[show abstract][hide abstract] ABSTRACT: Protein function is often regulated by conformational changes that occur in response to ligand binding or covalent modification such as phosphorylation. In many multidomain proteins these conformational changes involve reorientation of domains within the protein. Although X-ray crystallography can be used to determine the relative orientation of domains, the crystal-state conformation can reflect the effect of crystal packing forces and therefore may differ from the physiologically relevant form existing in solution. Here we demonstrate that the solution-state conformation of a multidomain protein can be obtained from its X-ray structure using an extensive set of dipolar couplings measured by triple-resonance multidimensional NMR spectroscopy in weakly aligning solvent. The solution-state conformation of the 370-residue maltodextrin-binding protein (MBP) loaded with β-cyclodextrin has been determined on the basis of one-bond 15N-HN, 15N-13C′, 13Cα-13C′, two-bond 13C′-HN, and three-bond 13Cα-HN dipolar couplings measured for 280, 262, 276, 262, and 276 residues, respectively. This conformation was generated by applying hinge rotations to various X-ray structures of MBP seeking to minimize the difference between the experimentally measured and calculated dipolar couplings. Consistent structures have been derived in this manner starting from four different crystal forms of MBP. The analysis has revealed substantial differences between the resulting solution-state conformation and its crystal-state counterpart (Protein Data Bank accession code 1DMB) with the solution structure characterized by an 11(±1)° domain closure. We have demonstrated that the precision achieved in these analyses is most likely limited by small uncertainties in the intradomain structure of the protein (ca 5° uncertainty in orientation of internuclear vectors within domains). In addition, potential effects of interdomain motion have been considered using a number of different models and it was found that the structures derived on the basis of dipolar couplings accurately represent the effective average conformation of the protein.
Journal of Molecular Biology 03/2000; · 3.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: A selective protonation strategy is described that uses [3-2H] 13C α-ketoisovalerate to introduce (1H-δ methyl)-leucine and
(1H-γ methyl)-valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine
and valine methyl groups is obtained by inclusion of ≈100mg/L α-ketoisovalerate in the bacterial growth medium. Addition
of [3,3-2H2] α-ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-δ1 methyl)-isoleucine
(>90% incorporation). 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with
this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling
agent and is more cost effective.
Journal of Biomolecular NMR 04/1999; 13(4):369-374. · 2.85 Impact Factor