NMR Insights into Folding and Self-Association of Plasmodium falciparum P2

The eukaryotic 60S-ribosomal stalk is composed of acidic ribosomal proteins (P1 and P2) and neutral protein P0, which are thought to be associated as a pentameric structure, [2P1, 2P2, P0]. Plasmodium falciparum P2 (PfP2) appears to play additional non-ribosomal functions associated with its tendency for homo-oligomerization. Recombinant bacterially expressed PfP2 protein also undergoes self-association, as shown by SDS-PAGE analysis and light scattering studies. Secondary structure prediction algorithms predict the native PfP2 protein to be largely helical and this is corroborated by circular dichroism investigation. The 1H-15N HSQC spectrum of native P2 showed only 43 cross peaks compared to the expected 138. The observed peaks were found to belong to the C-terminal region, suggesting that this segment is flexible and solvent exposed. In 9 M urea denaturing conditions the chain exhibited mostly non-native β structural propensity. 15N Relaxation data for the denatured state indicated substantial variation in ms-µs time scale motion along the chain. Average area buried upon folding (AABUF) calculations on the monomer enabled identification of hydrophobic patches along the sequence. Interestingly, the segments of slower motion in the denatured state coincided with these hydrophobic patches, suggesting that in the denatured state the monomeric chain undergoes transient hydrophobic collapse. The implications of these results for the folding mechanism and self-association of PfP2 are 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 characterization of the energy landscape of SUMO-1 in the native-state ensemble

Characterizing the low energy excited states in the energy landscape of a protein is one of the exciting and demanding problems in structural biology at the present time. These describe the adaptability of the protein structure to external perturbations. In this context, we used here non-linear dependence of amide proton chemical shifts on temperature to identify residues accessing alternative conformations in SUMO-1 in the native state as well as in the near-native states created by sub-denaturing concentrations of urea. The number of residues accessing alternative conformations increases and the profiles of curved temperature dependence also change with increasing urea concentration. In every case these alternative conformations lie within 2 kcal/mol from the ground state, and are separated from it by low energy barriers. The residues that access alternative conformations span the length of the protein chain but are located at particular regions on the protein structure. These include many of the loops, β2 and β5 strands, and some edges of the helices. We observed that some of the regions of the protein structure that exhibit such fluctuations coincide with the protein's binding surfaces with different substrate like GTPase effector domain (GED) of dynamin, SUMO binding motifs (SBM), E1 (activating enzyme, SAE1/SAE2) and E2 (conjugating enzyme, UBC9) enzymes of sumoylation machinery, reported earlier. We speculate that this would have significant implications for the binding of diversity of targets by SUMO-1 for the variety of functions it is involved in

Direct observation of (H8,H6)-Hr J-coupling correlations in oligonucleotides for unambiguous resonance assignments: use of J-scaling in two-dimensional correlated spectroscopy

Four-bond H8/H6-H1 scalar coupling correlations in two-dimensional correlated spectroscopy have been observed directly for the first time by using the J-scaled COSY [(1985) Chem. Phys. Lett. 116, 105-108] technique in a dinucleotide, cytidylyl(2'-5')guanosine (CpG). Unambiguous resonance assignment of nonexchangeable protons in CpG has been obtained using these H8/H6-H1' 4-bond correlations and the various 3- and 4-bond sugar ring proton correlations observed in the COSY and SUPER COSY experiments

Generation of serine/threonine check points in HN(C)N spectra

We describe here a simple modification of the HN(C)N experiment for the generation of serine/threonine check points in the three-dimensional experiment. The various 'triplet of residue' specific peak patterns in the spectra are documented for ease of analysis and sequential backbone resonance assignment. The performance of this experiment, referred to as HN(C)N-ST, is demonstrated using two proteins, one properly folded and the other completely denatured. It is noteworthy that, even in the denatured protein, where spectral dispersions are rather poor, about 90% of the sequential connectivities through the chain could be established from this single experiment. This would have great implications for structural genomics efforts

CD and NMR investigations on trifluoroethanol-induced step-wise folding of helical segment from scorpion neurotoxin

A 14 amino acid residue peptide from the helical region of Scorpion neurotoxin has been structurally characterized using CD and NMR spectroscopy in different solvent conditions. 2,2,2-Trifluoroethanol (TFE) titration has been carried out in 11 steps from 0 to 90% TFE and the gradual stabilization of the conformation to form predominantly α-helix covering all of the 14 residues has been studied by 1H and 13C NMR spectroscopy. Detailed information such as coupling constants, chemical shift indices, NOESY peak intensities and amide proton temperature coefficients at each TFE concentration has been extracted and analysed to derive the step-wise preferential stabilization of the helical segments along the length of the peptide. It was found that there is a finite amount of the helical conformation in the middle residues 5-11 even at low TFE concentrations. It was also observed that > 75% TFE (v/v) is required for the propagation of the helix to the N and C termini and for correct packing of the side chains of all of the residues. These observations are significant to understanding the folding of this segment in the protein and may throw light on the inherent preferences and side chain interactions in the formation of the helix in the peptide

Two-dimensional NMR for three-dimensional structure of nucleic acids: new techniques and novel results

The principles and the variety of two-dimensional (2D) NMR techniques useful for structure determination of nucleic acids have been described. Three new techniques namely, SUPERCOSY, J-scaled COSY and COSS which have been recently developed to overcome the problems of sensitivity and resolution have been described in greater detail. The strategies of resonance assignments and structure determination of nucleic acids have been discussed and the experimental results on four oligonucleotides longer than a complete turn of nucleic acid helix have been presented. The data indicate O I' -endo sugar geometry in a majority of the nucleotide units in two oligonucleotides namely, d-GAATTCGAATTC and d-GAA TTCCCGAATTC

Role of protein backbone in specific recognition of nucleic acid base sequences: a hypothesis

A novel H-bonding scheme of interaction between protein backbone and nucleic acid base pairs has been proposed. The importance of such an interaction in specific recognition of base sequences in double helical nucleic acids has been discussed. It is concluded that protein backbone can play very important role in specific recognition of base sequences