The structures of a dimeric mutant of the Lac repressor DNA-binding domain complexed with the auxiliary operators O2 and O3 have been determined using NMR spectroscopy and compared to the structures of the previously determined Lac-O1 and Lac-nonoperator complexes. Structural analysis of the Lac-O1 and Lac-O2 complexes shows highly similar structures with very similar numbers of specific and nonspecific contacts, in agreement with similar affinities for these two operators. The left monomer of the Lac repressor in the Lac-O3 complex retains most of these specific contacts. However, in the right half-site of the O3 operator, there is a significant loss of protein-DNA contacts, explaining the low affinity of the Lac repressor for the O3 operator. The binding mode in the right half-site resembles that of the nonspecific complex. In contrast to the Lac-nonoperator DNA complex where no hinge helices are formed, the stability of the hinge helices in the weak Lac-O3 complex is the same as in the Lac-O1 and Lac-O2 complexes, as judged from the results of hydrogen/deuterium experiments.
Background: A central question in translation initiation is how GTPase activity and fMet-tRNA positioning are connected. Results: NMR shows large structural rearrangements in the IF2-G2 subdomain upon nucleotide binding and considerable flexibility within the fMet-tRNA binding domain.
Conclusion:The GDP-induced rearrangements in G2 are not forwarded toward the fMet-tRNA binding C2 subdomain.Significance: There appears to be no structural relationship between GTP hydrolysis and fMet-tRNA positioning.
The presence of fluorine in an active pharmaceutical ingredient (API) can impart important pharmacological attributes with regards to metabolism, stability, and selectivity. As such, nearly one‐third of newly approved small molecule drugs contain at least one fluorine atom. 19F is 100% naturally abundant lending to virtually unimpeded detection of fluorine‐containing molecules, even in complex mixtures. Despite these promising characteristics, the thorough evaluation of a method for 19F qNMR (quantitative NMR) has been lacking. Herein we present experimental methodology and optimized parameters for uniform quantitative sampling of fluorine‐containing drugs and we compare results obtained using both gravimetric and absolute concentration methods for data reduction.
The precision of techniques and factors affecting the interpretation of residual dipolar couplings (RDCs) in analysis of spatial structures of partially aligned proteins are discussed. Experimental RDC values were obtained for pairs of 1H-15N nuclei of the protein barstar partially aligned in a liquid crystalline matrix of bicelles composed of dimiristoylphosphatidylcholine and dihexanoylphosphatidylcholine. The observed couplings agree well with the spatial structures of barstar determined earlier by X-ray and NMR methods. However, the differences between the experimental and calculated RDCs that were calculated on the basis of the known spatial structures of barstar, exceed the experimental errors three- to fourfold. These discrepancies can be explained by differences in the protein structures in solution and in crystal, a limited precision of the X-ray analysis, and the intramolecular mobility of the protein molecule. A comparison of the results of modeling of the molecular dynamics of barstar in solution, crystal structures, and the experimental RDCs showed that the methods of molecular dynamics provide for a reasonable description of the character and amplitudes of internal motions and they should be considered for the correct determination of protein spatial structures from NMR spectroscopic data.
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