Co-refolding of a functional complex of Dengue NS3 protease and NS2B co-factor domain and backbone resonance assignment by solution NMR.

A novel approach for separate expression of dengue virus NS3 protease and its NS2B cofactor domain is described in this paper. The two proteins are expressed in E.coli and purified separately and subsequently efficiently co-refolded to form a stable complex. This straightforward and robust method allows for separate isotope labeling of the two proteins, facilitating analysis by nuclear magnetic resonance (NMR) spectroscopy. Unlinked NS2B-NS3pro behaves better in NMR spectroscopy than linked NS2B-NS3pro, which has resulted in the backbone resonance assignment of the unlinked NS2B-NS3 complex bound to a peptidic boronic acid inhibitor.

Backbone Assignment of the MALT1 Paracaspase by Solution NMR.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a unique paracaspase protein whose protease activity mediates oncogenic NF-κB signalling in activated B cell-like diffuse large B cell lymphomas (ABC-DLBCLs). ABC-DLBCLs are aggressive lymphomas with high resistance to current chemotherapies. Low survival rate among patients emphasizes the urgent need for alternative treatment options. The characterization of the MALT1 will be an essential tool for developing new target-directed drugs against MALT1 dependent disorders. As the first step in the atomic-level NMR studies of the system, here we report, the (15)N/(13)C/(1)H backbone assignment of the apo form of the MALT1 paracaspase region together with the third immunoglobulin-like (Ig3) domain, 44 kDa, by high resolution NMR. In addition, the non-uniform sampling (NUS) based targeted acquisition procedure is evaluated as a mean of decreasing acquisition and analysis time for larger proteins.

pH-Dependent Interaction between C-Peptide and Phospholipid Bicelles.

C-peptide is the connecting peptide between the A and B chains of insulin in proinsulin. In this paper, we investigate the interaction between C-peptide and phospholipid bicelles, by circular dichroism and nuclear magnetic resonance spectroscopy, and in particular the pH dependence of this interaction. The results demonstrate that C-peptide is largely unstructured independent of pH, but that a weak structural induction towards a short stretch of ß-sheet is induced at low pH, corresponding to the isoelectric point of the peptide. Furthermore, it is demonstrated that C-peptide associates with neutral phospholipid bicelles as well as acidic phospholipid bicelles at this low pH. C-peptide does not undergo a large structural rearrangement as a consequence of lipid interaction, which indicates that the folding and binding are uncoupled. In vivo, local variations in environment, including pH, may cause C-peptide to associate with lipids, which may affect the aggregation state of the peptide.

NMR investigations of the dual targeting peptide of Thr-tRNA synthetase and its interaction with the mitochondrial Tom20 receptor in Arabidopsis thaliana.

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins containing an N-terminal targeting peptide and are imported into mitochondria through the import machineries, the translocase of the outer mitochondrial membrane (TOM) and the translocase of the inner mitochondrial membrane (TIM). The N-terminal targeting peptide of precursor proteins destined for the mitochondrial matrix is recognized by the Tom20 receptor and plays an important role in the import process. Protein import is usually organelle specific, but several plant proteins are dually targeted into mitochondria and chloroplasts using an ambiguous dual targeting peptide. We present NMR studies of the dual targeting peptide of Thr-tRNA synthetase and its interaction with Tom20 in Arabidopsis thaliana. Our findings show that the targeting peptide is mostly unstructured in buffer, with a propensity to form α-helical structure in one region, S6-F27, and a very weak ß-strand propensity for Q34-Q38. The α-helical structured region has an amphiphilic character and a φχχφφ motif, both of which have previously been shown to be important for mitochondrial import. Using NMR we have mapped out two regions in the peptide that are important for Tom20 recognition: one of them, F9-V28, overlaps with the amphiphilic region, and the other comprises residues L30-Q39. Our results show that the targeting peptide may interact with Tom20 in several ways. Furthermore, our results indicate a weak, dynamic interaction. The results provide for the first time molecular details on the interaction of the Tom20 receptor with a dual targeting peptide.

Membrane-perturbing properties of two Arg-rich paddle domains from voltage-gated sensors in the KvAP and HsapBK K(+) channels.

Voltage-gated K(+) channels are gated by displacement of basic residues located in the S4 helix that together with a part of the S3 helix, S3b, forms a "paddle" domain, whose position is altered by changes in the membrane potential modulating the open probability of the channel. Here, interactions between two paddle domains, KvAPp from the K(v) channel from Aeropyrum pernix and HsapBKp from the BK channel from Homo sapiens, and membrane models have been studied by spectroscopy. We show that both paddle domains induce calcein leakage in large unilamellar vesicles, and we suggest that this leakage represents a general thinning of the bilayer, making movement of the whole paddle domain plausible. The fact that HsapBKp induces more leakage than KvAPp may be explained by the presence of a Trp residue in HsapBKp. Trp residues generally promote localization to the hydrophilic-hydrophobic interface and disturb tight packing. In magnetically aligned bicelles, KvAPp increases the level of order along the whole acyl chain, while HsapBKp affects the morphology, also indicating that KvAPp adapts more to the lipid environment. Nuclear magnetic resonance (NMR) relaxation measurements for HsapBKp show that overall the sequence has anisotropic motions. The S4 helix is well-structured with restricted local motion, while the turn between S4 and S3b is more flexible and undergoes slow local motion. Our results indicate that the calcein leakage is related to the flexibility in this turn region. A possibility by which HsapBKp can undergo structural transitions is also shown by relaxation NMR, which may be important for the gating mechanism.

Structural characterization of AS1-membrane interactions from a subset of HAMP domains.

HAMP domains convert an extracellular sensory input into an intracellular signaling response in a wide variety of membrane-embedded bacterial proteins. These domains are almost invariably found adjacent to the inner leaflet of the cell membrane. We therefore examined the interaction of peptides corresponding to either AS1 or AS2 of four different, well-characterized HAMP domains with several membrane model systems. The proteins included an Archaeoglobus fulgidus protein (Af1503), the Escherichia coli osmosensor EnvZ(Ec), the E. coli nitrate/nitrite sensor NarX(Ec), and the aspartate chemoreceptor of E. coli (Tar(Ec)). Far-UV CD and NMR spectroscopy were used to monitor the induction of secondary structure upon association with neutral or acidic large unilamellar vesicles (LUVs) and bicelles. We observed significant increases in α-helicity within AS1 from NarX(Ec) and Tar(Ec) but not in AS1 from the other proteins. To characterize these interactions further, we determined the solution structure of AS1 from Tar(Ec) associated with acidic bicelles. The bulk of AS1 formed an amphipathic α-helix, whereas the N-terminal control cable, the region between TM2 and AS1, remained unstructured. We observed that the conserved prolyl residue found in AS1 of many membrane-adjacent HAMP domains defined the boundary between the unstructured and helical regions. In addition, two positively charged residues that flank the hydrophobic surface of AS1 are thought to facilitate electrostatic interactions with the membrane. We interpret these results within the context of the helix-interaction model for HAMP signaling and propose roles for AS1-membrane interactions during the membrane assembly and transmembrane communication of HAMP-containing receptors.

Solution structure of the HsapBK K+ channel voltage-sensor paddle sequence.

Voltage-gated potassium channels open and close in response to changes in the membrane potential. In this study, we have determined the NMR solution structure of the putative S3b-S4 voltage-sensor paddle fragment, the part that moves to mediate voltage gating, of the HsapBK potassium channel in dodecylphosphocholine (DPC) micelles. This paper presents the first structure of the S3b-S4 fragment from a BK channel. Diffusion coefficients as determined from PFG NMR experiments showed that a well-defined complex between the peptide and DPC molecules was formed. The structure reveals a helix-turn-helix motif, which is in agreement with crystal structures of other voltage-gated potassium channels, thus indicating that it is feasible to study the isolated fragment. The paddle motifs generally contain several basic residues, implicated in the gating. The critical Arg residues in this structure all reside on the surface, which is in agreement with crystal structures of K(v) channels. Similarities in the structure of the S3b-S4 fragment in BK and K(v) channels as well as important differences are seen, which may be important for explaining the details in paddle movement within a bilayer.