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. The "divide-and-conquer" method that has proven to be very powerful in applications to other systems produced assignments for approximately 60% of the observed methyl groups in TROSY maps of ATCase. By contrast, the combination of all approaches led to assignments for 86% of the methyls, providing a large number of probes of structure and dynamics. The derived assignments were used to interpret chemical shift changes of ATCase upon titration with the nucleotide ATP. Large shift changes in the N-terminal tails of the regulatory chain provide the first evidence for structural perturbations in a region that is known to play a critical role on the effect of nucleotide binding on distal catalytic sites of this allosteric enzyme.
Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8.
Algirdas
A
Velyvis
Howard K
HK
Schachman
Lewis E
LE
Kay
D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007532:Isoleucine / Q000737:chemistry*; D007930:Leucine / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000592:standards; D012015:Reference Standards; D014633:Valine / Q000737:chemistry*
D007532:Isoleucine; D001221:Aspartate Carbamoyltransferase; D007930:Leucine; D014633:Valine