P. M. Krishna Mohan

pH driven conformational dynamics and dimer‐to‐monomer transition in DLC8

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 15N 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.

NMR comparison of the native energy landscapes of DLC8 dimer and monomer

Characterization of the low energy excited states on the energy landscape of a protein is one of the exciting and challenging problems in structural biology today. In this context, we present here residue level NMR description of the low energy excited states representing locally different alternative conformations in the dynein light chain protein, in its dimeric as well as monomeric forms. Important differences have been observed between the two cases and these are not necessarily restricted to the dimer interface. Simulations indicate that the low energy excited states are within a free energy of 2-3 kcal/mol above the native state. In both the monomer and the dimer the energy landscape is very sensitive to small pH perturbations. Nearly 25% of the residues (total of residues at pH 3.0 and 3.5 for the monomer, and at pH 7.0 and 6.0 for the dimer) access alternative conformations. The observations have been rationalized on the basis of protonation-deprotonation equilibria in the side chains; histidines in the case of the dimer and aspartates/glutamates in the case of the monomer. The possible relationship of the observed ruggedness of the native energy landscape with the protein structure, and its implications to protein adaptability and unfolding have been discussed.

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

The detailed characterization of the structure, dynamics and folding process of a protein is crucial for understanding the biological functions it performs. Modern biophysical and nuclear magnetic resonance (NMR) techniques have provided a way to obtain accurate structural and thermodynamic information on various species populated on the energy landscape of a given protein. In this context, we review here the structure-function-folding relationship of an important protein, namely, dynein light chain protein (DLC8). DLC8, the smallest subunit of the dynein motor complex, acts as a cargo adaptor. The protein exists as a dimer under physiological conditions and dissociates into a pure monomer below pH 4. Cargo binding occurs at the dimer interface. Dimer stability and relay of perturbations through the dimer interface are anticipated to be playing crucial roles in the variety of functions the protein performs. NMR investigations have provided great insights into these aspects of DLC8 in recent years.

NMR investigations on residue level unfolding thermodynamics in DLC8 dimer by temperature dependent native state hydrogen exchange

Understanding protein stability at residue level detail in the native state ensemble of a protein is crucial to understanding its biological function. At the same time, deriving thermodynamic parameters using conventional spectroscopic and calorimetric techniques remains a major challenge for some proteins due to protein aggregation and irreversibility of denaturation at higher temperature values. In this regard, we describe here the NMR investigations on the conformational stabilities and related thermodynamic parameters such as local unfolding enthalpies, heat capacities and transition midpoints in DLC8 dimer, by using temperature dependent native state hydrogen exchange; this protein aggregates at high (>65°C) temperatures. The stability (free energy) of the native state was found to vary substantially with temperature at every residue. Significant differences were found in the thermodynamic parameters at individual residue sites indicating that the local environments in the protein structure would respond differently to external perturbations; this reflects on plasticity differences in different regions of the protein. Further, comparison of this data with similar data obtained from GdnHCl dependent native state hydrogen exchange indicated many similarities at residue level, suggesting that local unfolding transitions may be similar in both the cases. This has implications for the folding/unfolding mechanisms of the protein.

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

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 β‐sheets (β4 and β5) that form the hydrophobic core of the protein and the α2 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. Proteins 2009.

NMR characterization of structural and dynamics perturbations due to a single point mutation in drosophila DLC8 dimer: functional implications

Dynein light chain protein (DLC8), the smallest subunit of the dynein motor complex, acts as a cargo adaptor. The protein exists as a dimer under physiological conditions, and cargo binding occurs at the dimer interface. Dimer stability and relay of perturbations through the dimer interface can thus be anticipated to play crucial roles in the variety of functions the protein performs. Recent investigations point out that DLC8 also gets phosphorylated at Ser 88, which is located at the extreme C-terminal end. In this background, we investigate here by NMR the effects of a small perturbation by way of a single point mutation, S88A, on the structure, dynamics, and cargo binding efficacy of the DLC8 dimer. We observe that the perturbation travels far away along the sequence from the site of the mutation. This relay has been explained at the atomic level by looking into the packing of the side chains in the crystal structure of the protein. It follows that the interface is highly adaptable, which may account for the versatility of the dimers cargo binding ability. Binding studies with a peptide indicate that the mutation compromises binding efficacy. These observations show how remote residues that may not be directly bound to a target can still affect the affinity of the protein to the target. Furthermore, the S88A mutational perturbations seen here in Drosophila DLC8 are dramatically different from those of the same mutation in human DLC8 (also known as DLC1) ( Song, C. , et al., ( 2008) J. Biol. Chem, 283, 4004- 4013. ) which differs from Drosophila DLC8 at only five locations. All of these observations put together highlight the sensitivity of dynein light chain protein to small perturbations, and this would have great functional implications.

Differential native state ruggedness of the two Ca2+-binding domains in a Ca2+ sensor protein.

Characterization of near‐native excited states of a protein provides insights into various biological functions such as co‐operativity, protein–ligand, and protein–protein interactions. In the present study, we investigated the ruggedness of the native state of EhCaBP using nonlinear temperature dependence of backbone amide‐proton chemical shifts. EhCaBP is a two‐domain EF‐hand calcium sensor protein consisting of two EF‐hands in each domain and binds four Ca2+ ions. It has been observed that ∼30% of the residues in the protein access alternative conformations. Theoretical modeling suggested that these low‐energy excited states are within 2–3 kcal/mol from the native state. Further, it is interesting to note that the residues accessing alternative conformations are more dominated in the C‐terminal domain compared with its N‐terminal counterpart suggesting that the former is more rugged in its native state. These distinct characteristics of N‐ and C‐ terminal domains of a calcium sensor protein belonging to the super family of calmodulin would have implications for domain dependent Ca2+ signaling pathways. Proteins 2008.

Hierarchy of local structural and dynamics perturbations due to subdenaturing urea in the native state ensemble of DLC8 dimer

Local structural and dynamic modulations due to small environmental perturbations reflect the adaptability of the protein to different interactors. We have investigated here the preferential local perturbations in Dynein light chain protein (DLC8), a cargo adapter, by sub-denaturing urea concentrations. Equilibrium unfolding experiments by optical spectroscopic methods indicated a two state like unfolding of DLC8 dimer, with the transition mid-point occurring around 8.6M urea. NMR studies identified the β3 and β4 strands, N-, C- terminal regions, loops connecting β1 to α1, α1 to α2 and β3 to β4 as the soft targets of urea perturbation and thus indicated potential unfolding initiation sites. Native-state hydrogen exchange studies suggested the unfolding to traverse from the edges towards the centre of the secondary structural elements. At 6M urea the whole protein chain acts like a cooperative unit. These observations are expected to have important implications for the proteins multiple functions.

Hierarchy in guanidine unfolding of DLC8 dimer: regulatory functional implications.

Folding-unfolding caused by environmental changes play crucial regulatory roles in protein functions. To gain an insight into these for DLC8, a cargo adaptor in dynein motor complex, we investigated here the unfolding of homodimeric DLC8 by GdnHCl, a standard unfolding agent. Fluorescence spectroscopy revealed a three-state unfolding transition with midpoints at 1.5 and 4.0 M GdnHCl. The HSQC spectrum at 1.5 M GdnHCl displayed peaks belonging to a folded monomer. NMR chemical shift perturbations, line broadening effects and (15)N relaxation measurements at low GdnHCl concentrations identified a hierarchy in the unfolding process, with the dimer interface--the cargo binding site--being the most susceptible followed by the helices in the interior. Similar observations were made earlier for small pH perturbations and thus the early unfolding events appear to be intrinsic to the protein. These, by virtue of their location, influence target binding efficacies and thus have important regulatory implications.

pH dependent unfolding characteristics of DLC8 dimer: Residue level details from NMR.

Environment dependence of folding and unfolding of a protein is central to its function. In the same vein, knowledge of pH dependence of stability and folding/unfolding is crucial for many biophysical equilibrium and kinetic studies designed to understand protein folding mechanisms. In the present study we investigated the guanidine induced unfolding transition of dynein light chain protein (DLC8), a cargo adaptor of the dynein complex in the pH range 7-10. It is observed that while the protein remains a dimer in the entire pH range, its stability is somewhat reduced at alkaline pH. Global unfolding features monitored using fluorescence spectroscopy revealed that the unfolding transition of DLC8 at pH 7 is best described by a three-state model, whereas, that at pH 10 is best described by a two-state model. Chemical shift perturbations due to pH change provided insights into the corresponding residue level structural perturbations in the DLC8 dimer. Likewise, backbone (15)N relaxation measurements threw light on the corresponding motional changes in the dimeric protein. These observations have been rationalized on the basis of expected changes with increasing pH in the protonation states of the titratable residues on the structure of the protein. These, in turn provide an explanation for the change from three-state to two-state guanidine induced unfolding transition as the pH is increased from 7 to 10. All these results exemplify and highlight the role of environment vis-à-vis the sequence and structure of a given protein in dictating its folding/unfolding characteristics.