2D NMR Spectroscopy and 3D Structure Calculations

MoVIB and analogue peptides samples were prepared at 2 mg/ml in 90% H₂O/10% D₂O or 100% D₂O (pH 5.0) for Nuclear Magnetic Resonance (NMR) spectroscopy studies. Two dimensional (2D) homonuclear ¹H-¹H total correlation spectroscopy (TOCSY), nuclear Overhauser effect (NOESY) and exclusive correlation spectroscopy (ECOSY) datasets, and a 2D heteronuclear ¹H-¹³C HSQC were recorded at 900 MHz on a Bruker Avance II spectrometer, equipped with a cryogenically cooled probe and processed using Topsin 3.0 (Bruker). Homonuclear data were recorded with 2048 data points in the direct dimension and 512 increments in the indirect dimension over a sweep-width of 12 ppm. The HSQC spectrum was recorded with an indirect dimension sweep-width of 106 ppm. Data analysis were performed using the Computer Aided Resonance Assignment (CARA) software⁵⁶. Structural restraints derived from the NMR data included (i) Inter-proton distances derived from NOESY cross-peak intensities in spectra recorded in either H₂O or D₂O with a mixing time of 100 ms. (ii) Backbone dihedral angles (Phi and Psi) derived from a TALOS+^57,58 analysis of Cα, Cβ, Hα and HN chemical shifts. (iii) Side chain dihedral angles (χ1) derived from analysis of ³J_HαHβ coupling constants and intra residual NOE patterns (iv) Hydrogen-bond restraints derived from amide exchange rates and analysis of preliminary structures. Nuclear Overhauser effect (NOE) cross peaks were manually picked and subsequently calibrated and assigned automatically using the automatic assignment and structure calculation module of CYANA 3.0⁵⁹. For the final structures distance restraint lists from CYANA were used as input for simulated annealing and water minimization within CNS⁶⁰, using protocols from the RECOORD database⁶¹, modified as described previously⁶². In the final round 50 structures were calculated, and the best 20 based on energies and quality of packing and geometry as judged by MOLPROBITY⁶³ scores were chosen as representative of the solution structure of MoVIB. Figures were prepared using MOLMOL⁶⁴.