Anna E. Daily

Charged or aromatic anchor residue dependence of transmembrane peptide tilt.

The membrane-spanning segments of integral membrane proteins often are flanked by aromatic or charged amino acid residues, which may “anchor” the transmembrane orientation. Single spanning transmembrane peptides such as those of the WALP family, acetyl-GWW(LA)nLWWA-amide, furthermore adopt a moderate average tilt within lipid bilayer membranes. To understand the anchor residue dependence of the tilt, we introduce Leu-Ala “spacers” between paired anchors and in some cases replace the outer tryptophans. The resulting peptides, acetyl-GX2ALW(LA)6LWLAX22A-amide, have Trp, Lys, Arg, or Gly in the two X positions. The apparent average orientations of the core helical sequences were determined in oriented phosphatidylcholine bilayer membranes of varying thickness using solid-state 2H NMR spectroscopy. When X is Lys, Arg, or Gly, the direction of the tilt is essentially constant in different lipids and presumably is dictated by the tryptophans (Trp5 and Trp19) that flank the inner helical core. The Leu-Ala spacers are no longer helical. The magnitude of the apparent helix tilt furthermore scales nicely with the bilayer thickness except when X is Trp. When X is Trp, the direction of tilt is less well defined in each phosphatidylcholine bilayer and varies up to 70° among 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, and 1,2-dilauroyl-sn-glycero-3-phosphocholine bilayer membranes. Indeed, the X = Trp case parallels earlier observations in which WALP family peptides having multiple Trp anchors show little dependence of the apparent tilt magnitude on bilayer thickness. The results shed new light on the interactions of arginine, lysine, tryptophan, and even glycine at lipid bilayer membrane interfaces.

A Dynamic cpSRP43-Albino3 Interaction Mediates Translocase Regulation of Chloroplast Signal Recognition Particle (cpSRP)-targeting Components

The chloroplast signal recognition particle (cpSRP) and its receptor, chloroplast FtsY (cpFtsY), form an essential complex with the translocase Albino3 (Alb3) during post-translational targeting of light-harvesting chlorophyll-binding proteins (LHCPs). Here, we describe a combination of studies that explore the binding interface and functional role of a previously identified cpSRP43-Alb3 interaction. Using recombinant proteins corresponding to the C terminus of Alb3 (Alb3-Cterm) and various domains of cpSRP43, we identify the ankyrin repeat region of cpSRP43 as the domain primarily responsible for the interaction with Alb3-Cterm. Furthermore, we show Alb3-Cterm dissociates a cpSRP·LHCP targeting complex in vitro and stimulates GTP hydrolysis by cpSRP54 and cpFtsY in a strictly cpSRP43-dependent manner. These results support a model in which interactions between the ankyrin region of cpSRP43 and the C terminus of Alb3 promote distinct membrane-localized events, including LHCP release from cpSRP and release of targeting components from Alb3.

Is there a preferential interaction between cholesterol and tryptophan residues in membrane proteins

Recently, several indications have been found that suggest a preferential interaction between cholesterol and tryptophan residues located near the membrane-water interface. The aim of this study was to investigate by direct methods how tryptophan and cholesterol interact with each other and what the possible consequences are for membrane organization. For this purpose, we used cholesterol-containing model membranes of dimyristoylphosphatidylcholine (DMPC) in which a transmembrane model peptide with flanking tryptophans [acetyl-GWW(LA)8LWWA-amide], called WALP23, was incorporated to mimic interfacial tryptophans of membrane proteins. These model systems were studied with two complementary methods. (1) Steady-state and time-resolved Förster resonance energy transfer (FRET) experiments employing the fluorescent cholesterol analogue dehydroergosterol (DHE) in combination with a competition experiment with cholesterol were used to obtain information about the distribution of cholesterol in the bilayer in the presence of WALP23. The results were consistent with a random distribution of cholesterol which indicates that cholesterol and interfacial tryptophans are not preferentially located next to each other in these bilayer systems. (2) Solid-state 2H NMR experiments employing either deuterated cholesterol or indole ring-deuterated WALP23 peptides were performed to study the orientation and dynamics of both molecules. The results showed that the quadrupolar splittings of labeled cholesterol were not affected by an interaction with tryptophan-flanked peptides and, vice versa, that the quadrupolar splittings of labeled indole rings in WALP23 are not significantly influenced by addition of cholesterol to the bilayer. Therefore, both NMR and fluorescence spectroscopy results independently show that, at least in the model systems studied here, there is no evidence for a preferential interaction between cholesterol and tryptophans located at the bilayer interface.

NMR characterization of copper and lipid interactions of the C2B domain of synaptotagmin I—relevance to the non-classical secretion of the human acidic fibroblast growth factor (hFGF-1)

Human fibroblast growth factor (hFGF-1) is a approximately 17 kDa heparin binding cytokine. It lacks the conventional hydrophobic N-terminal signal sequence and is secreted through non-classical secretion routes. Under stress, hFGF-1 is released as a multiprotein complex consisting of hFGF-1, S100A13 (a calcium binding protein), and p40 synaptotagmin (Syt1). Copper (Cu(2+)) is shown to be required for the formation of the multiprotein hFGF-1 release complex (Landriscina et al. ,2001; Di Serio et al., 2008). Syt1, containing the lipid binding C2B domain, is believed to play an important role in the eventual export of the hFGF-1 across the lipid bilayer. In this study, we characterize Cu(2+) and lipid interactions of the C2B domain of Syt1 using multidimensional NMR spectroscopy. The results highlight how Cu(2+) appears to stabilize the protein bound to pS vesicles. Cu(2+) and lipid binding interface mapped using 2D (1)H-(15)N heteronuclear single quantum coherence experiments reveal that residues in beta-strand I contributes to the unique Cu(2+) binding site in the C2B domain. In the absence of metal ions, residues located in Loop II and beta-strand IV contribute to binding to unilamelar pS vesicles. In the presence of Cu(2+), additional residues located in Loops I and III appear to stabilize the protein-lipid interactions. The results of this study provide valuable information towards understanding the molecular mechanism of the Cu(2+)-induced non-classical secretion of hFGF-1.

Copper binding affinity of the C2B domain of synaptotagmin-1 and its potential role in the nonclassical secretion of acidic fibroblast growth factor

Fibroblast growth factor 1 (FGF1) is a heparin-binding proangiogenic protein. FGF1 lacks the conventional N-terminal signal peptide required for secretion through the endoplasmic reticulum (ER)-Golgi secretory pathway. FGF1 is released through a Cu(2+)-mediated nonclassical secretion pathway. The secretion of FGF1 involves the formation of a Cu(2+)-mediated multiprotein release complex (MRC) including FGF1, S100A13 (a calcium-binding protein) and p40 synaptotagmin (Syt1). It is believed that the binding of Cu(2+) to the C2B domain is important for the release of FGF1 into the extracellular medium. In this study, using a variety of biophysical studies, Cu(2+) and lipid interactions of the C2B domain of Syt1 were characterized. Isothermal titration calorimetry (ITC) experiments reveal that the C2B domain binds to Cu(2+) in a biphasic manner involving an initial endothermic and a subsequent exothermic phase. Fluorescence energy transfer experiments using Tb(3+) show that there are two Cu(2+)-binding pockets on the C2B domain, and one of these is also a Ca(2+)-binding site. Lipid-binding studies using ITC demonstrate that the C2B domain preferentially binds to small unilamellar vesicles of phosphatidyl serine (PS). Results of the differential scanning calorimetry and limited trypsin digestion experiments suggest that the C2B domain is marginally destabilized upon binding to PS vesicles. These results, for the first time, suggest that the main role of the C2B domain of Syt1 is to serve as an anchor for the FGF1 MRC on the membrane bilayer. In addition, the binding of the C2B domain to the lipid bilayer is shown to significantly decrease the binding affinity of the protein to Cu(2+). The study provides valuable insights on the sequence of structural events that occur in the nonclassical secretion of FGF1.

Response to Falk and Sinning: The C Terminus of Alb3 Interacts with the Chromodomains 2 and 3 of cpSRP43

This is a response to a letter by Falk and Sinning (1) We recently identified the ankyrin region of cpSRP43 as the primary domain responsible for binding Alb3-Cterm during light-harvesting chlorophyll-binding protein (LHCP) targeting, an interaction shown to facilitate cpSRP43-dependent stimulation of cpSRP GTPases by Alb3-Cterm (2). Falk et al. (3), using only protein interaction assays, report that CD2CD3 of cpSRP43 forms the Alb3-Cterm binding interface, which appears inconsistent with the fact that CD3 is not required for LHCP integration (4) and that CD2 is not required for LHCP integration by a cpSRP54/cpFtsY-independent pathway that relies on cpSRP43/Alb3 (5). We suggested that buffer choice, including the use of glycerol, may play a role in why Falk et al. (3) observed μm rather than nm affinity for cpSRP43 constructs (2). The use of high concentrations of glycerol in isothermal titration calorimetry (ITC) is known to cause experimental artifacts (6). Our control experiments clearly show that the use of glycerol, even at 5% v/v, causes significant background heat changes (Fig. 1). In their letter, Sinning and Falk report a 13 μm affinity even in the absence of glycerol, suggesting glycerol may not be the primary cause for the reported differences. Although species-specific differences in Alb3-Cterm could explain the observed affinity differences, comparing GTPase stimulation by Arabidopsis and Pisum sativum Alb3-Cterm does not support this possibility (Fig. 2). FIGURE 1. Isothermal titration calorimetry investigation of the influence of glycerol in various buffers in buffer to buffer experiments. ITC was conducted by injecting a specific buffer/glycerol formulation into a sample well containing the same buffer/glycerol ... FIGURE 2. Comparison of the ability of Pisum sativum and Arabidopsis thaliana Alb3-Cterm peptide to stimulate cpSRP43-dependent GTP hydrolysis by the cpSRP GTPases. The effect of Alb3-Cterm on the GTP hydrolysis activity of cpSRP54 and cpFtsY was examined in the ... Published reports (2, 4, 5) supporting the physiological relevance of high affinity protein interactions still suggest that the low affinity of cpSRP43 for Alb3-Cterm reported by Falk et al. (3) stems from assay conditions unfavorable for observing the primary targeting interaction that takes place between Alb3-Cterm and the Ank region of cpSRP43. Importantly, buffers used by Falk et al. (3) in ITC and size-exclusion experiments do not support LHCP integration (Fig. 3). In addition, Ank-containing cpSRP43 constructs, including those that lack CD2 and/or CD3, are able to prevent binding of radiolabeled cpSRP43 to Alb3 in salt-washed thylakoids whereas chromodomains do not (Fig. 4). FIGURE 3. Buffer influence on LHCP integration. Salt-washed thylakoids in IBM were incubated with 5 mm ATP, 1 mm GTP, 12.5 μl of radiolabeled pLHCP translation product, and recombinant cpSRP43, cpSRP54, and cpFtsY (2). The final volume was brought to 150 ... FIGURE 4. Competition for cpSRP43 binding to the C terminus of Alb3 in salt-washed thylakoids. Salt-washed thylakoids (equivalent to 75 μg of chlorophyll) and 8 nmol of recombinant cpSRP43 construct as indicated were incubated for 15 min at 25 °C ...