Figure 1. NOESY (a), ROESY (b), offset compensated ROESY (c), and EASY‐ROESY (d). 90° hard pulses (filled bars) and spin‐lock sequences (open rectangles) are applied along the x‐axes unless otherwise stated.

Figure 2. Example HMBC experiment: signals connect distal spin systems in the His and Trp side‐chains of MT‐II via correlations to C ^γ with the core spin system (H ^N, H ^α, H ^β′, H ^β″). The small letters a–j in the spectra designate correlations between distinct protons and C ^γ, as given in the chemical structures of both amino acid residues. Note that the γ carbon of Trp is strongly high‐field shifted.

Figure 3. Sequential assignment using an example HMBC spectrum of the cyclohexylalanine peptide cyclo(‐d‐Ala‐Ala‐Ala‐MeAla‐MeAla‐Ala‐). The small letters (k–o) in the spectrum correspond to the accordingly labeled correlations in the chemical structure. The resonances were first assigned to the individual spin systems via the intraresidual HMBC cross‐peaks k, l and m, n, respectively. The sequential assignment was derived from the inter‐residual HMBC cross‐peak o that links the spin systems in the given order.

Figure 4. Diastereotopic assignments of β hydrogen atoms. χ _I rotamers and the according and coupling constants of l‐ and d‐amino acid residues as considered in the assignment process. The prochirality of the β protons is inverted for residues Asn, Asp, Cys, His, Met, and Ser as for these residues, the priority of the more distal parts of the side‐chains is higher than the priority of the peptide backbone.

Figure 5. (a) Two different conformations that exchange fast on the chemical shift timescale. A low‐populated (p(1) = 20%) first conformation with a small interproton distance of r _ij(1) = 2 Å and a high‐populated second conformation (p(2) = 80%) with a large interproton distance of r _ij(2) = 6 Å are expected to give rise to a ROESY cross‐peak with an intensity that corresponds to an apparent distance of 2.6 Å. (b) Two neighboring N‐methylated residues d‐Trp and Lys. Their side‐chain dynamics in cyclo(‐MeA‐Tyr‐d‐MeTrp‐MeLys‐Val‐MeF‐) are reflected by the distributions of the distances d‐MeTrp H ^δ1 to MeK H ^γ′ and d‐MeTrp H ^δ1 to MeK H ^γ″ within a 50‐ms MD simulation. The ROESY cross‐peak intensities of both interactions correspond to distances of 2.8–3.6 Å.

Figure 6. (a) nQF‐COSY pulse sequence. Appropriate phase cycling can provide sums of 2QF‐ and 3QF‐COSY, resulting in characteristic E.COSY multiplet patterns as shown below for a N‐methylated Trp residue within (Ac‐Nle‐cyclo(5β → 10ɛ)(Asp5‐MeHis6‐d‐Phe7‐MeArg8‐MeTrp9‐MeLys10)‐NH ₂). Couplings are preferably read from appropriately chosen in‐phase splittings, as demonstrated in the example experiment here, not from antiphase splittings that would result in an overestimation of the couplings. (b) DISCO procedure for determining Asp5 couplings in the same peptide using the following cross‐sections of four 2QF‐COSY cross‐peaks: Left, solid line: H ^β′(f1)–H ^β″(f2), dashed line: H ^α(f1)–H ^β″(f2); right, solid line: H ^β″(f1)–H ^β′(f2), dashed line: H ^α(f1)–H ^β′(f2)). Cross‐sections were extracted along the direct dimension. Sums and differences of J couplings that are read from antiphase splittings in the sums (Σ) and differences (Δ) of these traces are given below. (c) Phase‐sensitive HMBC with gradient pulses. Multiplets like the H ^β–C′ (thin line) superimposed with reference spectra using different trial coupling constants of 1–7 Hz (bold line) clearly suggest a coupling constant of 3–4 Hz in the example shown here. (d) Part of a Gly H ^N(f1)–H ^α(f2) cross‐peak from a HETLOC spectrum of cyclo(‐d‐MeAla‐Ala‐Ala‐Ala‐Gly‐MeAla‐). The coupling is obtained as offset in the direct dimension (b) of two multiplet components that are split by the strong coupling in the direct dimension (a).

Figure 7. Process of subsequent distance geometry (DG) calculations with continuous restraint evaluation. If after many circles no homogeneous ensemble of almost identical conformations is obtained, flexibility of the peptide under consideration may be considered as the reason for continuous restraint violations.