12 research outputs found
UvA-DARE (Digital Academic Repository) Link to publication Citation for published version (APA): Identification and functional characterization of the Arabidopsis Snf1-related protein kinase SnRK2.4 phosphatidic acid-binding domain
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Identification and functional characterization of the Arabidopsisâ 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
Properties of Membrane-Incorporated WALP Peptides That Are Anchored on Only One End
Peptides of the âWALPâ family, acetyl-GWWÂ(LA)<sub><i>n</i></sub>LWWA-[ethanol]Âamide, have proven to be opportune
models for investigating lipidâpeptide 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-GGWWÂ(LA)<sub><i>n</i></sub>-ethanolamide and acetyl-(AL)<sub><i>n</i></sub>WWG-ethanolamide, in which <i>n</i> = 4
or 8, which we designate as âN-anchoredâ and âC-anchoredâ
peptides, respectively. Selected <sup>2</sup>H or <sup>15</sup>N labels
were incorporated for solid-state nuclear magnetic resonance (NMR)
spectroscopy. These peptides can be considered âhalfâ-anchored
WALP peptides, having only one pair of interfacial Trp residues near
either the amino or the carboxyl terminus. The hydrophobic lengths
of the (<i>n</i> = 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 (<i>n</i> = 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 ÎČ-structure
Proline Kink Angle Distributions for GWALP23 in Lipid Bilayers of Different Thicknesses
By using selected <sup>2</sup>H and <sup>15</sup>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<sup>5</sup>LALALALALALALW<sup>19</sup>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) <i>J. Biol. Chem. 285</i>, 31723â31730],
we utilize GWALP23 as a well-characterized framework for introducing
guest residues within a transmembrane sequence; for example, a central
proline yields acetyl-GGALW<sup>5</sup>LALALAP<sup>12</sup>ALALALW<sup>19</sup>LAGA-ethanolamide. We synthesized GWALP23-P12 with specifically
placed <sup>2</sup>H and <sup>15</sup>N labels for solid-state NMR
spectroscopy and examined the peptide orientation and segmental tilt
in oriented DMPC lipid bilayer membranes using combined <sup>2</sup>H GALA and <sup>15</sup>Nâ<sup>1</sup>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 âŒ34 ± 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 âŒ30 ± 5°, with an apparent helix unwinding
or âswivelâ angle of âŒ70°. In DLPC and DOPC,
on the basis of <sup>2</sup>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<sup>21</sup> in the phospholipids DMPC and DLPC yet remains intact
through Ala<sup>21</sup> 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