Inclusion complexes of benzene (Bz) with cyclodextrins (CD) have been investigated so far using non-NMR techniques in various solvents resulting in conflicting data. Here, the first application of NMR spectroscopy in combination with rigorous statistical analysis of the results has allowed us to determine accurately the stoichiometry of complexes and their association constants. Titration measurements have been performed by 1 H NMR spectroscopy in D 2 O at a magnetic field B 0 of 14.1 T. αCD and γCD host molecules form weak 1 : 1 complexes with Bz. In contrast, Bz and βCD build 1 : 1 and 2 : 1 complexes coexisting in solution with large binding constants. Binding of second benzene molecule is strongly cooperative.

Hosts, a key component of inclusion complexes, are cyclic oligomeric compounds containing a cavity in which another component of the complex is bound by non-covalent forces. Chiral hosts are particularly important and interesting because they allow the study of specific intermolecular interactions and molecular recognition. The most important classes of chiral hosts and their physicochemical properties are briefly reviewed. An important part of this Review is the description of selected concepts necessary to understand the properties and behavior of inclusion complexes studied by the most suitable analytical method for studying inclusion complexes—nuclear magnetic resonance.

The dynamics of the backbone and side-chains of protein are routinely studied by interpreting experimentally determined 15N spin relaxation rates. R1(15N), the longitudinal relaxation rate, reports on fast motions and encodes, together with the transverse relaxation R2, structural information about the shape of the molecule and the orientation of the amide bond vectors in the internal diffusion frame. Determining error-free 15N longitudinal relaxation rates remains a challenge for small, disordered, and medium-sized proteins. Here, we show that mono-exponential fitting is sufficient, with no statistical preference for bi-exponential fitting up to 800 MHz. A detailed comparison of the TROSY and HSQC techniques at medium and high fields showed no statistically significant differences. The least error-prone DD/CSA interference removal technique is the selective inversion of amide signals while avoiding water resonance. The exchange of amide with solvent deuterons appears to affect the rate R1 of solvent-exposed amides in all fields tested and in each DD/CSA interference removal technique in a statistically significant manner. In summary, the most accurate R1(15N) rates in proteins are achieved by selective amide inversion, without the addition of D2O. Importantly, at high magnetic fields stronger than 800 MHz, when non-mono-exponential decay is involved, it is advisable to consider elimination of the shortest delays (typically up to 0.32 s) or bi-exponential fitting.

Biomacromolecular dynamics studies based on 15N spin relaxation are well established among the atomic-level structural biology techniques, accessing molecular mobility at a wide range of time scales spanning over ten orders of magnitude. The 15N labelling constitutes the simplest, most accessible, stable isotope labelling strategy for numerous proteins and nucleic acids. Therefore, it has become a convenient method to experimentally target the atomic and sub-atomic levels of molecular mobility. Here we present the currently available experimental techniques to access the dynamics of the protein backbone and side-chains, and the numerical methods of interpretation. The complexity of molecular motional models has led to separate measurements and interpretation of the dynamics for well-folded globular proteins and domains, highly dynamic, flexible, intrinsically disordered regions, and entirely flexible, intrinsically disordered proteins. In addition, separate approaches have been developed to detect and interpret the mobility of various amino acid side-chains. The advantages and limitations of the available tools and the emerging need for a more in-depth understanding of molecular functional dynamics are discussed.

Inclusion complexes of naphthalene (NP) with cyclodextrins (CD) have been investigated so far using non-NMR techniques resulting in inconsistent data. Here, the first application of high-field NMR spectroscopy in combination with a precise analysis of the results has allowed us to determine accurately the stoichiometry of complexes and their association constants. Titration measurements have been performed by 1H NMR spectroscopy in D2O at a magnetic field B0 of 18.8 T. NP and αCD form a 1 : 2 complex in which a single NP molecule is closed in a capsule made up of two αCD macrocycles. NP and βCD build coexisting 2 : 1 and 2 : 2 complexes with large binding constants. Larger γCD host molecules form essentially similar complexes with NP as the βCD but corresponding binding constants are smaller.

Intramolecular motions in proteins are one of the important factors that determine their biological activity and interactions with molecules of biological importance. Magnetic relaxation of 15 N amide nuclei allows one to monitor motions of protein backbone over a wide range of time scales. 15 N{ 1 H} nuclear Overhauser effect is essential for the identification of fast backbone motions in proteins. Therefore, exact measurements of NOE values and their accuracies are critical for determining the picosecond time scale of protein backbone. Measurement of dynamic NOE allows for the determination of NOE values and their probable errors defined by any sound criterion of nonlinear regression methods. The dynamic NOE measurements can be readily applied for non-deuterated or deuterated proteins in both HSQC and TROSY-type experiments. Comparison of the dynamic NOE method with commonly implied steady-state NOE is presented in measurements performed at three magnetic field strengths. It is also shown that improperly set NOE measurement cannot be restored with correction factors reported in the literature.

Intramolecular motions in proteins are one of the important factors that determine their biological activity and interactions with molecules of biological importance. Magnetic relaxation of(15)N amide nuclei allows one to monitor motions of protein backbone over a wide range of time scales.N-15{H-1} nuclear Overhauser effect is essential for the identification of fast backbone motions in proteins. Therefore, exact measurements of NOE values and their accuracies are critical for determining the picosecond time scale of protein backbone. Measurement of dynamic NOE allows for the determination of NOE values and their probable errors defined by any sound criterion of nonlinear regression methods. The dynamic NOE measurements can be readily applied for non-deuterated or deuterated proteins in both HSQC and TROSY-type experiments. Comparison of the dynamic NOE method with commonly implied steady-state NOE is presented in measurements performed at three magnetic field strengths. It is also shown that improperly set NOE measurement cannot be restored with correction factors reported in the literature.

Intramolecular motions in proteins are one of the important factors that determine their biological activity and interactions with molecules of biological importance. Magnetic relaxation of 15N amide nuclei allows one to monitor motions of protein backbone over a wide range of time scales. 15N{1H} nuclear Overhauser effect is essential for the identification of fast backbone motions in proteins. Therefore, exact measurements of NOE values and their accuracies are critical for determining the picosecond time scale of protein backbone. Measurement of dynamic NOE allows for the determination of NOE values and their probable errors defined by any sound criterion of nonlinear regression methods. The dynamic NOE measurements can be readily applied for non-deuterated or deuterated proteins in both HSQC and TROSY-type experiments. Comparison of the dynamic NOE method with commonly implied steady-state NOE is presented in measurements performed at three magnetic field strengths. It is also shown that improperly set NOE measurement cannot be restored with correction factors reported in the literature.

Complex formation between quinine and natural cyclodextrins (CD) was studied using NMR spectroscopy. The strongest association was observed for complexes of neutral quinine molecules with βCD. Association constants for monocationic quinine were one order of magnitude smaller, while dicationic quinine did not bind to CDs. The distribution of complexation-induced shifts and ROESY spectra revealed bimodal quinine binding in complexes formed with βCD and γCD. Complex formation resulted in a decrease of the vicinal coupling constant between H2 and H9 protons owing to the rotation about the C2-C9 bond and in consequence in mutual reorientation of two main constituents of quinine: quinoline and quinuclidine. DFT calculations allowed establishing that H2 and H9 protons are antiperiplanar in the prevailing quinine conformer(s) in aqueous solution. Conformers with synclinal H2 and H9 protons participated in quinine complexation with CDs.

Simple and convenient method of protein dynamics evaluation from the insufficient experimental 15N relaxation data is presented basing on the ratios, products, and differences of longitudinal and transverse 15N relaxation rates obtained at a single magnetic field. Firstly, the proposed approach allows evaluating overall tumbling correlation time (nanosecond time scale). Next, local parameters of the model-free approach characterizing local mobility of backbone amide N–H vectors on two different time scales, S2 and R ex , can be elucidated. The generalized order parameter, S2, describes motions on the time scale faster than the overall tumbling correlation time (pico- to nanoseconds), while the chemical exchange term, R ex , identifies processes slower than the overall tumbling correlation time (micro- to milliseconds). Advantages and disadvantages of different methods of data handling are thoroughly discussed.

BacSp222 is a multifunctional bacteriocin produced by Staphylococcus pseudintermedius strain 222, an opportunistic pathogen of domestic animals. At micromolar concentrations, BacSp222 kills Gram-positive bacteria and is cytotoxic toward mammalian cells, while at nanomolar doses, it acts as an immunomodulatory factor, enhancing nitric oxide release in macrophage-like cell lines. The bacteriocin is a cationic, N-terminally formylated, 50-amino-acid-long linear peptide that is rich in tryptophan residues. In this study, the solution structure of BacSp222 was determined and compared to the currently known structures of similar bacteriocins. BacSp222 was isolated from a liquid culture medium in a uniformly 13C- and 15N-labeled form, and NMR data were collected. The structure was calculated based on NMR-derived constraints and consists of a rigid and tightly packed globular bundle of four alpha-helices separated by three short turns. Although the amino acid sequence of BacSp222 has no significant similarity to any known peptide or protein, a 3D structure similarity search indicates a close relation to other four-helix bundle-motif bacteriocins, such as aureocin A53, lacticin Q and enterocins 7A/7B. Assuming similar functions, biology, structure and physicochemical properties, we propose to distinguish the four-helix bundle bacteriocins as a new Type A in subclass IId of bacteriocins, containing linear, non-pediocin-like peptides.

Gibbs energies of complex formation between enantiomers of bicyclic terpenoid, fenchone, and naturally occurring cyclodextrins, βCD and γCD, were determined by means of 13C and 1H nuclear magnetic resonance (NMR) titration data. These results were compared with the corresponding data obtained previously for the diastereomeric fenchone/αCD complexes. The size of the inner cavity of host molecules significantly influences stoichiometry, association constants, and enantiomeric differentiation of the studied complexes. These complementary data allow us to discuss qualitatively the influence of the host size on the guest–host interactions. A method of the simultaneous use of titration data collected for several resonances of different isotopes in the determination of association constants was worked out and thoroughly analyzed. Comparison of the results of global data analyses with weighted means of individual ones revealed that both these approaches are equally trustworthy.

This article describes new deltorphin I analogs in which phenylalanine residues were replaced by the corresponding (R) or (S)‐α‐benzyl‐β‐azidoalanine, α‐benzyl‐β‐(1‐pyrrolidinyl)alanine, α‐benzyl‐β‐(1‐piperidinyl)alanine, and α‐benzyl‐β‐(4‐morpholinyl)‐alanine residues. The potency and selectivity of the new analogs were evaluated by a competitive receptor binding assay in the rat brain using [3H]DAMGO (a μ ligand) and [3H]DELT (a δ ligand). The affinity of analogs containing (R) or (S)‐α‐benzyl‐β‐azidoalanine in position 3 to δ‐receptors strongly depended on the chirality of the α,α‐disubstituted residue. The conformational behavior of peptides modified with (R) or (S)‐α‐benzyl‐β‐(1‐piperidinyl)Ala, which displays the opposite selectivity, was analyzed by 1H and 13C NMR. The μ‐selective Tyr‐d‐Ala‐(R)‐α‐benzyl‐β‐(1‐piperidinyl)Ala‐Asp‐Val‐Val‐Gly‐NH2 lacks the helical conformation observed in the δ‐selective Tyr‐d‐Ala‐(S)‐α‐benzyl‐β‐(1‐piperidinyl)Ala‐Asp‐Val‐Val‐Gly‐NH2. Our results support the proposal that differences between δ‐ and μ‐selective opioid peptides are attributable to the presence or absence of a spatial overlap between the N‐terminal message domain and the C‐terminal address domain.

The synthesis of new dermorphin analogues is described. The (R)‐alanine or phenylalanine residues of natural dermorphin were substituted by the corresponding α‐methyl‐β‐azidoalanine or α‐benzyl‐β‐azido(1‐piperidinyl)alanine residues. The potency and selectivity of the new analogues were evaluated by a competitive receptor binding assay in rat brain using [3H]DAMGO (a μ ligand) and [3H]DELT (a δ ligand). The most active analogue in this series, Tyr‐(R)‐Ala‐(R)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH2 and its epimer were analysed by 1H and 13C NMR spectroscopy and restrained molecular dynamics simulations. The dominant conformation of the investigated peptides depended on the absolute configuration around Cα in the α‐benzyl‐β‐azidoAla residue in position 3. The (R) configuration led to the formation of a type I β‐turn, whilst switching to the (S) configuration gave rise to an inverse β‐turn of type I′, followed by the formation of a very short β‐sheet. The selectivity of Tyr‐(R)‐Ala‐(R) and (S)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH2 was shown to be very similar; nevertheless, the two analogues exhibited different conformational preferences. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed analysis of the structural and thermodynamic effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small β-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation constant of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is critical for the tight association of protein subunits.

Nuclear magnetic relaxation provides a powerful method giving insight into molecular motions at atomic resolution on a broad time scale. Dynamics of biological macromolecules has been widely exploited by measuring N-15 and C-13 relaxation data. Interpretation of these data relies almost exclusively on the use of the model-free approach (MFA) and its extended version (EMFA) which requires no particular physical model of motion and a small number of parameters. It is shown that EMFA is often unable to cope with three different time scales and fails to describe slow internal motions properly. In contrast to EMFA, genuine MFA with two time scales can reproduce internal motions slower than the overall tumbling. It is also shown that MFA and simplified EMFA are equivalent with respect to the values of the N-H bond length and chemical shift anisotropy. Therefore, the vast majority of N-15 relaxation data for proteins can be satisfactorily interpreted solely with MFA.

Anika Lasota, Oliwia Frączak, Adriana Muchowska, Aleksandra Misicka, Michal Nowakowski, Maciej Maciejczyk, Andrzej Ejchart , and Aleksandra Olma Institute of Organic Chemistry, Lodz University of Technology, Zeromskiego, 116,90-924, Lodz; Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawinskiego 5, 01-793, Warsaw; Centre of New Technologies, University of Warsaw, Zwirki i Wigury 93, 02-089, Warsaw; Chair of Physics and Biophysics, University of Warmia and Mazury ul. Oczapowskiego 4, 10-719, Olsztyn; Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106, Warsaw, Poland

New analogues of deltorphin I (DT I, Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 ), with the D-Ala residue in position 2 replaced by α-methyl-β-azido(amino, 1-pyrrolidinyl, 1-piperidinyl or 4-morpholinyl)alanine, were synthesized by a combination of solid-phase and solution methods. All ten new analogues were tested for receptor affinity and selectivity to μ- and δ-opioid receptors. The affinity of analogues containing (R) or (S)-α-methyl-β-azidoalanine in position 2 to δ-receptors strongly depended on the chirality of the α,α-disubstituted residue. Peptide II, containing (S)-α-methyl-β-azidoalanine in position 2, displayed excellent δ-receptor selectivity with its δ-receptor affinity being only three times lower than that of DT I.

The transient folding of domain 4 of an RNA polymerase subunit () induced by an increasing concentration of 2,2,2-trifluoroethanol (TFE) in an aqueous solution was monitored by means of CD and heteronuclear NMR spectroscopy. NMR data, collected at a 30 % TFE, allowed the estimation of the population of a locally folded structure (CSI descriptors) and of local backbone dynamics (N relaxation). The spontaneous organization of the helical regions of the initially unfolded protein into a TFE-induced 3D structure was revealed from structural constraints deduced from N- to C-edited NOESY spectra. In accordance with all the applied criteria, three highly populated α-helical regions, separated by much more flexible fragments, form a transient HLHTH motif resembling those found in PDB structures resolved for homologous proteins. All the data taken together demonstrate that TFE induces a transient native-like structure in the intrinsically disordered protein.

The transient folding of domain 4 of an E. coli RNA polymerase σ⁷⁰ subunit (rECσ₄⁷⁰) induced by an increasing concentration of 2,2,2-trifluoroethanol (TFE) in an aqueous solution was monitored by means of CD and heteronuclear NMR spectroscopy. NMR data, collected at a 30% TFE, allowed the estimation of the population of a locally folded rECσ₄⁷⁰ structure (CSI descriptors) and of local backbone dynamics ((15)N relaxation). The spontaneous organization of the helical regions of the initially unfolded protein into a TFE-induced 3D structure was revealed from structural constraints deduced from (15)N- to (13)C-edited NOESY spectra. In accordance with all the applied criteria, three highly populated α-helical regions, separated by much more flexible fragments, form a transient HLHTH motif resembling those found in PDB structures resolved for homologous proteins. All the data taken together demonstrate that TFE induces a transient native-like structure in the intrinsically disordered protein.