Solution NMR Studies of a 42 KDa Escherichia Coli Maltose Binding Protein/β-Cyclodextrin Complex:  Chemical Shift Assignments and Analysis

Abstract

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 Cβ 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.

Structural Basis for Capping Protein Sequestration by Myotrophin (V-1)

“…The most probable cause of missing peaks was incomplete exchange of amide deuterium atoms back to hydrogen after protein production in D 2 O, especially in the case of residues in the hydrophobic core of the protein. In the case of larger proteins, partial unfolding followed by refolding in an H 2 O environment can increase the amount of reprotonation (52,61,62). However, even partial denaturation of CP in the presence of 2 M guanidine hydrochloride rendered the protein insoluble, preventing attempts at partial unfolding and refolding.…”

The Maltodextrin System ofEscherichia coli: Metabolism and Transport

“…There are two modes of ligand binding by MBP. High affinity requires a free reducing end of the maltodextrin molecule, while binding to extended polysaccharide chains is of lower affinity (29). Maltodextrins whose reducing end is blocked by residues larger than a methyl group (21), even though bound well by the binding protein, cannot be transported, indicating a polarity in the mechanism in which linear dextrins are channeled into the transporter (25,52,62).…”

Mars – robust automatic backbone assignment of proteins

“…MARS has been tested on 14 proteins ranging in size from the 71-residue Z domain of Staphylococcal protein A to 723-residue malate synthase G (Alattia et al, 2000;Gardner et al, 1998;Garrett et al, 1997;Ikura et al, 1991;Liu et al, 2000;Schwaiger et al, 1998;Tashiro et al, 1997;Tugarinov et al, 2002;Vathyam et al, 1999;Wang et al, 1995). Special focus was put on proteins that are challenging with respect to assignment either by their size or because chemical shifts are missing for a substantial portion of residues (Table 1).…”