Determination of a high precision structure of a novel protein, Linum usitatissimum trypsin inhibitor (LUTI), using computer-aided assignment of NOESY cross-peaks.
The solution structure of a novel 69 residue proteinase inhibitor, Linum usitatissimum trypsin inhibitor (LUTI), was determined using a method based on computer aided assignment of nuclear Overhauser enhancement spectroscopy (NOESY) data. The approach applied uses the program NOAH/DYANA for automatic assignment of NOESY cross-peaks. Calculations were carried out using two unassigned NOESY peak lists and a set of determined dihedral angle restraints. In addition, hydrogen bonds involving amide protons were identified during calculations using geometrical criteria and values of HN temperature coefficients. Stereospecific assignment of beta-methylene protons was carried out using a standard procedure based on nuclear Overhauser enhancement intensities and 3J(alpha)(beta) coupling constants. Further stereospecific assignment of methylene protons and diastereotopic methyl groups were established upon structure-based method available in the program GLOMSA and chemical shift calculations. The applied algorithm allowed us to assign 1968 out of 2164 peaks (91%) derived from NOESY spectra recorded in H2O and 2H2O. The final experimental data input consisted of 1609 interproton distance restraints, 88 restraints for 44 hydrogen bonds, 63 torsion angle restraints and 32 stereospecifically assigned methylene proton pairs and methyl groups. The algorithm allowed the calculation of a high precision protein structure without the laborious manual assignment of NOESY cross-peaks. For the 20 best conformers selected out of 40 refined ones in the program CNS, the calculated average pairwise rmsd values for residues 3 to 69 were 0.38 A (backbone atoms) and 1.02 A (all heavy atoms). The three-dimensional LUTI structure consists of a mixed parallel and antiparallel beta-sheet, a single alpha-helix and shows the fold of the potato 1 family of proteinase inhibitors. Compared to known structures of the family, LUTI contains Arg and Trp residues at positions P6' and P8', respectively, instead of two Arg residues, involved in the proteinase binding loop stabilization. A consequence of the ArgTrp substitution at P8' is a slightly more compact conformation of the loop relative to the protein core.
Cierpicki
T
T
Laboratory of Protein Engineering, Institute of Biochemistry and Molecular Biology, University of Wroclaw, Wroclaw, Poland.
Otlewski
J
J
D000595:Amino Acid Sequence; D001331:Automation / Q000379:methods; D001665:Binding Sites; D004220:Disulfides / Q000737:chemistry; D019597:Flax / Q000737:chemistry*; D006860:Hydrogen Bonding; D007090:Image Interpretation, Computer-Assisted; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010940:Plant Proteins / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011522:Protons; D016415:Sequence Alignment; D012984:Software; D011198:Solanum tuberosum / Q000737:chemistry; D013696:Temperature; D014361:Trypsin Inhibitors / Q000737:chemistry*
D004220:Disulfides; C414684:LUTI protein, Linum usitatissimum; D010940:Plant Proteins; D011522:Protons; D014361:Trypsin Inhibitors