Assignment of Ile, Leu, and Val methyl correlations in supra-molecular systems: an application to aspartate transcarbamoylase.

A number of complementary approaches for the assignment of Ile, Leu, and Val methyl groups in Methyl-TROSY spectra of supra-molecular protein complexes are presented and compared. This includes the transfer of assignments from smaller fragments to the complex using a "divide-and-conquer" approach, assignment transfer via exchange spectroscopy, or, alternatively, generating assignments of the complex through the measurement of pseudocontact shifts, facilitated by the introduction of paramagnetic probes. The methodology is applied to the assignment of the regulatory chains in the 300 kDa enzyme aspartate transcarbamoylase, ATCase.

Application of methyl-TROSY NMR to test allosteric models describing effects of nucleotide binding to aspartate transcarbamoylase.

Aspartate transcarbamoylase has emerged as a textbook example of an allosteric enzyme whose binding of active-site substrates can be explained on the basis of the classical Monod-Wyman-Changeux (MWC) model of allostery. There is still debate, however, regarding the mode of action of ATP and cytidine triphosphate (CTP)--allosteric effectors that bind at regulatory sites 60 A away from the nearest active site. A large body of data for nucleotide binding is consistent with the MWC model, including a previous NMR study showing a shift in the allosteric equilibrium between R and T states that is predicted by this scheme.

A solution NMR study showing that active site ligands and nucleotides directly perturb the allosteric equilibrium in aspartate transcarbamoylase.

The 306-kDa aspartate transcarbamoylase is a well studied regulatory enzyme, and it has emerged as a paradigm for understanding allostery and cooperative binding processes. Although there is a consensus that the cooperative binding of active site ligands follows the Monod-Wyman-Changeux (MWC) model of allostery, there is some debate about the binding of effectors such as ATP and CTP and how they influence the allosteric equilibrium between R and T states of the enzyme. In this article, the binding of substrates, substrate analogues, and nucleotides is studied, along with their effect on the R-T equilibrium by using highly deuterated, (1)H,(13)C-methyl-labeled protein in concert with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR.