J.L. Nieto

1H NMR and CD evidence of the folding of the isolated ribonuclease 50-61 fragment

In our search for potential folding intermediates we have prepared and characterized the fragment of RNase A corresponding to residues 50–61. Proton chemical shift variations with temperature, addition of stabilizing (TFE) or denaturing agents (urea) provide a strong experimental basis for concluding that in aqueous solution this RNase fragment forms an α‐helix structure similar to that in the intact RNase A crystal. This conclusion lends strong support to the idea that elements of secondary structure (mainly α‐helices) can be formed in the absence of tertiary interactions and act as nucleation centers in the protein folding process.

Low‐temperature 1H‐NMR evidence of the folding of isolated ribonuclease S‐peptide

The temperature (−7°C to 45°C, pH 5.4) and pH (0°C) dependence of 1H chemical shifts of ribonuclease S‐peptide (5 mM, 1 M NaCl) has been measured at 360 MHz. The observed variations evidence the formation of a partial helical structure, involving the fragment Thr‐3—Met‐13. Two salt‐bridges stabilize the helix: those formed by Glu‐9−…His‐12+ and Glu‐2−…Arg‐10+. The structural features deduced from the 1H‐NMR at low temperature for the isolated S‐peptide are compatible with the structure shown by the same molecule in the ribonuclease S crystal.

On the fundamental role of the Glu 2- ... Arg 10+ salt bridge in the folding of isolated ribonuclease A S-peptide.

The fundamental role of the Glu 2- ... Arg 10+ salt bridge in the folding of isolated S-peptide (1-19 N-terminal fragment of Ribonuclease A) is demonstrated from the comparison of the helix contents, at 0 degrees C, of S-peptide and related peptides. Helix contents have been determined from the analysis of proton chemical shift vs. temperature curves. The observed data can be accounted for by assuming that two side-chain interactions contribute to stabilize the 3-13 helix of S-peptide, the salt bridges Glu 2- ... Arg 10+ and Glu 9-... His 12+, the former being more effective. The salt bridge Glu 9- ... Arg 10+ turns to a weaker interaction, a hydrogen bond Glu 2 (C delta = 0) ... Arg 10+, on protonation or esterification of the Glu 2 carboxylate.

A study of the NH NMR signals of Gly-Gly-X-Ala tetrapeptides in H2O at low temperature.

Abstract The effect of temperature, pH and addition of denaturing agents on the amide and side chain NH δ values of a series of random coil linear tetrapeptides (Gly-Gly-X-Ala with X=Glu, Asp, His, Trp, Arg, Gly, Pro, Asn, Gln) has been measured in dilute aqueous solutions at two temperatures (24 and 0°C). Amide shift temperature coefficients were within the −5.8 to −9.1 ppb/K range. Amide δ changes following urea addition were ⩽ 0.03 ppm, with the exception of the Ala terminal residue of all peptides, and the His residue as well. Signs of a non-random structure near the COO − terminus were found for the Gly-Gly-His-Ala tetrapeptide.

Characterization of low populated peptide helical structures in solution by means of NMR proton conformational shifts

A NOE independent NMR method is proposed to characterize unambiguously residues involved in low populated isolated peptide helices. The method is based on the comparison of amide and H alpha chemical shift changes originated upon the addition of stabilizing or denaturing agents with true helical conformational shifts that have been measured for the first time using an isolated model peptide helix, the one formed by Ac-(Leu-Lys-Lys-Leu)3-NHEt in aqueous solution.

Conformational properties of the isolated 1–23 fragment of human hemoglobin α-chain

Abstract With the purpose of establishing whether, as a general rule, regions of a protein chain that are helical in the native structure maintain, at least partially, the same helical structure when isolated in solution, we have prepared the 1–23 fragment of human hemoglobin α-chain, and studied its conformational properties in aqueous solution by CD and 1 H-NMR. From the analysis of CD and NMR spectral changes with temperature, salt and addition of trifluoroethanol (TFE) it can be concluded that the 1–23 peptide forms a measurable population (18% at 22°C (pH 5.6) TFE/H 2 O, 30:70 (v/v)) of an α-helix structure that spans the same residues that are helical in the native protein (namely, 6 to 17). These results, taken together with similar ones obtained previously in the 1–19, 21–42 and 50–61 RNAase fragments, support the idea that no helices other than the native ones are actually formed in solution by protein fragments. This implies that the final helical structure of a protein is present from the very beginning of the folding process, and also that such elements of secondary structure can act as primary nucleation centers.

Amide 1H n.m.r. study of the folding of ribonuclease C-peptide

Abstract The folding of ribonuclease A 1–13 (C-peptide) in H 2 O near 0°C has been monitored by means of the amide and side chain NH proton resonances. The C-peptide carboxylate at low temperature forms, in a significant amount, a folded structure similar to the one that the 1–19 S-peptide adopts in the same conditions (3–13 α-helix). A quantitative comparison between helix stabilities of the lactone and carboxylate forms of C-peptide and S-peptide is reported. It is concluded that the proposed His 12 + … Hse 13 (COO − salt bridge, which competes with the one-turn stabilizing salt bridge His 12 + … Glu 9 − in the C-peptide carboxylate, does not suppress helix formation as previously suggested but it merely reduces its stability. The behaviour of the N 5 -H resonance of the Arg 10 + side chain provides evidence for its implication in a further stabilizing interaction, most probably with Glu 2 − .

Refined solution structure of bovine pancreatic Ribonuclease A by1H NMR methods. Sidechain dynamics

An extension of the assignment of the1H NMR spectra of bovine pancreatic Ribonuclease A has been carried out at 600 MHz covering all sidechain protons, including those belonging to long sidechain residues. Vicinal coupling constants,3JHNα and3Jαβ and3Jαβ′ have been measured with the purpose of imposing torsional constraints on the backbone ϕ angle as well as to introduceHββ′ specific assignments. On the basis of distance constraints evaluated from 1285 assigned NOEs, a first restrained molecular dynamics calculation has been carried out. The resulting root-mean-square deviation for the backbone atoms is 1.5 Å as compared to 1.8 Å for the 360 MHz NOE basis. For heavy atoms in sidechains, these numbers were, respectively, 3.0 and 2.5 Å A second calculation was carried out by introducing 37 dihedral angle constraints on backbone ϕ, torsions and five onX1 angular torsions as well as with stereospecific assignments for nearly 50 residues. Backbone and sidechain RMS deviations were, respectively, 1.2 and 2.0 Å. Sidechains have been classified in terms of their dynamical behavior aroundX1 after considering jointly the coupling information and that resulting from the second set of calculated structures. Results are discussed in relation to the crystal structure.

The homologous angiogenin and ribonuclease N-terminal fragments fold into very similar helices when isolated

The solution structure of the N-terminal hexadecapeptide of human angiogenin, a protein of unknown tertiary structure, has been precisely delineated by the combined use of CD, NOE and secondary shift data. A helix that starts just after Ser 3 and ends at Asp 14 was stabilized in 30% trifluoroethanol. This helix is strikingly similar in origin and length to the one formed by its homologous, the S-peptide of Ribonuclease (conformationally reexamined here), despite their quite different sequences (only four conserved residues). These results support the idea that individual start and stop signals indeed govern the location and size of natural isolated helices.

Quantum-chemical calculations of a proposed PHEn-HISn+4 stabilizing interaction in peptide α-helices

Abstract The interaction between the sidechains of the Phe (n) and His (n+4) which has been proposed as a stabilizing effect for α-helix formation has been modelled by means of totuene: 4-Me-imidazole. Intermolecular potentials and their extrema are calculated at several degrees of approximation for neutral and protonated forms of the imidazole partner. A marked increase in stability for the charged complex relative to the neutral one is found. The interaction energies are used to reparameterize an analytical atom-atom potential for C, N and H contacts.