Dynein light chain protein, a part of the cytoplasmic motor assembly, is a homodimer at physiological pH and dissociates below pH 4.5 to a monomer. The dimer binds to a variety of cargo, whereas the monomer does not bind any of the target proteins. We report here the pH induced stepwise structural and motional changes in the protein, as derived from line broadening and 15 N transverse relaxation measurements. At pH 7 and below until 5, partial protonation and consequent interconversion between molecules carrying protonated and neutral histidines, causes conformational dynamics in the dimeric protein and this increases with decreasing pH. Enhanced dynamics in turn leads to partial loosening of the structure. This would have implications for different efficacies of binding by target proteins due to small variations in pH in different parts of the cell, and hence for cargo trafficking from one part to another. Below pH 5, enhanced charge repulsions, partial loss of hydrophobic interactions, and destabilization of H-bonds across the dimer interface cause further loosening of the dimeric structure, leading eventually to the dissociation of the dimer. Keywords: dynein light chain protein;15 N transverse relaxation; conformational exchange; line broadening; pH driven conformational transitions; nuclear magnetic resonance; dimer-monomer equilibrium DLC8 is known to interact with several proteins of diverse biological functions and presumably plays a primary role as a cargo adaptor in the transport of several organelles (Jaffrey and Snyder 1996;Fan et al. 1998;Liang et al. 1999;Puthalakath et al. 1999Puthalakath et al. , 2001Espindola et al. 2000;Naisbitt et al. 2000;Schnorrer et al. 2000;Fuhrmann et al. 2002;Day et al. 2004;Vadlamudi et al. 2004;Lo et al. 2005;Yang et al. 2005). DLC8 is highly conserved throughout the evolution and its structure has been determined both by X-ray crystallography (Liang et al. 1999) and by NMR ; the two structures are essentially identical. At the physiological pH of 7.0 the protein exists as a homodimer, whereas below pH 4.5 it has been reported to exist as a monomer (Barbar et al. 2001). The structure of the monomer has also been determined to high resolution (Wang et al. 2003;Makokha et al. 2004). The NMR structures of the dimer and the monomer are shown in Figure 1. In the dimer, the b-strands form the core while the helices are on the surface. The protruding b3 strand of one monomer pairs in an antiparallel fashion with the Abbreviations: NMR, nuclear magnetic resonance; HSQC, heteronuclear single quantum coherence; DLC8, dynein light chain protein.Article published online ahead of print. Article and publication date are at

Folding studies on proteases by the conventional hydrogen exchange experiments are severely hampered because of interference from the autolytic reaction in the interpretation of the exchange data. We report here NMR identification of the hierarchy of early conformational transitions (folding propensities) in HIV-1 protease by systematic monitoring of the changes in the state of the protein as it is subjected to different degrees of denaturation by guanidine hydrochloride. Secondary chemical shifts, H N -H ␣ coupling constants, 1 H-15 N nuclear Overhauser effects, and 15 N transverse relaxation parameters have been used to report on the residual structural propensities, motional restrictions, conformational transitions, etc., and the data suggest that even under the strongest denaturing conditions (6 M guanidine) hydrophobic clusters as well as different native and non-native secondary structural elements are transiently formed. These constitute the folding nuclei, which include residues spanning the active site, the hinge region, and the dimerization domain. Interestingly, the proline residues influence the structural propensities, and the small amino acids, Gly and Ala, enhance the flexibility of the protein. On reducing the denaturing conditions, partially folded forms appear. The residues showing high folding propensities are contiguous along the sequence at many locations or are in close proximity on the native protein structure, suggesting a certain degree of local cooperativity in the conformational transitions. The dimerization domain, the flaps, and their hinges seem to exhibit the highest folding propensities. The data suggest that even the early folding events may involve many states near the surface of the folding funnel.The folding of a protein is conceptually described in terms of a folding funnel (1-6). The narrow end of the funnel represents the folded native state, and the broad end represents the unfolded state consisting of millions of rapidly inter-converting conformers. As a protein folds from an unfolded state, it goes through one or more partially folded intermediates, which need to be characterized for elucidation of its folding pathways. Experimentally, this is a very challenging task. The most common and direct approach relies on kinetic pulse labeling experiments of amide protons coupled with hydrogen exchange at different time points along the folding reaction of the protein (reviewed in Ref. 7). However, as has been pointed out (8), this also has limitations. First, it is limited by the necessity of detectable protection against exchange. Second, any lack of protection does not necessarily imply absence of structure. Third, it biases the interpretation of the structure in intermediates toward the native state. Even so, useful information has been obtained in many protein systems (7). However, this approach is complicated in the case of proteases with autolytic property, because the autolytic reaction interferes with interpretation of the hydrogen exchange data. Therefore, in these sys...

Calcium binding proteins carry out various signal transduction processes upon binding to Ca2+. In general, these proteins perform their functions in a high background of Mg2+. Here, we report the role of Mg2+ on a calcium sensor protein from Entamoeba histolytica (EhCaBP), containing four Ca2+-binding sites. Mg2+-bound EhCaBP exists as a monomer with a conformation different from that of the holo- and apo-EhCaBP. NMR and biophysical data on EhCaBP demonstrate that Mg2+ stabilizes the closed conformation of the apo form. In the presence of Mg2+, the partially collapsed apo-EhCaBP gains stability and structural integrity. Mg2+ binds to only 3 out of 4 calcium binding sites in EhCaBP. The Ca2+ binding affinity and cooperativity of the conformational switching from the "closed" to the "open" state is significantly modulated by the presence of Mg2+. This fine-tuning of the Ca2+ concentration to switch its conformation is essential for CaBPs to carry out the signal transduction process efficiently.

No 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.