Residue‐wise conformational stability of DLC8 dimer from native‐state hydrogen exchange

Abstract

Dynein light chain (DLC8) is the smallest subunit of the dynein motor complex, which is known to act as a cargo adaptor in intracellular trafficking. The protein exists as a pure dimer at physiological pH and a completely folded monomer below pH 4. Here, we have determined the energy landscape of the dimeric protein using a combination of optical techniques and native-state hydrogen exchange of amide groups, the former giving the global features and the latter yielding the residue level details. The data indicated the presence of intermediates along the equilibrium unfolding transition. The hydrogen exchange data suggested that the molecule has differential stability in its various segments. We deduce from the free energy data that the antiparallel beta-sheets (beta4 and beta5) that form the hydrophobic core of the protein and the alpha2 helix, all of which are highly protected with regard to hydrogen exchange, contribute significantly to the initial step of the protein folding mechanism. Denaturant-dependent hydrogen exchange indicated further that some amides exchange via local fluctuations, whereas there are others which exchange via global unfolding events. Implications of these to cargo adaptability of the dimer are discussed.

Structure-function-folding relationships and native energy landscape of dynein light chain protein: nuclear magnetic resonance insights

“…Further, between the two helices, the stability index suggests that the α2-helix is more stable than the α1-helix. The higher stability of the α2-helix, β4 and β5 sheets compared with those of the remaining structural elements is consistent with the presence of some residues in there whose peaks did not lose intensity even after 4 months in the NHX experiment (Mohan et al 2009a).…”

“…One more noticeable feature is the increase in unfolding free energy (ΔG uf ) values, i.e. upward slope (m-value) with an increase in the GdnHCl concentration for the residues T26 and Q27 in the α1-helix, suggesting a different type of stability in this segment (Brorsson et al 2006;Mohan et al 2009a) (fi gure 7C right panel). In the case of β-strands, both local and global mechanisms do exist.…”

“…In the former case (0 M GdnHCl), the protein exists as a pure dimer, whereas DLC8-H55K is known to exist as a pure monomer (Nyarko et al 2005). Further, recently reported native-state hydrogenexchange (NHX) experiments using NMR spectroscopy have delineated the structural stability and unfolding characteristics of the individual residues in fi ner detail (Mohan et al 2009a). NHX analysis of the DLC8 dimer has provided valuable insights into the stabilities of the various structural elements of the protein (fi gure 7A).…”

“…The GdnHCl-dependent NHX experiments provided insights into the nature of unfolding of the individual residues (Mohan et al 2009a). These experiments suggested that the unfolding landscape of the DLC8 dimer is dominated both by global and local fl uctuations.…”

NMR Investigations on Ruggedness of Native State Energy Landscape in Folded Proteins

“…It is evident that Ser 88 in WT dimer does show a curved temperature dependence of amide proton chemical shifts. Thus, it can be concluded that the amide proton in Ser 88 in WT DLC8 dimer is transiently H-bonded; that it is not a stable H-bond was independently inferred from deuterium exchange studies Mohan et al 2008a). Moreover, the dimeric structure suggests that the amide group of Gly 89 is very close (~ 2.6 Ǻ) to the backbone nitrogen of Ser 88 suggesting a possibility of potential transient H-bonding.…”