Nuclear Overhauser Enhancement Spectroscopy Experiments Research Articles

The interactions of neuropeptides with membrane model systems: a case study.

The interactions between the positively charged neuropeptides substance P (SP), bradykinin (BK), and zwitterionic Met-enkephalin (ME) neuropeptides, and negatively charged SDS and zwitterionic lysophosphatidylcholine (LPC) membrane model systems, have been investigated using one- and two-dimensional nmr experiments. Proton longitudinal relaxation studies were used to characterize these interactions as intrinsic or extrinsic. An extrinsic interaction are similar to those observed for extrinsic membrane proteins. An intrinsic interaction are similar to those observed for intrinsic membrane proteins, and would require that the hydrophobic residues penetrate or insert into the hydrophobic core of the membrane. The interactions between both SP and BK and SDS, based on nmr results, may be characterized as intrinsic, and the interaction between ME and SDS may be characterized as extrinsic. Two-dimensional nuclear Overhauser enhancement spectroscopy experiments proved the insertion of the phenylalanine residues on both SP and BK into the hydrophobic core of SDS micelles. The interaction between SP and BK with LPC based on nmr results are characterized as extrinsic, with the interaction between ME and SDS characterized as weakly intrinsic.

1H‐ and natural abundance 15N‐nmr studies of a derivative of a rabies glycoprotein fragment

Using a combination of one- and two-dimensional methods, 1H- and 15N-nmr spectroscopy has been employed to perform the complete assignment and the structural determination of the immunogenic undecapeptide CTTTNSRGTTT in DMSO solution. Nuclear Overhauser enhancement spectroscopy experiments indicated the presence of secondary structures, mainly turn-like structures, which only represent a family, albeit a dominant one, of an ensemble of conformations available to the peptide. Since reverse turns may play an important role as intermediates in protein folding, the experimental observations described here may link the immunological and theoretical approaches to protein folding.

The solution structure of the lantibiotic gallidermin

The 21-peptide amide antibiotic gallidermin is a potential therapeutic against acne disease. It belongs to the class of polycyclic lanthionine and alpha,beta-didehydroamino acids containing polypeptides, which were named "lantibiotics." The structural gene of the recently elucidated lantibiotic gallidermin encodes a precursor peptide containing Ser, Thr, and Cys residues in the C-terminal prolantibiotic part, and an unusually hydrophilic leader peptide. The ribosomally synthesized pregallidermin is posttranslationally modified and processed to a complex peptide antibiotic with four sulfide rings and two unsaturated residues. The complete solution structure of gallidermin was determined in trifluoroethanol: water (95:5) and dimethylsulfoxide by two-dimensional 1H-nmr at 500 MHz, using a combination of double quantum filtered correlated spectroscopy, homonuclear Hartman-Hahn, and nuclear Overhauser enhancement spectroscopy experiments. Using a total number of 152 distance constraints from NOEs and 14 torsional constraints, derived from coupling constants, we obtained a screwlike solution structure of gallidermin. Restrained molecular dynamics simulations yielded a set of five converging structures with an atomic rms difference of 1.7 A for the backbone atoms, not dependent on the starting structure. The spatial structure model is in excellent agreement with the amphiphilic and channel-forming properties of gallidermin on membranes and its tryptic cleavage at the exposed site between residues 13 and 14.

Double-quantum NOESY. Coupled coherent and incoherent transfer of magnetization

The application of 2D Ff NMR to the structural analysis of biological macromolecules has revolutionized the field. On the one hand networks of scalar coupled spins in complicated molecules such as proteins and nucleic acids can be analyzed by making use of coherent magnetization transfer in two-dimensional correlated spectroscopy (COSY) (l-4), on the other hand the proximity of specific nuclei in such molecules can be investigated by detecting incoherent magnetization transfer by means of nuclear Overhauser enhancement spectroscopy (NOESY) (5-7). During the last five years the number of techniques using coherent magnetization transfer has grown substantially: examples are relayed coherence spectroscopy and doublequantum spectroscopy (8-13). The latter techniques were shown to be a prerequisite in the unraveling of networks of scalar coupled spins which, due to ambiguities arising from extensive overlap, could not be resolved within COSY or SECSY (14-16). The process of incoherent transfer of magnetization, taking place via dipolar cross relaxation, is fundamentally different from coherent magnetization transfer which occurs via the J coupling between spins. In methods developed so far, the two phenomena have been employed strictly independently from one another; this is true even in the so-called COCONOSY experiment (I 7). However, there is no reason why a 2D FT NMR experiment should be based solely on either coherent or incoherent magnetization transfer (18). In this paper an experiment is described which couples coherent and incoherent transfer. It will be shown that such an experiment can be used as an aid in resolving ambiguities that may arise in NOESY spectra. To illustrate the basic principles of this new experiment, which we call doublequantum NOESY (DQ NOESY), we shall compare it with the conventional NOESY experiment (see Table 1). Both experiments can be divided in six periods and both experiments start by creating coherences which are frequency labeled during the evolution period I,. In a conventional NOESY experiment a 7r/2 pulse is used to create in-phase single-quantum coherence (i.e., transverse magnetization (5)). In DQ NOESY the preparation sequence of double-quantum spectroscopy (II, 19, 20), namely r/2-r-r-r--1r/2 is used, which gives rise to double-quantum coherences that are frequency labeled during the evolution period t, . After the evolution period,