Solution NMR-derived global fold of a monomeric 82-kDa enzyme

Abstract

The size of proteins that can be studied by solution NMR spectroscopy has increased significantly because of recent developments in methodology. Important experiments include those that make use of approaches that increase the lifetimes of NMR signals or that define the orientation of internuclear bond vectors with respect to a common molecular frame. The advances in NMR techniques are strongly coupled to isotope labeling methods that increase sensitivity and reduce the complexity of NMR spectra. We show that these developments can be exploited in structural studies of highmolecular-weight, single-polypeptide proteins, and we present the solution global fold of the monomeric 723-residue (82-kDa) enzyme malate synthase G from Escherichia coli, which has been extensively characterized by NMR in the past several years.isotope labeling ͉ protein NMR ͉ transverse relaxation-optimized spectroscopy N MR spectroscopy is one of the most powerful techniques for the study of protein structure and dynamics (1). In addition, NMR spectroscopy has emerged as an important tool for the investigation of protein-ligand interactions (2), and their quantification in terms of structure, dynamics, kinetics, and thermodynamics. However, a drawback of the methodology is the size limitation of the molecules that can be studied. The short lifetimes of NMR signals and the complexity of spectra generated in applications involving high-molecular-weight systems still limit many NMR applications to studies of relatively small biomolecules.In this regard, important advances have been made in the past several years that have significantly extended the range of molecules that are now amenable to investigation. Large gains in both the sensitivity and the resolution of NMR spectra of large molecules can be achieved by using the so-called transverse relaxation-optimized spectroscopy (TROSY) approach (3), in which only the slowly decaying components of nuclear magnetization contribute to the final signal. Since the original pioneering developments involving studies of amide (3) and aromatic moieties (4), more recent applications with methyl (5) and methylene groups (6) have appeared. A second major advance has involved the ''reintroduction'' of magnetic interactions that would normally average to zero in isotropic solution by means of the use of media leading to a weak alignment of the macromolecule of interest (7). The resulting orientational restraints, such as dipolar couplings and changes in chemical shifts that are generated by such alignment, are extremely valuable in structural studies, in particular for large proteins in which the number of restraints per residue is significantly less than what is normally obtained in studies of small (Յ30-kDa) proteins. A third important contribution has been the development of isotopic-labeling approaches, such as those involving uniform 15 N, 13 C labeling along with high levels of deuteration, which maximize the lifetimes of NMR signals and optimize the HON TROSY effect. NMR experiments that have emerged ...

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