Peptides relaxation measurements

Nicolau, Cl., Dreeskamp, H., and Schultc-Frohlinde, D. (1974). FEBS Lett. 43, 148. I3C Nuclear Magnetic Resonance Relaxation Measurements of tt-I.eci-thin-Peptide Interaction in Model Membranes. 

Specific enrichment of individual carbon positions in amino acids facilitates relaxation measurements which are typically time consuming and enables studies to be carried out at reduced concentration. This is of particular value in view of the growing literature documenting intermolecular assciation of peptides.(46-48) Nlccolai, et al.(4q) have compared the rotational correlation times of N-acetyl allolsoleuclne in H2O as determined by (high concentration) and H (lower... 

Relaxation measurements can be conducted both under static and MAS condition. MAS technique was introduced by Pines and coworkers [119] and further developed by others [120-122]. Application MAS results in splitting of the quadrupolar tensor into system of the spinning sidebands separated by the spinning frequency. The shape of the envelope is comparable with the shape of the static spectrum but the S/N ratio is significantly higher. Except ID techniques, 2D experiments can be applied. Spiess and coworkers [123] introduced 2D NMR approach to study the slow motions, in the 1 ms up to 1 s regime. Very detailed, concise and comprehensive review of techniques applied in MAS SS NMR of peptides and proteins was published by Reif and coworkers [82,124]. 

Finally, structural investigations of a human calcitonin-derived carrier peptide in a membrane enviromnent by solid-state NMR have been reported. The typical axially symmetric powder patterns of NMR spectra were used to confirm the presence of lamellar bilayers in the samples studied. The chemical shift anisotropy of the NMR spectra was monitored in order to reveal weak interaction of the peptide with the lipid headgroups. In addition, paramagnetic enhancement of relaxation rates and NMR order parameters of the phospholipid fatty acid chains in the absence and presence of the carrier peptide were measured. All peptide signals were resolved and fully assigned in 2D proton-driven spin diffusion experiments. The isotropic chemical shifts of CO, C and provided information about the secondary structure of the carrier peptide. In addition, dipolar eoupling measurements indicated rather high amplitudes of motion of the peptide. 

Specific models for internal motions can be used to interpret heteronuclear relaxation, such as restricted diffusion and site-jump models. However, model-free formal methods are preferable, at least for the initial analysis, since available experimental data generally are insufficient to completely characterize complex internal motions or to uniquely determine a specific motional model. The model-free approach of Lipari and Szabo for the analysis of relaxation data has been used for proteins and even for peptides. It attempts to reproduce relaxation rates by a weighted product of spectral density functions with different correlation times The weighting factors are identified as order parameters for the molecular rotational correlation time and optional further local correlation times r. The term (1-S ) would then be proportional to the amplitude of the corresponding internal motion. However, the Lipari-Szabo approach is based on the assumption that molecular and local correlation times are not coupled, i.e. they should be distinct enough (e.g. differing by at least a factor of 10 in time) to allow for this separation. However, in small molecules the rates of these different processes are of the same order of magnitude, and the requirements of the Lipari-Szabo approach may not be fulfilled. Molecular dynamics simulation provide a complementary approach for the interpretation of relaxation measurements. 

The concept of molecular structure implies a reduction in the freedom of motion for the involved atoms. Thus an indirect strategy for identifying structured segments is to search for restricted motion for contiguous sets of amino acid residues. Relaxation of the 15N nucleus in the peptide bond provides a quantitative measure of the rates and angular range of motion experienced by individual amino acids under equilibrium conditions (Palmer, 2001). 

Solid-phase peptide synthesis offers a fast and convenient route for many peptides when isotope-enriched compounds are not required. Classical synthesis additionally permits the use of non-natural amino acids and allows site-specific isotope labeling. Although Fmoc protected 15N-labeled amino adds are commercially available, the cost of such synthesis is usually prohibitive, and the peptides from chemical synthesis require perdeuterated detergents and unfortunately exclude investigation of internal dynamics through measurement of 15N relaxation. 

Fig. 2 HN(CO)CA-derived experiment for the measurement of /nhc dipole-CSA CCR-rates. Various ratios of NBD peptide and NEMO were used. A 200-fold excess of ligand optimal conditions. B 100-fold excess auto relaxation and cross relaxation are too fast. The resonance assignment of the peptide is indicated...

A suitable CCR-rate to determine the backbone torsion angle 0 by CCR is the /NHCHcf dipole-dipole CCR-rate that conveniently can be measured by an HNCA-derived experiment [44]. Alternatively, like for the torsion angle 0, the FcuaCii-i) dipole-CSA CCR can be measured by a triple-resonance experiment that is derived from a combination of HNCA and HNCO experiments [45]. Also, CCR experiments for which the rate depends on 0 and

dipole-dipole CCR experiment can be used [46]. Unfortunately for the peptide under investigation, we were not able to successfully record any of these spectra, possibly due to the relatively strong auto relaxation. 

Fan, P., Li, M., Brodsky, B., and Baum, J. (1993). Backbone dynamics of (Pro-Hyp-Gly) 10 and a designed collagen-like triple-helical peptide by 15N NMR relaxation and hydrogen-exchange measurements. Biochemistry 32, 13299-13309. 

The best explanation of the good results for peptide syntheses in ice-water mixtures are based on the freeze-concentration-model, which just provides for a volume-reducing function for the ice while the liquid aqueous part is still the only relevant phase for the reaction. All observed enhancements of reaction rate would then have to be attributed to an increase in effective concentration. H-NMR relaxation time measurements have been used to determine the amount of unfrozen water in partially frozen systems, thus quantifying the extent of the freeze-concentration effect (Ullmann, 1997). Comparative studies in ice and at room temperature verify the importance of freeze-concentration which, however, is not sufficient for a complete understanding of the observed effects. 

The applications of 13C nuclear relaxation in the peptide field have been reviewed very recently (Deslauriers and Smith, 1976). We can therefore restrict the discussion here to a few comments. It was pointed out that the general assumption that relaxation of all carbon atoms of a peptide are dipolar is not necessarily true (Cutnell et al., 1975). It is emphasized that NOE measurements should be obligatory before motional assumptions are made from 7j data. The kind of information one obtains from relaxation work for small peptides of perhaps up to 10 amino-acid residues may be seen from the... 

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