its inherent toxicity and limited availability, nickel homeostasis is maintained through a network of pathways that are coordinated by the nickel-responsive transcription factor NikR. Nickel binding to H. pylori NikR (HpNikR) induces an allosteric response favoring a conformation that can bind specific DNA motifs, thereby serving to either activate or repress transcription of specific genes involved in nickel homeostasis and acid adaptation. Here, we examine how nickel induces this response using 19F-NMR, which reveals conformational and dynamic changes associated with nickel-activated DNA complex formation. HpNikR adopts an equilibrium between an open state and DNA-binding competent states regardless of nickel binding, but a higher level of dynamics is observed in the absence of metal. Nickel binding shifts the equilibrium toward the binding-competent states and decreases the mobility of the DNA-binding domains. The nickel-bound protein is then able to adopt a single conformation upon binding a target DNA promoter. Zinc, which does not promote high-affinity DNA binding, is unable to induce the same allosteric response as nickel. We propose that the allosteric mechanism of nickel-activated DNA binding by HpNikR is driven by conformational selection.

Detergent- and phospholipid-based reconstitution systems have differential effects on constitutive activity of G-protein-coupled receptors.

A hallmark of G-protein-coupled receptors (GPCRs) is the conversion of external stimuli into specific cellular responses. In this tightly-regulated process, extracellular ligand binding by GPCRs promotes specific conformational changes within the seven transmembrane helices, leading to the coupling and activation of intracellular "transducer" proteins, such as heterotrimeric G proteins. Much of our understanding of the molecular mechanisms that govern GPCR activation is derived from experiments with purified receptors reconstituted in detergent micelles. To elucidate the influence of the phospholipid bilayer on GPCR activation, here we interrogated the functional, pharmacological, and biophysical properties of a GPCR, the ß2-adrenergic receptor (ß2AR), in high-density lipoprotein (HDL) particles. Compared with detergent-reconstituted ß2AR, the ß2AR in HDL particles had greatly enhanced levels of basal (constitutive) activity and displayed increased sensitivity to agonist activation, as assessed by activation of heterotrimeric G protein and allosteric coupling between the ligand-binding and transducer-binding pockets. Using 19F NMR spectroscopy, we directly linked these functional differences in detergent- and HDL-reconstituted ß2AR to a change in the equilibrium between inactive and active receptor states. The contrast between the low levels of ß2AR constitutive activity in cells and the high constitutive activity observed in an isolated phospholipid bilayer indicates that ß2AR basal activity depends on the reconstitution system and further suggests that various cellular mechanisms suppress ß2AR basal activity physiologically. Our findings provide critical additional insights into GPCR activation and reveal how dramatically reconstitution systems can impact membrane protein function.

Understanding Protein Function Through an Ensemble Description: Characterization of Functional States by 19F NMR.

Protein function is a consequence of a complex and dynamic equilibrium between allosterically coupled functional states. However, it is often difficult to distinguish the representative members of an ensemble by spectroscopic means. 19F NMR is particularly useful in this regard owing to the sensitivity of its chemical shift to subtle differences in environment. Here, we address aspects of 19F NMR relevant to the study of ensembles. In particular, we discuss current trends toward: (1) 19F-reporters that can be biosynthetically incorporated into proteins, (2) Approaches to chemical tagging of proteins by 19F reporters, (3) Improving delineation of states by 19F NMR, (4) Distinguishing states by (19F NMR-based) topology measurements that focus on solvent exposure and hydrophobicity, (5) Relaxation experiments and simple approaches to delineating states in fast and slow exchange, (6) Extending resolution of states by 19F NMR, and (7) Validating 19F NMR spectroscopy by computational methods. Many of these advances are demonstrated through recent 19F NMR studies of a homodimeric enzyme, fluoroacetate dehalogenase.

Site-Specific Labeling of Protein Lysine Residues and N-Terminal Amino Groups with Indoles and Indole-Derivatives.

Indoles and indole-derivatives can be used to site-specifically label proteins on lysine and N-terminal amino groups under mild, nondenaturing reaction conditions. Hen egg white lysozyme (HEWL) and α-lactalbumin were labeled with indole, fluoroindole, or fluoroindole-2-carboxylate via electrophilic aromatic substitutions to lysine side chain Nε- and N-terminal amino imines, formed in situ in the presence of formaldehyde. The reaction is highly site-selective, easily controlled by temperature, and does not eliminate the native charge of the protein, unlike many other common lysine-specific labeling strategies. (19)F NMR was used to monitor reaction progression, and in the case of HEWL, unique resonances for each labeled side chain could be resolved. We demonstrate that the indole tags are highly selective for primary amino groups. (19)F NMR demonstrates that each lysine exhibits a different rate of conjugation to indoles making it possible to employ these tags as a means of probing surface topology by NMR or mass spectrometry. Given the site-specificity of this tagging method, the mildness of the reaction conditions (aqueous, buffered, or unbuffered) and the low stoichiometry required for the reaction, indole-derivatives should serve as a valuable addition to the bioconjugation toolkit. We propose that labeling lysine side chains and N-terminal amino groups with indoles is a versatile and general strategy for bioconjugations with substituted indoles having broad implications for protein functionalization.