Functional Binding Surface of a β‐Hairpin VEGF Receptor Targeting Peptide Determined by NMR Spectroscopy in Living Cells

In this study, the functional interaction of HPLW peptide with VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) was determined by using fast ¹⁵N‐edited NMR spectroscopic experiments. To this aim, ¹⁵N uniformly labelled HPLW has been added to Porcine Aortic Endothelial Cells. The acquisition of isotope‐edited NMR spectroscopic experiments, including ¹⁵N relaxation measurements, allowed a precise characterization of the in‐cell HPLW epitope recognized by VEGFR2.     

Unique Identification of Supramolecular Structures in Amyloid Fibrils by Solid‐State NMR Spectroscopy

The fibril structure formed by the amyloidogenic fragment SNNFGAILSS of the human islet amyloid polypeptide (hIAPP) is determined with 0.52 Å resolution. Symmetry information contained in the easily obtainable resonance assignments from solid‐state NMR spectra (see picture), along with long‐range constraints, can be applied to uniquely identify the supramolecular organization of fibrils.

     

Combining Topology and Sequence Design for the Discovery of Potent Antimicrobial Peptide Dendrimers against Multidrug‐Resistant Pseudomonas aeruginosa

Multidrug‐resistant opportunistic bacteria, such as Pseudomonas aeruginosa, represent a major public health threat. Antimicrobial peptides (AMPs) and related peptidomimetic systems offer an attractive opportunity to control these pathogens. AMP dendrimers (AMPDs) with high activity against multidrug‐resistant clinical isolates of P. aeruginosa and Acinetobacter baumannii were now identified by a systematic survey of the peptide sequences within the branches of a distinct type of third‐generation peptide dendrimers. Combined topology and peptide sequence design as illustrated here represents a new and general strategy to discover new antimicrobial agents to fight multidrug‐resistant bacterial pathogens.     

Engineering a β‐Helical d,l‐Peptide for Folding in Polar Media

β Helices—helices formed by alternating d,l ‐peptides and stabilized by β‐sheet hydrogen bonding—are found naturally in only a handful of highly hydrophobic peptides. This paper explores the scope of β‐helical structure by presenting the first design and biophysical characterization of a hydrophilic d,l ‐peptide, 1 , that forms a β helix in methanol. The design of 1 is based on the β‐hairpin/β helix—a new supersecondary that had been characterized previously only for hydrophobic peptides in nonpolar solvents. Incorporating polar residues in 1 provided solubility in methanol, in which the peptide adopts the expected β‐hairpin/β‐helical structure, as evidenced by CD, analytical ultracentrifugation (AUC), NMR spectroscopy, and NMR‐based structure calculations. Upon titration with water (at constant peptide concentration), the structure in methanol ( 1 m ) transitions cooperatively to an extended conformation ( 1 w ) resembling a cyclic β‐hairpin; observation of an isodichroic point in the solvent‐dependent CD spectra indicates that this transition is a two‐state process. In contrast, neither 1 m nor 1 w show cooperative thermal melting; instead, their structures appear intact at temperatures as high as 65 °C; this observation suggests that steric constraint is dominant in stabilizing these structures. Finally, the ¹H NMR CαH spectroscopic resonances of 1 m are downfield‐shifted with respect to random‐coil values, a hitherto unreported property for β helices that appears to be a general feature of these structures. These results show for the first time that an appropriately designed β‐helical peptide can fold stably in a polar solvent; furthermore, the structural and spectroscopic data reported should prove useful in the future design and characterization of water‐soluble β helices.     

Design,Synthesis, and Application of an Optimized Monofluorinated Aliphatic Label for Peptide Studies by Solid‐State 19F NMR Spectroscopy

A conformationally restricted monofluorinated α‐amino acid, (3‐fluorobicyclo[1.1.1]pentyl)glycine (F‐Bpg), was designed as a label for the structural analysis of membrane‐bound peptides by solid‐state ¹⁹F NMR spectroscopy. The compound was synthesized and validated as a ¹⁹F label for replacing natural aliphatic α‐amino acids. Calculations suggested that F‐Bpg is similar to Leu/Ile in terms of size and lipophilicity. The ¹⁹F NMR label was incorporated into the membrane‐active antimicrobial peptide PGLa and provided information on the structure of the peptide in a lipid bilayer.     

Crystal‐Structure Determination of Powdered Paramagnetic Lanthanide Complexes by Proton NMR Spectroscopy

Shifts for crystals : Solid‐state NMR spectroscopy can be used for structure determination of microcrystalline paramagnetic solids at natural isotopic abundance. The protocol makes use of paramagnetic effects, measured on suitably recorded ¹H NMR spectra, to define the conformation of a molecule in the lattice and the intermolecular packing in the solid phase. The method is illustrated with a family of lanthanide compounds (see picture).

     

Structure Determination of a Flexible Cyclic Peptide Based on NMR and MD Simulation 3J‐Coupling

Molecular dynamics (MD) simulations, in which experimental values such as nuclear Overhauser effects (NOEs), dipolar couplings, ³J‐coupling constants or crystallographic structure factors are used to bias the values of specific molecular properties towards experimental ones, are often carried out to study the structure refinement of peptides and proteins. However, ³J‐coupling constants are usually not employed because of the multiplicity of torsional angle values (φ) corresponding to each ³J‐coupling constant value. Here, we apply the method of adaptively enforced restraining using a local‐elevation (LE) biasing potential energy function in which a memory function penalizes conformations in case both the average <³J> and the current ³J‐values deviate from the experimental target value. Then, the molecule is forced to sample other parts of the conformational space, thereby being able to cross high energy barriers and to bring the simulated average <³J> close to the measured <³J> value. Herein, we show the applicability of this method in structure refinement of a cyclo‐β‐tetrapeptide by enforcing the ³J‐value restraining with LE on twelve backbone torsional angles. The resulting structural ensemble satisfies the experimental ³J‐coupling data better than the NMR model structure derived using conventional single‐structure refinement based on these data. Thus, application of local‐elevation search MD simulation in combination with biasing towards ³J‐coupling makes it possible to use ³J‐couplings quantitatively in structure determination of peptides.     

The G‐Protein‐Coupled Neuropeptide Y Receptor Type 2 is Highly Dynamic in Lipid Membranes as Revealed by Solid‐State NMR Spectroscopy

In spite of the recent success in crystallizing several G‐protein‐coupled receptors (GPCRs), a comprehensive biophysical characterization of these molecules under physiological conditions also requires the study of the molecular dynamics of these proteins. The molecular mobility of the human neuropeptide Y receptor type 2 reconstituted into dimyristoylphosphatidylcholine (DMPC) membranes was investigated by means of solid‐state NMR spectroscopy. Static ¹⁵N NMR spectra show that the receptor performs axially symmetric motions in the membrane, and several residues undergo large amplitude fluctuations. This was confirmed by quantitative measurements of the motional ¹H,¹³C order parameter of the CH, CH₂, and CH₃ groups. In directly polarized ¹³C NMR experiments, these order parameters showed astonishingly low values of S_CH=0.55, S=0.33, and S=0.17, which corresponds to segmental amplitudes of approximately 50° in the backbone and approximately 50–60° in the side chain. At physiological temperature, ²H NMR spectra of the deuterated receptor showed a narrow component that is indicative of molecular order parameters of S≤0.3 superimposed with a very broad spectrum that could stem from the transmembrane α‐helices. These results suggest that the crystal structures of GPCRs only represent a static snapshot of these highly mobile molecules, which undergo significant structural fluctuations with relatively large amplitudes in a liquid‐crystalline membrane at physiological temperature.     

Recovering Invisible Signals by Two‐Field NMR Spectroscopy

Nuclear magnetic resonance (NMR) studies have benefited tremendously from the steady increase in the strength of magnetic fields. Spectacular improvements in both sensitivity and resolution have enabled the investigation of molecular systems of rising complexity. At very high fields, this progress may be jeopardized by line broadening, which is due to chemical exchange or relaxation by chemical shift anisotropy. In this work, we introduce a two‐field NMR spectrometer designed for both excitation and observation of nuclear spins in two distinct magnetic fields in a single experiment. NMR spectra of several small molecules as well as a protein were obtained, with two dimensions acquired at vastly different magnetic fields. Resonances of exchanging groups that are broadened beyond recognition at high field can be sharpened to narrow peaks in the low‐field dimension. Two‐field NMR spectroscopy enables the measurement of chemical shifts at optimal fields and the study of molecular systems that suffer from internal dynamics, and opens new avenues for NMR spectroscopy at very high magnetic fields.     

Characterization of G‐Quadruplex/Hairpin Transitions of RNAs by 19F NMR Spectroscopy

2′‐O‐[(4‐Trifluoromethyl‐triazol‐1‐yl)methyl] reporter groups have been incorporated into guanosine‐rich RNA models (including a known bistable Qd/Hp RNA and two G‐rich regions of mRNA of human prion protein, PrP) and applied for the ¹⁹F NMR spectroscopic characterization of plausible G‐quadruplex/hairpin (Qd/Hp) transitions in these RNA structures. For the synthesis of the CF₃‐labeled RNAs, phosphoramidite building blocks of 2′‐O‐[(4‐CF₃‐triazol‐1‐yl)methyl] nucleosides (cytidine, adenosine, and guanosine) were prepared and used as an integral part of the standard solid‐phase RNA synthesis. The obtained ¹⁹F NMR spectra supported the usual characterization data (obtained by UV‐ and CD‐melting profiles and by ¹H NMR spectra of the imino regions) and additionally gave more detailed information on the Qd/Hp transitions. The molar fractions of the secondary structural species (Qd, Hp) upon thermal denaturation and under varying ionic conditions could be determined from the intensities and shifts of the ¹⁹F NMR signals. For a well‐behaved Qd/Hp transition, thermodynamic parameters could be extracted.     

Targeting Bacterial Membranes: NMR Spectroscopy Characterization of Substrate Recognition and Binding Requirements of D‐Arabinose‐5‐Phosphate Isomerase

Lipopolysaccharide (LPS) is an essential component of the outer membrane of Gram‐negative bacteria and consists of three elements: lipid A, the core oligosaccharide, and the O‐antigen. The inner‐core region is highly conserved and contains at least one residue of 3‐deoxy‐D ‐manno‐octulosonate (Kdo). Arabinose‐5‐phosphate isomerase (API) is an aldo–keto isomerase catalyzing the reversible isomerization of D ‐ribulose‐5‐phosphate (Ru5P) to D ‐arabinose‐5‐phosphate (A5P), the first step of Kdo biosynthesis. By exploiting saturation transfer difference (STD) NMR spectroscopy, the structural requirements necessary for API substrate recognition and binding were identified, with the aim of designing new API inhibitors. In addition, simple experimental conditions for the STD experiments to perform a fast, robust, and efficient screening of small libraries of potential API inhibitors, allowing the identification of new potential leads, were set up. Due to the essential role of API enzymes in LPS biosynthesis and Gram‐negative bacteria survival, by exploiting these data, a new generation of potent antibacterial drugs could be developed.     

Proton‐Detected Solid‐State NMR Spectroscopy of Natural‐Abundance Peptide and Protein Pharmaceuticals

The natural way : A sensitive NMR spectroscopic method is developed to obtain well‐resolved two‐dimensional spectra (¹⁵N–¹H and ¹³C–¹H) for natural‐abundance (that is, without the need for isotopic enrichment) large‐molecule samples, such as biopharmaceuticals. This method gives structural insights on two lyophilized aprotinin samples and three insulin samples in lyophilized, microcrystalline suspension formulation (red; see picture) and fibril (green) forms.

     

Structure‐Based Approach To Improve a Small‐Molecule Inhibitor by the Use of a Competitive Peptide Ligand

Structural information about the target–compound complex is invaluable in the early stage of drug discovery. In particular, it is important to know into which part of the initial compound additional interaction sites could be introduced to improve its affinity. Herein, we demonstrate that the affinity of a small‐molecule inhibitor for its target protein could be successfully improved by the constructive introduction of the interaction mode of a competitive peptide. The strategy involved the discrimination of overlapping and non‐overlapping peptide–compound pharmacophores by the use of a ligand‐based NMR spectroscopic approach, INPHARMA. The obtained results enabled the design of a new compound with improved affinity for the platelet receptor glycoprotein VI (GPVI). The approach proposed herein efficiently combines the advantages of compounds and peptides for the development of higher‐affinity druglike ligands.     

Matrix‐Free DNP‐Enhanced NMR Spectroscopy of Liposomes Using a Lipid‐Anchored Biradical

Magic‐angle spinning dynamic nuclear polarization (MAS‐DNP) has been proven to be a powerful technique to enhance the sensitivity of solid‐state NMR (SSNMR) in a wide range of systems. Here, we show that DNP can be used to polarize lipids using a lipid‐anchored polarizing agent. More specifically, we introduce a C16‐functionalized biradical, which allows localization of the polarizing agents in the lipid bilayer and DNP experiments to be performed in the absence of excess cryo‐protectant molecules (glycerol, dimethyl sulfoxide, etc.). This constitutes another original example of the matrix‐free DNP approach that we recently introduced.