Solution NMR studies of a 42 KDa Escherichia coli maltose binding protein/β-cyclodextrin complex: Chemical shift assignments and analysis

The use of deuteration in concert with uniform ¹⁵N,¹³C-labeling has been critical for the chemical shift assignment of several proteins and protein complexes over 30 kDa. Unfortunately, deuteration reduces the number of interproton distance restraints available for structure determination, compromising the precision and accuracy of the NMR-derived structures determined from these samples. We have recently described an isotopic labeling strategy that addresses this problem by generating proteins labeled uniformly with ¹⁵N, ¹³C, and extensively with ²H with high levels of protonation at exchangeable sites and the methyl groups of Val, Leu, and Ile (δ1 only) (Gardner, K. H.; Kay, L. E. J. Am. Chem. Soc. 1997, 119, 7599- 7600). This labeling pattern maintains the high efficiency of triple resonance methods while retaining sufficient protons to establish long-range NOEs between secondary structure elements. We demonstrate the utility of samples labeled in this manner by presenting the chemical shift assignments of one of the largest monomeric proteins assigned to date, the 370 residue Escherichia coli maltose binding protein in complex with β-cyclodextrin (42 kDa). The high level of Cα and C β deuteration provided by our labeling scheme enabled the collection of triple resonance data with high sensitivity and resolution, allowing assignment of over 95% of the backbone ¹⁵N, ¹³Cα, ¹HN, and side chain ¹³Cβ nuclei. By using a combination of existing experiments and a new pulse scheme described here for correlating methyl chemical shifts with ¹³Cβ (Val), ¹³Cγ (Leu), or ¹³Cγ1 (Ile) carbons, over 98% of methyl ¹³C and ¹H assignments from Val, Leu, and Ile (Cδ1 only) have been obtained. Analysis of the backbone chemical shifts and qualitative HN exchange data have confirmed that the MBP/β-cyclodextrin complex has a secondary structure similar to that previously observed in a 1.8 Å crystal structure.