title: Global folds of proteins with low densities of NOEs using residual dipolar couplings: application to the 370-residue maltodextrin-binding protein.
abstract: The global fold of maltose-binding protein in complex with the substrate beta-cyclodextrin was determined by solution NMR methods. The two-domain protein is comprised of a single polypeptide chain of 370 residues, with a molecular mass of 42 kDa. Distance information in the form of H(N)-H(N), H(N)-CH(3) and CH(3)-CH(3) NOEs was recorded on (15)N, (2)H and (15)N, (13)C, (2)H-labeled proteins with methyl protonation in Val, Leu, and Ile (C(delta1) only) residues. Distances to methyl protons, critical for the structure determination, comprised 77 % of the long-range restraints. Initial structures were calculated on the basis of 1943 NOEs, 48 hydrogen bond and 555 dihedral angle restraints. A global pair-wise backbone rmsd of 5.5 A was obtained for these initial structures with rmsd values for the N and C domains of 2.4 and 3.8 A, respectively. Direct refinement against one-bond (1)H(N)-(15)N, (13)C(alpha)-(13)CO, (15)N-(13)CO, two-bond (1)H(N)-(13)CO and three-bond (1)H(N)-(13)C(alpha) dipolar couplings resulted in structures with large numbers of dipolar restraint violations. As an alternative to direct refinement against measured dipolar couplings we have developed an approach where discrete orientations are calculated for each peptide plane on the basis of the dipolar couplings described above. The orientation which best matches that in initial NMR structures calculated from NOE and dihedral angle restraints exclusively is used to refine further the structures using a new module written for CNS. Modeling studies from four different proteins with diverse structural motifs establishes the utility of the methodology. When applied to experimental data recorded on MBP the precision of the family of structures generated improves from 5.5 to 2.2 A, while the rmsd with respect to the X-ray structure (1dmb) is reduced from 5.1 to 3.3 A.
authors.affiliation: Protein Engineering Network Centers of Excellence, Department of Medical Genetics, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
authors.forename: G A
authors.initials: GA
authors.lastname: Mueller
authors.forename: W Y
authors.initials: WY
authors.lastname: Choy
authors.forename: D
authors.initials: D
authors.lastname: Yang
authors.forename: J D
authors.initials: JD
authors.lastname: Forman-Kay
authors.forename: R A
authors.initials: RA
authors.lastname: Venters
authors.forename: L E
authors.initials: LE
authors.lastname: Kay
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D003505:Cyclodextrins / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D033902:Periplasmic Binding Proteins; D011487:Protein Conformation; D017510:Protein Folding; D011522:Protons; D012680:Sensitivity and Specificity; D012984:Software; D012996:Solutions; D047392:beta-Cyclodextrins
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D003505:Cyclodextrins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D033902:Periplasmic Binding Proteins; D011522:Protons; D012996:Solutions; D047392:beta-Cyclodextrins; C031215:betadex