A new strategy for structure determination of large proteins in solution without deuteration

Abstract

So far high-resolution structure determination by nuclear magnetic resonance (NMR) spectroscopy has been limited to proteins <30 kDa, although global fold determination is possible for substantially larger proteins. Here we present a strategy for assigning backbone and side-chain resonances of large proteins without deuteration, with which one can obtain high-resolution structures from (1)H-(1)H distance restraints. The strategy uses information from through-bond correlation experiments to filter intraresidue and sequential correlations from through-space correlation experiments, and then matches the filtered correlations to obtain sequential assignment. We demonstrate this strategy on three proteins ranging from 24 to 65 kDa for resonance assignment and on maltose binding protein (42 kDa) and hemoglobin (65 kDa) for high-resolution structure determination. The strategy extends the size limit for structure determination by NMR spectroscopy to 42 kDa for monomeric proteins and to 65 kDa for differentially labeled multimeric proteins without the need for deuteration or selective labeling.

Stability of Peroxide Antimalarials in the Presence of Human Hemoglobin

“…The lack of reactivity is not surprising, considering the tertiary structure of the globin protein around the heme residues in Hb. X-ray crystal structures of oxyHb (26) (and the solution nuclear magnetic resonance structure of carboxyHb [37]) show that the iron center of each heme residue is not exposed to the bulk solution and is in close proximity to the His58␣, Phe43␣, and Val62␣ (or His63␤, Phe42␤, and Val67␤) amino acid side chains. This structure allows tight binding of diatomic molecules (O 2 , CO, and CN); however, only minimal binding occurs for larger isocyanides (25,35) and monocyclic ligands (33) due to steric factors.…”

Solution and Crystal Structures of mRNA Exporter Dbp5p and Its Interaction with Nucleotides

“…The backbone and side-chain chemical shift assignments were obtained using the strategy recently developed in our laboratory. 26,27 For the NTD, 26 residues did not yield observable HSQC signals. These residues are located mainly in the N-terminal region, including Y71-L77, D79, L88-A91, F94, E96, K111, F112, K117, Q119-R121, Q137, and S138.…”

“…The NMR experiments used on each sample are 2D HSQC, 3D TROSY (transverse relaxation optimized spectroscopy)-HNCA, 3D MQ-CCH-TOCSY (total correlated spectroscopy), 34,35 and 4D 13 C, 15 N time-shared NOESY (NOE spectroscopy). 26,27 All data were processed with NMRPipe 36 and analyzed with SPARKY. 37 The distance restraints were obtained by classifying the NOE cross peaks into three categories: strong (1.8-2.9 Å), medium (1.8-3.5 Å), and weak (1.8-5.0 Å).…”

Structure–function analysis of full-length midkine reveals novel residues important for heparin binding and zebrafish embryogenesis

“…All data were processed with NMRpipe and analysed with in-house NMRspy and an XYZ4D extension (http://yangdw.science.nus.edu.sg/Software&Scripts/ XYZ4D/index.htm). Resonance assignment was achieved using the four-dimensional NOESY-based strategy [33].…”