Peptides conformation-dependent chemical shifts

Complementary to the analysis of conformation-dependent chemical shifts, two-dimensional experiments that correlate anisotropic interactions may be used to study backbone conformation by MAS NMR. This is possible in particular for dipolar interactions, whose orientations are along the intemuclear vector, and for the carbonyl CSAs, which generally adopt a particular orientation with respect to the peptide plane (Fig. 3a). Under MAS, these interactions may be recoupled during the evolution and/or detection period of a two-dimensional correlation experiment, as discussed earlier. The resulting spectrum will contain cross peaks whose pattern is characteristic of the relative orientation of the two interactions being correlated. An alternative approach is to excite a state of 2QC between two neighbouring nuclei, which then evolves under the influence of two anisotropic interactions. 

The structure of silk fibroin from a wild silkworm was examined by using solution and solid state NMR methods. The structural transition of the silk fibroin in aqueous solution was monitored by using solution NMR spectroscopy as a function of temperature. The torsion angles of several Ala and Gly residues in the model peptide, GGAGGGYGGDGG(A)i2GGA-GDGY-GAG, were determined by the conformation-dependent chemical shifts, REDOR and 2D spin-diffusion techniques in the solid state. 

A novel approach based on the application of solid-state NMR spectroscopy has been reported that permits the rapid determination of 3D molecular structure with a single uniformly isotope labelled sample. Analogy with the solution NMR spectroscopic investigations is used, which rely on the detection of short distances between hydrogen atoms providing the principal source of information about the 3D fold of the protein. Since the 2D H, H-correlation methods are of limited use for solid-state NMR spectroscopy due to the restricted spectral resolution, the indirect detection and structural analysis of interactions via C, C-correlation spectroscopy is proposed. It has been shown that combined with dihedral-angle constraints, which can be derived from conformation-dependent chemical shifts, the characterisation of the 3D molecular structure from a single protein sample becomes possible. The new approach has been demonstrated on kaliotoxin, a 38-residue peptide. 

Hanessian studied the solution structure of tetra-, hexa-, and octa-y-peptide analogues of the sequence (-Ala-Val-).236 All three of these y4-peptides derived from L-amino acids adopted stable right-handed helical conformations in solution. The helical parameters were identical to those found by the Seebach group 2.6 residues per turn stabilized by S(14) H-bonds. Temperature-dependent chemical shifts suggested that these intrastrand interactions are strong. As also noted by Seebach, CD did not reveal a pattern diagnostic of secondary structure. The obvious but important lesson from these reports is that CD cannot be used alone as a means of screening for secondary structure. 

Solid-state NMR study of structural heterogeneity in peptides containing both polyalanine and repeated GGA sequences as a local structural model of Nephila clavipes dragline silk (Spidroin 1) has been reported. Solvent treatments prior to the NMR measurements were shown to induce a structural change in these model peptides. Conformation-dependent NMR chemical shifts were used to determine the local structure, including the evaluation of the fraction of several conformations. 

Furthermore, in flexible linear peptides the chemical shifts are typical of random structures similar to nonfolded proteins. Deviation from these random shifts sometimes identifies specific conformational preferences. NH-proton chemical shifts depend strongly on external influences (solvent, temperature, concentration, specific sequence). Random coil shifts for these protons correlate less well than chemical shifts of the a-protons or a-carbonsJ19-261 Not only are the shift differences of different heterotopic protons similar, but also those of diastereotopic P-protons. A preferred side-chain conformation is normally only found when there is also a preferred backbone conformation. 

Spin-lattice relaxation times and 13C chemical shifts were used to study conformational changes of poly-L-lysine, which undergoes a coil-helix transition in a pH range from 9 to 11. In order to adopt a stable helical structure, a minimum number of residues for the formation of hydrogen bonds between the C = 0 and NH backbone groups is necessary therefore for the polypeptide dodecalysine no helix formation was observed. Comparison of the pH-dependences of the 13C chemical shifts of the carbons of poly-L-lysine and (L-Lys)12 shows very similar values for both compounds therefore downfield shifts of the a, / and peptide carbonyl carbons can only be correlated with caution with helix formation and are mainly due to deprotonation effects. On the other hand, a sharp decrease of the 7] values of the carbonyl and some of the side chain carbons is indicative for helix formation [854]. 

Chemical shifts (CSs) are the most common NMR parameters obtained as the result of the assignment process as a prerequisite of all subsequent analyses. As a consequence, large data sets of CSs and their analyses are available therefore, their relation to protein structure and dynamics is well documented. The basic observation is that CSs, especially Ha and Ca values depend heavily on the local conformation of the peptide chain. This relationship can be further refined by additional factors such as the spatial environment of these atoms. Currently, applications such as SHIFTX47 are capable of calculating CSs from 3D protein structures and, as a reversal, protein folds can be reconstructed solely on the basis of CS information.48 49 Moreover, internal dynamics of proteins can be estimated solely on the basis of CSs.50 51... 

Highly accurate interatomic distances (ultimately 0.05 A) may be obtained from REDOR experiments [49], which are therefore an attractive tool for studies of hydrogen bonding. This technique has been used recently to characterise ex-helix structures in polypeptides by measuring 13C=0---H-15N hydrogen bond lengths [50]. The intrachain 13C- 15N interatomic distances, measured for a number of different samples, were found to be 4.5 0.1 A. This finding was used as evidence for the a-helix structure, which is consistent with the conformation dependent displacements of 13C chemical shifts of the Ca, Cp and carbonyl carbons of the peptide unit [51]. 

Metaldi ef o . 112) nthesized a cyclic pentapeptide, Cyclo Qly-Ala-Gly-Gly Pro), and invest ted the 220-MHz NMR spectra in DMSO-d. Each proton gave two sets of resonance signals, so that the existence of two different conformations was considered, a nu r conformation being represented by M and a minor conformation by m. Hie chemical shift and the temperature dependence of the resonance signal, and die oinqparison with model peptides suggested the structures shown in Fig. 19... 

It has been demonstrated that the isotropic chemical shifts of systems, such as (Gly) (polyglycine(PG)), (Ala) , (Leu) (poly(L-leucine)), (Ileu) -(poly(L-isoleucine)), (Val) (poly(L-valine)), (Phe) (poly(L-phenylalanine)), (Glu(OMe)) (poly(y-methyl L-glutamate)), (Asp(OBzl)) (poly()8-benzyl l-aspartate)) and (Pro) in the peptide backbone of polypeptides in the solid state, exhibit a significant conformation-dependent change [5,6]. Both experimental observation and theoretical calculations confirm this. The Sjso for the a-helix form (97.0-99.2 ppm) appears to low frequencies by about 1.2-... 

For this purpose, it has been demonstrated that the high resolution solid-state NMR approach provides one with an alternative and convenient means to distinguish a variety of crystalline polymorphs and to reveal the secondary structures of biological macromolecules, because the chemical shifts of backbone carbons are displaced (up to 8 ppm) [1, 2] depending on their respective conformations. In addition, it is emphasized that this type of empirical approach can be used as a very valuable constraint to construct the three-dimensional structure of biological molecules, such as peptides and proteins, based on a set of accurately determined interatomic distances measured by a partial dipolar recoupling method, such as REDOR (rotational echo double resonance) [3-6]. 

We demonstrate here how the NMR approach is a very useful means to reveal the conformation and dynamics of biological macromolecule with reference to the conformation-dependent displacements of peaks, with illustrative examples from polysaccharides, structural and membrane proteins and biologically active peptides. It is emphasized here that careful examination of the displacements of or N chemical shifts can serve as an excellent probe when referred to an accumulated data base of reference samples of known secondary structure. 

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