The solution behavior of poly-L-proline: I. Light scattering studies in water and concentrated aqueous neutral salt solutions

The solution bebavior of poly-L -proline Form II has been studied in water and aqueous salt solutions by both elastic and quasi-elastic light -scattering techniques. The results of this study suggest that polyproline Form II can exist in water at 24 °C as an associated polymer complex and that certain salts which do not appear to affect the helix integrity, e.g., guanidinium-HCl, resutl in dissociation of the aggregate. Other neutral salts, of the variety effective in mediating unfolding of the Form II helix (e.g., 4M NaClO₄) also induce aggregate dissociation, but 4M CaCl₂ results in enhanced aggregation of polyproline. Kinetic experiments indicate that a time of 20 hours is necessary for the completion of the “large” to “small” transformation (at 22°C) which is induced by the addition of 4M NaClO₄. Thus it appears that neutral salts additives in aqueous solutions of polyproline influence both the state of aggregation and the conformation of this polymer.     

Two-dimensional NMR studies and secondary structure of cobrotoxin in aqueous solution

The 1H-NMR spectra of cobrotoxin, a neurotoxic protein isolated from Formosan cobra Naja naja atra, have been studied by two-dimensional NMR techniques. Of 62 amino acid residues in cobrotoxin, the complete assignments of 58 residues have been made. The resonances from several of the remaining residues have been identified but not yet specifically assigned. The secondary structure of an antiparallel triple- and double-stranded beta-sheet has also been determined by observing the NOE.     

Two dimensional NMR studies on the solution structure of d-CTCGAGCTCGAG

Two dimensional (2D) FT-NMR investigations have been carried out on the self-complementary dodecanucleotide d-CTCGAGCTCGAG, which has cleavage sites for the restriction enzyme Xho I (between C and T). The central TCG portion is also known to show a preference for DNAase activity. Complete resonance assignments have been obtained for the non-exchangeable sugar and base protons of the oligonucleotide. Information regarding sugar geometries, glycosidic torsion angles and other structural parameters has been obtained from the relative intensities of the cross peaks in the COSY and NOESY spectra. The results indicate that deoxyribose rings of C1 and C7 adopt a conformation different from the remaining sugars in the double helical oligonucleotide. The central TCG portion also exhibits variations in the backbone structure. The base stacking in the double helix shows interesting sequence dependent effects suggesting that the sequence effects are not localised to nearest neighbours but extended over longer stretches.     

Proton NMR studies of angiotensin II and its analogs in aqueous solution

The¹H nuclear magnetic resonance (NMR) spectra of angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) and five of its octapeptide analogs as well as angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) and angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Phe) in aqueous solutions (90% H₂O/10% D₂O) were completely assigned by two-dimensional COSY and ROESY experiments. All of the peptides give rise to two distinct sets of signals. The minor set accounts for about 5% of the total population belowpH 5.5 and increases to 12–20% aroundpH 7.0. The two sets of signals result from acis-trans isomerization of the His-Pro peptide bond with the major resonances arising from thetrans isomer. One analog in which the Pro is replaced with a D-Pro displays a very different isomerization behavior. The measured coupling constants J_NH-CH, the temperature dependence of the amide proton shifts and the relative intensities of the intraresidue and sequential NH-CH ROEs, are all indicative of an extended backbone conformation for ANGII. However, some evidence for the existence of conformers with local structure involving preferred sidechain positions for the Tyr, His, Phe, and the carboxyl group of the Phe was found, particularly in the ROESY andpH-titration experiments. Moreover,pH effects and the unusual amide exchange behavior of the Arg NH suggests the presence of interactions between the Asp and Arg sidechains of ANGII. At low temperatures the Arg guanidinium NH₂ protons were detected as two broad peaks which are related by sizeable exchange peaks in ROESY experiments. This behavior could be useful as a general probe for the study of Arg sidechain mobility and accessibility in other peptides and proteinsPreliminary results of this work have been presented at the XIIth International Conference on Magnetic Resonance in Biological Systems in abstract form (1988).     

Expression and purification of CB2 for NMR studies in micellar solution

We demonstrate feasibility of biophysical characterization of the peripheral cannabinoid receptor CB2 produced by heterologous expression in E. coli membranes. Recombinant receptor was purified by affinity chromatography, and NMR diffusion experiments performed on CB2 solubilized in dodecylphosphocholine (DPC) micelles. Circular dichroism spectroscopy indicated high alpha-helical content (49 %) of CB2.     

NMR studies of L-dopa melanin-manganese(II) complex in water solution

The study of water relaxation rates of solutions of melanins with paramagnetic metal ions provides a powerful tool for investigating the binding sites and for obtaining useful structural and dynamic information. The measure of 1H- and 2H-longitudinal and transverse relaxation rates at a single, high-magnetic field for H2O/D2O (80:20) solutions allows the determination of tau c, tau R, tau e, tau M, r, q, and Z (the outer sphere contribution to the overall relaxation rate) for Mn(II)-L-Dopa melanin system.     

On choosing a detergent for solution NMR studies of membrane proteins

Translational diffusion coefficients and catalytic activities were measured for the integral membrane protein diacylglycerol kinase (DAGK) in a variety of types of detergent micelles. Despite the structural diversity of the detergents examined, the translational diffusion coefficients observed for DAGK spanned a fairly limited range of values: 2.7 to 4.7 (× 10^-7cm²/s). No general correlation was observed between the diffusion coefficients for the detergent-DAGK aggregates and the sizes of the corresponding protein-free micelles. These results indicate that the effective molecular weights of the DAGK-detergent aggregates were determined more by the structural properties of the protein than by the properties of the detergents. The catalytic activity of DAGK in detergents having medium-length alkyl chains such as dodecylphosphocholine or decylmaltoside was usually observed to be substantially higher than in short-chain detergents such as octylphosphocholine or octylglucoside. Taken together, the diffusion and activity results indicate that medium-chain detergents are generally preferred for use in NMR studies of complex membrane proteins because they are no worse than short-chained detergents in terms of increasing the effective molecular weight of the protein of interest while they are considerably better at maintaining native-like protein conformation. Among the 10 detergents examined, only sodium dodecylsulfate was observed to be unable to support DAGK activity under any conditions examined, suggesting that this well-known protein denaturant should be used with care in studies of complex membrane proteins.     

Theoretical optical properties of poly-L-proline

A non-perturbational technique is used to calculate the circular dichroism and absorption spectra of polypeptide chains having conformations similar to that of poly-L -proline II. The method employs a Bogoliubov exciton formalism, from which the various optical terms associated with parallel and perpendicular components of the exciton band are obtained. A simple model for the peptide unit, consisting of three Gaussian absorption bands, leads to reasonable results for the polymer spectra, provided the lowest energy peptide π → π* transition is taken at 207 mμ and the value of the Ramachandran angle Ψ is taken to be 390°. The calculations suggest that the polymer circular dichroism spectrum is the resultant of strong interference among the two Gaussian exciton terms and the non-Gaussian helix term. Consequently, the CD spectrum is very sensitive to the value of Ψ. It is found that the small positive CD band in the vicinity of 230 mμ arises partly from the effect of the static (crystal) field interactions on the n → π* CD band.     

NMR studies of lantibiotics. The structure of nisin in aqueous solution.

Nisin is a posttranslationally modified protein of 34 amino acids, and is a member of the class of bacteriocidal polypeptides known as lantibiotics, that contain the unusual amino acid lanthionine. Its structure in aqueous solution has been determined on the basis of NMR data, i.e. interproton distance constraints derived from nuclear Overhauser enhancement spectroscopy and torsion angle constraints derived from double-quantum-filtered correlated spectroscopy. Translation of the NMR constraints into a three-dimensional structure was carried out with the distance-geometry program DISMAN, followed by restrained energy minimization using CHARMm. The internal mobility of the peptide chain prohibited the determination of a precise overall folding of the molecule, but parts of the structure could be obtained, albeit sometimes with low resolution. The structure of nisin can best be defined as follows. The outermost N-terminal and C-terminal regions of nisin appear quite flexible, the remainder of the molecule consists of an amphiphilic N-terminal fragment (residues 3-19), joined by a flexible 'hinge' region to a rigid double-ring fragment formed by residues 23-28. The latter fragment has the appearance of a somewhat overwound alpha-helix. It is suggested, by assuming the presence of a (transient) alpha-helical structure in this part of prenisin, that the coupling between residues 23 and 26, as well as between 25 and 28, by thioether bridges, and the inversion of the C alpha chiralities at positions 23 and 25, can be rationalized.     

Structural flexibility and fast proton transfer reflected by the dielectric properties of poly-L-proline in aqueous solution

Dielectric dispersion curves for the helix-11 form of poly-L-proline in aqueous solution have been determined for various pH in the acid range of zwitterion formation. The results could be excellently described by means of a Cole-Cole dispersion function involving the three parameters Δ?⁰ (total dielectric increment), τ_r (effective rotational relaxation time) and h (characterizing the width of the dispersion region). The quantities τ_r, and h were found to be clearly independent of pH and added inert electrolyte. An analysis of the data permits an evaluation of the dipole moments and leads to the conclusion that the molecule cannot be considered to be a completely stretched rigid rod but must be more or less bent. Addition of formic acid slightly below pH 4 caused a distinct broadening of the experimental curves which could be quantitatively interpreted by a second dielectric relaxation process due to orientation of zwitterions by means of fast proton transfer.     

Fluorescent probe and NMR studies of the aggregation of bile salts in aqueous solution

The binding of the fluorescent probes 1-anilino-8-naphthalene sulfonate and dansyl cadaverine to the sodium salts of cholic, deoxycholic and dehydrocholic acids has been investigated. Enhanced probe solubilisation accompanies aggregation. Monitoring of fluorescence intensities as a function of bile salt concentration permits the detection of primary micelle formation, as well as secondary association. The transition concentrations obtained by fluorescence are in good agreement with values determined for the critical micelle concentrations, by other methods. Differences in the behaviour of cholate and deoxycholate have been noted. Fluorescence polarisation studies of 1,6-diphenyl-1,3,5-hexatriene solubilised in bile salt micelles suggest a higher microviscosity for the interior of the deoxycholate micelle as compared to cholate. ¹H NMR studies of deoxycholate over the range 1–100 mg/ml suggest that micelle formation leads to a greater immobilisation of the C₁₈ and C₁₉ methyl groups as compared to the C₂₁ methyl group. Well resolved ¹³C resonances are observed for all three steroids even at high concentration. Both fluorescence and NMR studies confirm that dehydrocholate does not aggregate.     

NMR studies on solution structure of single-stranded oligonucleotides causing line broadening.

Unusual line broadening of 1H-NMR lines attributable to the proton (8H) of guanine residues was observed for all tetradeoxyoligonucleotides tested here which have a specific base sequence of dGXXG (X = A or T). For the same samples, line broadening was also obtained in the 31P-NMR spectra. These broadened signals did not become sharp up to 60 degrees C. This unusual spectral phenomenon has been attributed by 2D-NMR and differential NOE to the compact solution structure of the oligonucleotides.     

Conformation of secretin in dimethyl sulfoxide solution. NMR studies and restrained molecular dynamics

The solution conformation of the 27-residue polypeptide hormone secretin in dimethyl sulfoxide has been determined on the basis of 1H-NMR measurements. The experimental data set used in the structure determination consisted of 98 nuclear-Overhauser-enhancement-derived interproton and dihedral angle restraints from coupling constants. The NH-NH and H alpha-NH NOEs were determined from build-up rates, while the remaining distances were classified in a qualitative manner. The structure calculations consisted of two phases. First, dynamical simulated annealing calculations were carried out to find conformations of the peptide which satisfy NOE and phi dihedral restraints. The convergence of ten calculated structures was good except for those regions of the molecule where NOE data were not unambiguous. From the calculated set another initial structure was built which was again minimized in several 5-ps calculations now employing the full empirical energy function. The resulting structures of secretin reveal conformationally well-defined regions, but not a single uniform secondary structure. The structure is different from the calculated structure from trifluoroethanol/water measurements.     

NMR studies of the solution conformation of the sex peptide from Drosophila melanogaster

The insect sex peptide (SP) elicits a variety of biological responses upon transfer to the mated female. SP contains 36 amino acids, including a tryptophan-rich N-terminal region, a central region containing five hydroxyproline (Hyp) residues, and a C-terminal region enclosed by a disulfide bridge. The solution structure of SP, studied here using NMR spectroscopy, includes a motif WPWN that adopts a type I β-turn in the N-terminal Trp-rich region. This turn region is connected to the central Hyp-rich region, which adopts extended and/or PPII-like conformations. The C-terminal disulfide-bonded loop populates helical turns or nascent helical structure. Overall, the results reveal a rather flexible peptide that lacks a compact folded structure in solution.