NMR studies of inclusion complexes: naphthalene and natural cyclodextrins.

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 ßCD build coexisting 2 : 1 and 2 : 2 complexes with large binding constants. Larger γCD host molecules form essentially similar complexes with NP as the ßCD but corresponding binding constants are smaller.

Dynamic 15N{1H} NOE measurements: a tool for studying protein dynamics.

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.

Shape adaptation of quinine in cyclodextrin cavities: NMR studies.

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 ßCD. 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 ßCD 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.

Fast evaluation of protein dynamics from deficient 15N relaxation data.

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.

Size makes a difference: Chiral recognition in complexes of fenchone with cyclodextrins studied by means of NMR titration.

Gibbs energies of complex formation between enantiomers of bicyclic terpenoid, fenchone, and naturally occurring cyclodextrins, ßCD and γCD, were determined by means of 13 C and 1 H 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.

The impact of ß-azido(or 1-piperidinyl)methylamino acids in position 2 or 3 on biological activity and conformation of dermorphin analogues.

The synthesis of new dermorphin analogues is described. The (R)-alanine or phenylalanine residues of natural dermorphin were substituted by the corresponding α-methyl-ß-azidoalanine or α-benzyl-ß-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 [(3) H]DAMGO (a µ ligand) and [(3) H]DELT (a δ ligand). The most active analogue in this series, Tyr-(R)-Ala-(R)-α-benzyl-ß-azidoAla-Gly-Tyr-Pro-Ser-NH2 and its epimer were analysed by (1) H and (13) C NMR spectroscopy and restrained molecular dynamics simulations. The dominant conformation of the investigated peptides depended on the absolute configuration around C(α) in the α-benzyl-ß-azidoAla residue in position 3. The (R) configuration led to the formation of a type I ß-turn, whilst switching to the (S) configuration gave rise to an inverse ß-turn of type I', followed by the formation of a very short ß-sheet. The selectivity of Tyr-(R)-Ala-(R) and (S)-α-benzyl-ß-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.

Analogues of deltorphin I containing conformationally restricted amino acids in position 2: structure and opioid activity.

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-ß-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-ß-azidoalanine in position 2 to δ-receptors strongly depended on the chirality of the α,α-disubstituted residue. Peptide II, containing (S)-α-methyl-ß-azidoalanine in position 2, displayed excellent δ-receptor selectivity with its δ-receptor affinity being only three times lower than that of DT I.

NMR structural studies of the first catalytic half-domain of ubiquitin activating enzyme.

We report a high resolution NMR structure and (15)N relaxation studies of the first catalytic cysteine half-domain (FCCH) of the mouse ubiquitin-activating enzyme E1, together with interaction studies of FCCH and the other catalytic E1 subdomain - SCCH (second catalytic cysteine half-domain). In solution, mouse FCCH forms a well-defined six-stranded antiparallel ß-barrel structure, a common fold for many proteins with a variety of cellular functions. (15)N relaxation data reveal FCCH complex backbone dynamics and indicate which residues experience slow intramolecular motions. Some of these residues make contacts with the polar face of ubiquitin in the co-crystal structure of yeast E1 and ubiquitin. However, the titration of FCCH with ubiquitin does not show any visible chemical shift changes in the 2D (1)H/(15)N HSQC spectra of the FCCH. The 2D (1)H/(15)N HSQC experiments performed both for each catalytic half-domain individually and for their equimolar mixture in the milimolar concentration range display no detectable chemical shift perturbation, suggesting a lack of interaction between the two subdomains unless they are covalently linked via the adenylation domain.

The TFE-induced transient native-like structure of the intrinsically disordered σ47° domain of Escherichia coli RNA polymerase.

The transient folding of domain 4 of an E. coli RNA polymerase σ7° subunit (rECσ47°) 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 rECσ47° 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.

Complex formation of fenchone with α-cyclodextrin: NMR titrations.

13C NMR titration studies of inclusion complexes of bicyclic terpenoid, fenchone enantiomers with α-cyclodextrin revealed their 1:2 guest-host stoichiometry. Sequential binding constants were determined indicating a strong binding cooperativity of two α-cyclodextrin to fenchone. The overall association constants were used to calculate the Gibbs free energies of diastereomeric complex formation, which might be used as a measure of chiral recognition of fenchone by α-cyclodextrin. These results were compared with corresponding data derived for camphor, which is an isomeric bicyclic terpenoid.

Impact of calcium binding and thionylation of S100A1 protein on its nuclear magnetic resonance-derived structure and backbone dynamics.

S100 proteins play a crucial role in multiple important biological processes in vertebrate organisms acting predominantly as calcium signal transmitters. S100A1 is a typical representative of this family of proteins. After four Ca(2+) ions bind, it undergoes a dramatic conformational change, resulting in exposure, in each of its two identical subunits, a large hydrophobic cleft that binds to target proteins. It has been shown that abnormal expression of S100A1 is strongly correlated with a number of severe human diseases: cardiomyopathy and neurodegenerative disorders. A few years ago, we found that thionylation of Cys 85, the unique cysteine in two identical S100A1 subunits, leads to a drastic increase of the affinity of the protein for calcium. We postulated that the protein activated by thionylation becomes a more efficient calcium signal transmitter. Therefore, we decided to undertake, using nuclear magnetic resonance methods, a comparative study of the structure and dynamics of native and thionylated human S100A1 in its apo and holo states. In this paper, we present the results obtained for both forms of this protein in its holo state and compare them with the previously published structure of native apo-S100. The main conclusion that we draw from these results is that the increased calcium binding affinity of S100A1 upon thionylation arises, most probably, from rearrangement of the hydrophobic core in its apo form.

Selective diagonal-free (13)C, (13)C-edited aliphatic-aromatic NOESY experiment with non-uniform sampling.

A band-selective aromatic-aliphatic C,C-edited four-dimensional NOESY experiment is proposed here. Its key advantage is the absence of auto-correlation signals which makes it very attractive for joint use with non-uniform sampling. It is demonstrated here that the sensitivity of the experiment is not significantly affected by utilization of selective pulses (for either aromatic-13C or aliphatic-13C spins). The method was applied to the sample of E32Q mutant of human S100A1 protein, a homodimer of total molecular mass ~20 kDa. High-resolution 4D spectra were obtained from ~1.5 % of sampling points required conventionally. It is shown that superior resolution facilitates unambiguous assignment of observed aliphatic-aromatic cross-peaks. Additionally, the addition of aliphatic-13C dimension enables to resolve peaks with degenerated aliphatic (1)H chemical shifts. All observed cross-peaks were validated against previously determined 3D structure of E32Q mutant of S100A1 protein (PDB 2LHL). The increased reliability of structural constraints obtained from the proposed high-resolution 4D 13C(ali),13C(aro)-edited NOESY can be exploited in the automated protocols of structure determination of proteins.

Structure and dynamics of the first archaeal parvulin reveal a new functionally important loop in parvulin-type prolyl isomerases.

Parvulins are a group of peptidyl-prolyl isomerases (PPIases) responsible for important biological processes in all kingdoms of life. The PinA protein from the psychrophilic archaeon Cenarchaeum symbiosum is a parvulin-like PPIase. Due to its striking similarity to the human parvulins Pin1 and Par14, PinA constitutes an interesting subject for structural and functional studies. Here, we present the first high resolution NMR structure of an archaeal parvulin, PinA, based on 1798 conformational restraints. Structure calculation yields an ensemble of 20 convergent low energy structures with a backbone r.m.s.d. value of 0.6 Å within the secondary structure elements. The overall fold of PinA comprises the ß-α(3)-ß-α-ß(2) fold typical for all parvulin structures known so far, but with helix III being a short 3(10)-helix. A detailed comparison of this high resolution structure of the first archaeal PinA protein with bacterial and eukaryotic parvulin PPIase structures reveals an atypically large catalytic binding site. This feature provides an explanation for cold-adapted protein function. Moreover, the residues in and around 3(10)-helix III exhibit strong intramolecular dynamics on a microsecond to millisecond timescale and display structural heterogeneity within the NMR ensemble. A putative peptide ligand was found for PinA by phage display and was used for (1)H-(15)N-HSQC titrations. Again, the flexible region around 3(10)-helix III as well as residues of the peptide binding pocket showed the strongest chemical shift perturbations upon peptide binding. The local flexibility of this region also was modulated by ligand binding. A glycine and two positively charged residues are conserved in most parvulin proteins in this flexible loop region, which may be of general functional importance for parvulin-type PPIases.

Solution NMR structure and dynamics of human apo-S100A1 protein.

S100A1 belongs to the EF-hand superfamily of calcium binding proteins. It is a representative of the S100 protein family based on amino acid sequence, three-dimensional structure, and biological function as a calcium signal transmitter. It is a homodimer of noncovalently bound subunits. S100A1, like most of other members of the S100 protein family, is a multifunctional, regulatory protein involved in a large variety of biological processes and closely associated with several human diseases. The three-dimensional structure of human apo-(i.e. calcium free)-S100A1 protein was determined by NMR spectroscopy (PDB 2L0P) and its backbone dynamics established by ¹5N magnetic relaxation. Comparison of these results with the structure and backbone dynamics previously determined for bovine apo-S100A1 protein modified by disulfide formation with ß-mercaptoethanol at Cys 85 revealed that the secondary structure of both these proteins was almost identical, whereas the global structure of the latter was much more mobile than that of human apo-S100 protein. Differences between the structures of human and rat apo-S100A1 are also discussed.

Synthesis and receptor binding of opioid peptide analogues containing beta3-homo-amino acids.

beta-Amino acids containing hybrid peptides and beta-peptides show great potential as peptidomimetics. In this paper we describe the synthesis and affinity toward the micro- and delta-opioid receptors of beta-peptides, analogues of Leu-enkephalin, deltorphin I, dermorphin and alpha,beta-hybrides, analogues of deltorphin I. Substitution of alpha-amino acid residues with beta(3)-homo-amino acid residues, in general resulted in decrease of affinity to opioid receptors. However, the incorporation beta(3)h-D-Ala in position 2 or beta(3)hPhe in position 3 of deltorphin I resulted in potent and selective ligand for delta-opioid receptor. The NMR studies of beta-deltorphin I analogue suggest that conformational motions in the central part of the peptide backbone are partially restricted and some conformational preferences can be expected.

Spatial attributes of the four-helix bundle group of bacteriocins - The high-resolution structure of BacSp222 in solution.

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.

15N magnetic relaxation study of backbone dynamics of the ribosome-associated cold shock response protein Yfia of Escherichia coli.

In the solution structure of the ribosome-associated cold shock response protein Yfia of Escherichia coli in the free state two structural segments can be distinguished: a well structured, rigid N-terminal part displaying a betaalphabetabetabetaalpha topology and a flexible C-terminal tail comprising last 20 amino-acid residues. The backbone dynamics of Yfia protein was studied by (15)N nuclear magnetic relaxation at three magnetic fields and analyzed using model-free approach. The overall diffusional tumbling of the N-terminal part is strongly anisotropic with a number of short stretches showing increased mobility either on a subnanosecond time scale, or a micro- to millisecond time scale, or both. In contrast, the unstructured polypeptide chain of the C-terminal part, which cannot be regarded as a rigid structure, shows the predominance of fast local motions over slower ones, both becoming faster closer to the C-terminus.

Determination of association constants at moderately fast chemical exchange: complexation of camphor enantiomers by alpha-cyclodextrin.

Association constants in weak molecular complexes can be determined by analysis of chemical shifts variations resulting from changes of guest to host concentration ratio. In the regime of very fast exchange, i.e., when exchange rate is several orders of magnitude larger than the Larmor angular frequency difference of the observed resonance in free and complexed molecule, the apparent position of averaged resonance is a population-weighted mean of resonances of particular forms involved in the equilibrium. The assumption of very fast exchange is often, however, tacitly admitted in literature even in cases where the process of interest is much slower than required. We show that such an unjustified simplification may, under certain circumstances, lead to significant underestimation of association constant and, in consequence, to non-negligible errors in Gibbs free energy under determination. We present a general method, based on iterative numerical NMR line shape analysis, which allows one for the compensation of chemical exchange effects, and delivers both the correct association constants and the exchange rates. The latter are not delivered by the other mentioned method. Practical application of our algorithm is illustrated by the case of camphor-alpha-cyclodextrin complexes.

Synthesis, Biological Activity, and NMR-Based Structural Studies of Deltorphin I Analogs Modified in Message Domain with a New α,α-Disubstituted Glycines.

This article describes new deltorphin I analogs in which phenylalanine residues were replaced by the corresponding (R) or (S)-α-benzyl-ß-azidoalanine, α-benzyl-ß-(1-pyrrolidinyl)alanine, α-benzyl-ß-(1-piperidinyl)alanine, and α-benzyl-ß-(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 [(3) H]DAMGO (a µ ligand) and [(3) H]DELT (a δ ligand). The affinity of analogs containing (R) or (S)-α-benzyl-ß-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-ß-(1-piperidinyl)Ala, which displays the opposite selectivity, was analyzed by (1) H and (13) C NMR. The µ-selective Tyr-d-Ala-(R)-α-benzyl-ß-(1-piperidinyl)Ala-Asp-Val-Val-Gly-NH2 lacks the helical conformation observed in the δ-selective Tyr-d-Ala-(S)-α-benzyl-ß-(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.

Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly.

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 ß-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.