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

Folding regulates autoprocessing of HIV-1 protease precursor

Autoprocessing of HIV-1 protease (PR) precursors is a crucial step in the generation of the mature protease. Very little is known regarding the molecular mechanism and regulation of this important process in the viral life cycle. In this context we report here the first and complete residue level investigations on the structural and folding characteristics of the 17-kDa precursor TFR-PR-Cnn (161 residues) of HIV-1 protease. The precursor shows autoprocessing activity indicating that the solution has a certain population of the folded active dimer. Removal of the 5-residue extension, Cnn at the C-terminal of PR enhanced the activity to some extent. However, NMR structural characterization of the precursor containing a mutation, D25N in the PR at pH 5.2 and 32 °C under different conditions of partial and complete denaturation by urea, indicate that the precursor has a high tendency to be unfolded. The major population in the ensemble displays some weak folding propensities in both the TFR and the PR regions, and many of these in the PR region are the non-native type. As both D25N mutant and wild-type PR are known to fold efficiently to the same native dimeric form, we infer that TFR cleavage enables removal of the non-native type of preferences in the PR domain to cause constructive folding of the protein. These results indicate that intrinsic structural and folding preferences in the precursor would have important regulatory roles in the autoprocessing reaction and generation of the mature enzyme

Crystallization of a scRIP-gelonin isolated from plant seeds Gelonium multiforum

Single crystals of the protein gelonin isolated from the seeds of Gelonium multiforum have been grown at room temperature by vapor diffusion method. The crystals are monclinic with a = 49.4 Å, b = 44.9 Å, c = 137.4 Å, and β = 98.3°. The space group is P21, with two molecules in the asymmetric unit which are related by a noncrystallographic 2-fold axis along ψ =13° and φ =88°. The crystals diffract X-rays to high resolution, making it possible to obtain an accurate structure of this single chain ribosome inactivating protein

NMR elucidation of early folding hierarchy in HIV-1 protease

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, HN-Hα coupling constants, 1H-15N nuclear Overhauser effects, and 15N 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

NMR observation of a novel A-tetrad in a telomeric DNA segment in aqueous solution

NMR studies on the telomeric DNA segment d-AG3T show that the molecule exists as a mixture of several multistranded structures whose stabilities are dependent on added salt concentrations. There is one major conformation which can be selectively studied at low salt concentration and the NMR data indicate that this is a parallel stranded quadruplex with a novel A-tetrad at the 5' -end, The A-bases adopt the syn glycosidic conformation as against the anti conformation adopted by the G - bases in the three G - tetrads

The DNA-binding domain of Drosophila melanogaster c-Myb undergoes a multistate denaturation

The DNA-binding domain of Drosophila c-Myb protein has been studied using different spectroscopic probes, namely CD, fluorescence, acrylamide quenching and NMR, to determione the structure of some of its sub-domains and their relative stabilities in aqueous solutions. While CD and fluorescence spectroscopy showed that the protein had completely lost its tertiary and secondary structures in approximately 3 M urea, solvent accessibility of the tryptophan residues was still partial, as determined by acrylamide quenching. This suggested the presence of significant amounts of residual structure which persisted until the urea concentration was raised to approximately 6.0 M. Thermal-denaturation experiments also indicated the presence of an intermediate in the unfolding pathway. The experimental data could be fitted assuming a minimum of three states in both modes of denaturation. The thermodynamic parameters for the apparent three-state transition have been determined. From the protein stability curve, we have determined that Drosophila melanogaster Myb R123 has maximal stability at 16°C and pH 7.0

The single-atom box: bosonic staircase and effects of parity

We have developed a theory of a Josephson junction formed by two tunnel-coupled Bose-Einstein condensates in a double-well potential in the regime of strong atom-atom interaction for an arbitrary total number $N$ of bosons in the condensates. The tunnel resonances in the junction are shown to be periodically spaced by the interaction energy, forming a single-atom staircase sensitive to the parity of $N$ even for large $N$. One of the manifestations of the staircase structure is the periodic modulation with the bias energy of the visibility of the interference pattern in lattices of junctions.Comment: 5 pages, 4 included figues, published versio

NMR studies on the Myb-DNA system

NMR structural studies on a bacterially produced DNA binding domain of Drosophila c-Myb protein as well as its cognate DNA sequence carried out in our laboratory have been surveyed. The structure of a self-complementary dodecamer DNA containing the Myb responsive element (TAACGG) has been determined to atomic resolution by the combined use of two-dimensional NMR, spectral simulations, restrained energy minimization and distance geometry calculations. The structure is seen to possess novel features which may play important roles during its interaction with the Myb protein, The DNA binding domain of c-Myb protein was seen to have a hydrophobic core and we have identified the types of residues contributing to its formation. Residues contributing to the hydrophobic core formation are seen to be well spread out over the whole length of the 160 residues in the protein and include isoleucines, valines, leuclnes, alanines threonines, aromatic residues, glutamines and possibly aspartates. Our experimental data in combination with those of others indicate that some of the amino acid residues which form the helical motifs that directly interact with DNA may also be a part of the hydrophobic core

X-ray structure of gelonin and gelonin-AMP complex

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Exploring 4D Quantum Hall Physics with a 2D Topological Charge Pump

The discovery of topological states of matter has profoundly augmented our understanding of phase transitions in physical systems. Instead of local order parameters, topological phases are described by global topological invariants and are therefore robust against perturbations. A prominent example thereof is the two-dimensional integer quantum Hall effect. It is characterized by the first Chern number which manifests in the quantized Hall response induced by an external electric field. Generalizing the quantum Hall effect to four-dimensional systems leads to the appearance of a novel non-linear Hall response that is quantized as well, but described by a 4D topological invariant - the second Chern number. Here, we report on the first observation of a bulk response with intrinsic 4D topology and the measurement of the associated second Chern number. By implementing a 2D topological charge pump with ultracold bosonic atoms in an angled optical superlattice, we realize a dynamical version of the 4D integer quantum Hall effect. Using a small atom cloud as a local probe, we fully characterize the non-linear response of the system by in-situ imaging and site-resolved band mapping. Our findings pave the way to experimentally probe higher-dimensional quantum Hall systems, where new topological phases with exotic excitations are predicted