Identification and functional characterization of the Arabidopsis†Snf1-related protein kinase SnRK2.4 phosphatidic acid-binding domain.

Phosphatidic acid (PA) is an important signalling lipid involved in various stress-induced signalling cascades. Two SnRK2 protein kinases (SnRK2.4 and SnRK2.10), previously identified as PA-binding proteins, are shown here to prefer binding to PA over other anionic phospholipids and to associate with cellular membranes in response to salt stress in Arabidopsis roots. A 42 amino acid sequence was identified as the primary PA-binding domain (PABD) of SnRK2.4. Unlike the full-length SnRK2.4, neither the PABD-YFP fusion protein nor the SnRK2.10 re-localized into punctate structures upon salt stress treatment, showing that additional domains of the SnRK2.4 protein are required for its re-localization during salt stress. Within the PABD, five basic amino acids, conserved in class 1 SnRK2s, were found to be necessary for PA binding. Remarkably, plants overexpressing the PABD, but not a non-PA-binding mutant version, showed a severe reduction in root growth. Together, this study biochemically characterizes the PA-SnRK2.4 interaction and shows that functionality of the SnRK2.4 PABD affects root development.

Cell behavior on surface modified polydimethylsiloxane (PDMS).

Designing complex tissue culture systems requires cell alignment and directed extracellular matrix (ECM) and gene expression. Here, a micro-rough, polydimethylsiloxane (PDMS) surface, that also integrates a micro-pattern of 50 µm wide lines of fibronectin (FN) separated by 60 µm wide lines of bovine serum albumin (BSA), is developed. Human fibroblasts cultured on the rough, patterned substrate have aligned growth and a significant change in morphology when compared to cells on a flat, patterned surface. The rough PDMS topography significantly decreases cell area and induces the upregulation of several ECM related genes by two-fold when compared to cells cultured on flat PDMS. This study describes a simple surface engineering procedure for creating surface architecture for scaffolds to design and control the cell-surface interface.

Liposome-binding assays to assess specificity and affinity of phospholipid-protein interactions.

Protein-lipid interactions play an important role in cellular protein relocation, activation and signal transduction. The liposome-binding assay is a simple and inexpensive method to examine protein-lipid binding in vitro. The phospholipids used for liposome production are dried and hydrated. Subsequent extrusion of the phospholipid mixture ensures the production of large unilamellar vesicles (LUV) filled with raffinose. Those LUVs can be easily separated from the aqueous solution by centrifugation. By incubating a protein of interest with the LUVs and subsequent centrifugation steps, the bound protein fraction can be determined using Western Blot or Coomassie staining. This technique enables analysis of protein-lipid binding affinity and specificity.

Properties of membrane-incorporated WALP peptides that are anchored on only one end.

Peptides of the "WALP" family, acetyl-GWW(LA)(n)LWWA-[ethanol]amide, have proven to be opportune models for investigating lipid-peptide interactions. Because the average orientations and motional behavior of the N- and C-terminal Trp (W) residues differ, it is of interest to investigate how the positions of the tryptophans influence the properties of the membrane-incorporated peptides. To address this question, we synthesized acetyl-GGWW(LA)(n)-ethanolamide and acetyl-(AL)(n)WWG-ethanolamide, in which n = 4 or 8, which we designate as "N-anchored" and "C-anchored" peptides, respectively. Selected (2)H or (15)N labels were incorporated for solid-state nuclear magnetic resonance (NMR) spectroscopy. These peptides can be considered "half"-anchored WALP peptides, having only one pair of interfacial Trp residues near either the amino or the carboxyl terminus. The hydrophobic lengths of the (n = 8) peptides are similar to that of WALP23. These longer half-anchored WALP peptides incorporate into lipid bilayers as α-helices, as reflected in their circular dichroism spectra. Solid-state NMR experiments indicate that the longer peptide helices assume defined transmembrane orientations with small non-zero average tilt angles and moderate to high dynamic averaging in bilayer membranes of 1,2-dioleoylphosphatidylcholine, 1,2-dimyristoylphosphatidylcholine, and 1,2-dilauroylphosphatidylcholine. The intrinsically small apparent tilt angles suggest that interactions of aromatic residues with lipid headgroups may play an important role in determining the magnitude of the peptide tilt in the bilayer membrane. The shorter (n = 4) peptides, in stark contrast to the longer peptides, display NMR spectra that are characteristic of greatly reduced motional averaging, probably because of peptide aggregation in the bilayer environment, and CD spectra that are characteristic of ß-structure.

Proline kink angle distributions for GWALP23 in lipid bilayers of different thicknesses.

By using selected (2)H and (15)N labels, we have examined the influence of a central proline residue on the properties of a defined peptide that spans lipid bilayer membranes by solid-state nuclear magnetic resonance (NMR) spectroscopy. For this purpose, GWALP23 (acetyl-GGALW(5)LALALALALALALW(19)LAGA-ethanolamide) is a suitable model peptide that employs, for the purpose of interfacial anchoring, only one tryptophan residue on either end of a central α-helical core sequence. Because of its systematic behavior in lipid bilayer membranes of differing thicknesses [Vostrikov, V. V., et al. (2010) J. Biol. Chem. 285, 31723-31730], we utilize GWALP23 as a well-characterized framework for introducing guest residues within a transmembrane sequence; for example, a central proline yields acetyl-GGALW(5)LALALAP(12)ALALALW(19)LAGA-ethanolamide. We synthesized GWALP23-P12 with specifically placed (2)H and (15)N labels for solid-state NMR spectroscopy and examined the peptide orientation and segmental tilt in oriented DMPC lipid bilayer membranes using combined (2)H GALA and (15)N-(1)H high-resolution separated local field methods. In DMPC bilayer membranes, the peptide segments N-terminal and C-terminal to the proline are both tilted substantially with respect to the bilayer normal, by ~34 ± 5° and 29 ± 5°, respectively. While the tilt increases for both segments when proline is present, the range and extent of the individual segment motions are comparable to or smaller than those of the entire GWALP23 peptide in bilayer membranes. In DMPC, the proline induces a kink of ~30 ± 5°, with an apparent helix unwinding or "swivel" angle of ~70°. In DLPC and DOPC, on the basis of (2)H NMR data only, the kink angle and swivel angle probability distributions overlap those of DMPC, yet the most probable kink angle appears to be somewhat smaller than in DMPC. As has been described for GWALP23 itself, the C-terminal helix ends before Ala(21) in the phospholipids DMPC and DLPC yet remains intact through Ala(21) in DOPC. The dynamics of bilayer-incorporated, membrane-spanning GWALP23 and GWALP23-P12 are less extensive than those observed for WALP family peptides that have more than two interfacial Trp residues.