Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary

Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine β-, γ- and δ-hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs α-β³, β³-γ³, β²-γ², γ²-δ², and γ³-δ³ (2.3 Å) are shorter than those in the pairs α-β², β²-γ³, β³-γ², γ²-δ³, and γ³-δ² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the β³-γ² and γ²-δ³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the α-β², β²-γ³, and γ³-δ² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.