Figure 1. Diagram outlining the course of a protein structure determination by NMR in solution (see text).

Figure 2. Three-dimensional ¹⁵N-correlated [¹H,¹H]-NOESY spectrum of uniformly ¹⁵N-labelled E. coli glutaredoxin.

Figure 3. (a) Sequential resonance assignment based on sequential ¹H–¹H NOEs. In the dipeptide segment –Ala–Val– the dotted lines indicate ¹H–¹H relations that can be established by scalar, through-bond spin–spin couplings seen in homonuclear ¹H NMR experiments. The broken arrows indicate pairs of protons in sequentially neighbouring residues that can be related by ¹H–¹H NOEs that manifest short sequential distances d_N (between CH and the amide proton of the following residue) and d_NN (between the amide protons of neighbouring residues). (b) Segment of a uniformly ¹³C/¹⁵N-labelled polypeptide chain with indication of the scalar spin–spin couplings (in Hz) that provide the basis for obtaining sequential assignments by triple-resonance experiments (see text).

Figure 4. Protein structure determination based on upper distance constraints measured by ¹H–¹H nuclear Overhauser effects (NOEs). Part of a 2D [¹H,¹H]-NOESY spectrum of the Antennapedia homeodomain is shown, and at the top there is a line, NC that represents a polypeptide chain. Two ¹H atoms of the polypeptide are identified by circles, and straight arrows point from the corresponding peaks on the diagonal in the NOESY spectrum to these protons (these arrows represent the result of the sequential assignment). The presence of a NOESY cross-peak outside of the diagonal, which is connected with the two aforementioned diagonal positions by a vertical and a horizontal line, shows that these two protons are separated by a short distance (£ 4.0 Å), and therefore the observation of this single NOE imposes a looped structure on the polypeptide chain (top right). Each additional NOESY cross-peak defines a similar loop. The NMR structure corresponds to the molecular geometry that contains simultaneously all the loops defined by the NOESY data.

Figure 5. Stereo view of the polypeptide backbone of 20 energy-refined conformers selected to represent the NMR solution structure of the 60-residue tick anticoagulant protein from Ornithodoros moubata. The numbers identify selected sequence positions.