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

Structural characterization of the large soluble oligomers of the GTPase effector domain of dynamin

Dynamin, a protein playing crucial roles in endocytosis, oligomerizes to form spirals around the necks of incipient vesicles and helps their scission from membranes. This oligomerization is known to be mediated by the GTPase effector domain (GED). Here we have characterized the structural features of recombinant GED using a variety of biophysical methods. Gel filtration and dynamic light scattering experiments indicate that in solution, the GED has an intrinsic tendency to oligomerize. It forms large soluble oligomers (molecular mass > 600 kDa). Interestingly, they exist in equilibrium with the monomer, the equilibrium being largely in favour of the oligomers. This equilibrium, observed for the first time for GED, may have regulatory implications for dynamin function. From the circular dichroism measurements the multimers are seen to have a high helical content. From multidimensional NMR analysis we have determined that about 30 residues in the monomeric units constituting the oligomers are flexible, and these include a 17 residue stretch near the N-terminal. This contains two short segments with helical propensities in an otherwise dynamic structure. Negatively charged SDS micelles cause dissociation of the oligomers into monomers, and interestingly, the helical characteristics of the oligomer are completely retained in the individual monomers. The segments along the chain that are likely to form helices have been predicted from five different algorithms, all of which identify two long stretches. Surface electrostatic potential calculation for these helices reveals that there is a distribution of neutral, positive and negative potentials, suggesting that both electrostatic and hydrophobic interactions could be playing important roles in the oligomer core formation. A single point mutation, I697A, in one of the helices inhibited oligomerization quite substantially, indicating firstly, a special role of this residue, and secondly, a decisive, though localized, contribution of hydrophobic interaction in the association process

NMR of unfolded proteins

In the post-genomic era, as more and more genome sequences are becoming known and hectic efforts are underway to decode the information content in them, it is becoming increasingly evident that flexibility in proteins plays a crucial role in many of the biological functions. Many proteins have intrinsic disorder either wholly or in specific regions. It appears that this disorder may be important for regulatory functions of the proteins, on the one hand, and may help in directing the folding process to reach the compact native state, on the other. Nuclear magnetic resonance (NMR) has over the last two decades emerged as the sole, most powerful technique to help characterize these disordered protein systems. In this review, we first discuss the significance of disorder in proteins and then describe the recent developments in NMR methods for their characterization. A brief description of the results obtained on several disordered proteins is presented at the end

Imido-P(V) trianion supported enantiopure neutral tetrahedral Pd(II) cages

An enantiomeric pair of chiral tetrahedral cages (1-R and 1-S) were synthesized which show chiral separation of small racemic organic molecules such as epichlorohydrin, beta-butyrolactone, 3-methyl cyclopentanone, and 3-ethyl cyclopentanone.</p

Characterizing RNA Dynamics at Atomic Resolution Using Solution-state NMR Spectroscopy

Many recently discovered non-coding RNAs do not fold into a single native conformation, but rather, sample many different conformations along their free energy landscape to carry out their biological function. Unprecedented insights into the RNA dynamic structure landscape are provided by solution-state NMR techniques that measure the structural, kinetic, and thermodynamic characteristics of motions spanning picosecond to second timescales at atomic resolution. From these studies a basic description of the RNA dynamic structure landscape is emerging, bringing new insights into how RNA structures change to carry out their function as well as applications in RNA-targeted drug discovery and RNA bioengineering

Spectroscopic labeling of A, S/T in the <SUP>1</SUP>H-<SUP>15</SUP>N HSQC spectrum of uniformly (<SUP>15</SUP>N-<SUP>13</SUP>C) labeled proteins

A new triple resonance two-dimensional experiment, termed (HC)NH, has been described to generate specific labels on the peaks of alanines and serines/threonines, separately, in the 1H-15N HSQC spectrum of a protein. The performance of the pulse sequence has been demonstrated with a 151 residue protein. The method permits the investigation of local environments around those specific residues without actually having to obtain complete resonance assignments for the entire protein. With this one can envisage use of the technique for studying large protein systems from different points of view

Equilibrium refolding transitions driven by trifluoroethanol and by guanidine hydrochloride dilution are similar in GTPase effector domain: implications to sequence-self-association paradigm

Protein folding transitions starting from a denatured state play crucial roles in deciding the final fate of a protein. A fundamental question in this regard is the role of the amino acid sequence of the protein. In this context, we have investigated here the equilibrium refolding to a partially folded state of the GTPase effector domain (GED) of dynamin driven by addition of increasing amounts of trifluoroethanol (TFE) and compared it with that driven by progressive dilution of the guanidine hydrochloride (Gdn-HCl) denaturant, which has been reported recently [ (2008) Protein Science17, 1319-1325]. The structural and dynamics changes as the molecule refolds starting from the Gdn-HCl denatured state have been monitored by circular dichroism, fluorescence, and NMR. The molecule remains a monomer in the TFE limiting case, whereas in the Gdn-HCl case, the molecule self-associates as the denaturant is removed. Even so, the two equilibrium transitions seem to have many similarities. The limiting helical contents are similar, and the regions of progressive increase in millisecond time scale motions, suggestive of slow conformational transitions, are largely the same. Though in the guanidine dilution case the partially folded molecules self-associate and there is multimer-monomer equilibrium, the very high concentration (~6 M) of guanidine prevents self-association in the case of TFE created species. Taken together, the observations under the drastically different solvation conditions suggest that the GED sequence is designed to self-assemble via helices leading to formation of a fully folded megadalton size assembly. The present observations may also have implications for the folding and association mechanism of the protein. These are important from the point of view of dynamin function

Pockets of short-range transient order and restricted topological heterogeneity in the guanidine-denatured state ensemble of GED of dynamin

The nature and variety in the denatured state of a protein, a non-native state under a given set of conditions, has been a subject of intense debate. Here, using multidimensional NMR, we have characterized the 6 M Gdn-HCl-denatured state of GED, the assembly domain of dynamin. Even under such strongly denaturing conditions, we detected the presence of conformations in slow exchange on the NMR chemical shift time scale. Although the GED oligomer as well as the SDS-denatured monomeric GED were seen to be predominantly helical [Chugh et al. (2006) FEBS J. 273, 388-397], the 6 M Gdn-HCl-denatured GED has largely &#946; -structural preferences. However, against such a background, we could detect the presence of a population with a short helical stretch (Arg42-Ile47) in the ensemble. The 1H-1H NOEs suggested presence of pockets of transient short-range order along the chain. Put together these segments may lead to a rather small number of interconverting topologically distinguishable ensembles. Spectral density analysis of 15N relaxation rates and {1H}-15N NOE, measured at 600 and 800 MHz, and comparison of J(0) with hydrophobic patches calculated using AABUF approach, indicated presence of four domains of slow motions. These coincided to a large extent with those showing significant Rex. Additionally, a proline residue in the connection between two of these domains seems to cause a fast hinge motion. These observations help enhance our understanding of protein denatured states, and of folding concepts, in general

Tuning the HNN experiment: generation of serine-threonine check points

We describe here the tunability of the HNN experiment to obtain certain residue specific peak patterns in the spectra of (15N, 13C) labeled proteins. This is achieved by tuning a band-selective 180&#176; pulse on the carbon channel in the pulse sequence, whereby one can tamper with the C&#945;-C&#946; coupling evolutions for the different residues. Specifically, we generate distinctive peak patterns for serine and threonine and their neighbors in the different planes of the three dimensional spectrum. These provide useful anchor points during sequential assignment of backbone resonances. The performance of this experiment, referred to as HNN-ST here, is demonstrated using two proteins, one properly folded and the other completely denatured. With the availability of high field spectrometers, techniques such as TROSY, and ever increasing sensitivities in the probes, this experiment with its large number of check points has a great potential for rapid and unambiguous backbone resonance assignment in large proteins

Comparison of NMR structural and dynamics features of the urea and guanidine-denatured states of GED

Denatured states of proteins, the starting points as well as the intermediates of folding in vivo, play important roles in biological function. In this context, we describe here urea unfolding and characterization of the denatured state of GTPase effector domain (GED) of dynamin created by 9.7 M urea. These are compared with similar data for guanidine induced denaturation reported earlier. The unfolding characteristics in the two cases, as measured by the optical probes, are significantly different, urea unfolding proceeding via an intermediate. The structural and motional characteristics, determined by NMR, of the two denatured states are also strikingly different. The urea-denatured state shows a combination of &#945;- and &#946;-preferences in contrast to the entirely &#946;-preferences in the guanidine-denatured state. Higher 15N transverse relaxation rates suggest higher folding propensities in the urea-denatured state. The implications of these to GED folding are discussed