We describe an integrated workflow that robustly identifies cross-links from endogenous protein complexes in human cellular lysates. Our approach is based on the application of mass spectrometry (MS)-cleavable cross-linkers, sequential collision-induced dissociation (CID)-tandem MS (MS/MS) and electron-transfer dissociation (ETD)-MS/MS acquisitions, and a dedicated search engine, XlinkX, which allows rapid cross-link identification against a complete human proteome database. This approach allowed us to detect 2,179 unique cross-links (1,665 intraprotein cross-links at a 5% false discovery rate (FDR) and 514 interprotein cross-links at 1% FDR) in HeLa cell lysates. We validated the confidence of our cross-linking results by using a target-decoy strategy and mapping the observed cross-link distances onto existing high-resolution structures. Our data provided new structural information about many protein assemblies and captured dynamic interactions of the ribosome in contact with different elongation factors.

Solid-state NMR methods employing (2)H NMR and geometric analysis of labeled alanines (GALA) were used to study the structure and orientation of the transmembrane alpha-helical peptide acetyl-GWW(LA)(8)LWWA-amide (WALP23) in phosphatidylcholine (PC) bilayers of varying thickness. In all lipids the peptide was found to adopt a transmembrane alpha-helical conformation. A small tilt angle of 4.5 degrees was observed in di-18:1-PC, which has a hydrophobic bilayer thickness that approximately matches the hydrophobic length of the peptide. This tilt angle increased slightly but systematically with increasing positive mismatch to 8.2 degrees in di-C12:0-PC, the shortest lipid used. This small increase in tilt angle is insufficient to significantly change the effective hydrophobic length of the peptide and thereby to compensate for the increasing hydrophobic mismatch, suggesting that tilt of these peptides in a lipid bilayer is energetically unfavorable. The tilt and also the orientation around the peptide axis were found to be very similar to the values previously reported for a shorter WALP19 peptide (GWW(LA)(6)LWWA). As also observed in this previous study, the peptide rotates rapidly around the bilayer normal, but not around its helix axis. Here we show that these properties allow application of the GALA method not only to macroscopically aligned samples but also to randomly oriented samples, which has important practical advantages. A minimum of four labeled alanine residues in the hydrophobic transmembrane sequence was found to be required to obtain accurate tilt values using the GALA method.

Phosphatidic acid (PA) is a minor but important phospholipid that, through specific interactions with proteins, plays a central role in several key cellular processes. The simple yet unique structure of PA, carrying just a phosphomonoester head group, suggests an important role for interactions with the positively charged essential residues in these proteins. We analyzed by solid-state magic angle spinning 31 P NMR and molecular dynamics simulations the interaction of low concentrations of PA in model membranes with positively charged side chains of membrane-interacting peptides. Surprisingly, lysine and arginine residues increase the charge of PA, predominantly by forming hydrogen bonds with the phosphate of PA, thereby stabilizing the protein-lipid interaction. Our results demonstrate that this electrostatic/hydrogen bond switch turns the phosphate of PA into an effective and preferred docking site for lysine and arginine residues. In combination with the special packing properties of PA, PA may well be nature's preferred membrane lipid for interfacial insertion of positively charged membrane protein domains. Phosphatidic acid (PA)7 is a minor but important bioactive lipid involved in at least three essential and likely interrelated processes in a typical eukaryotic cell. PA is a key intermediate in the biosynthetic route of the main membrane phospholipids and triglycerides (1); it is involved in membrane dynamics, i.e. fission and fusion (2-4), and has important signaling functions (5-7). The role of PA in membrane dynamics and signaling is most likely 2-fold, either via an effect on the packing properties of the membrane lipids or via the specific binding of effector proteins (4, 7-9). The origin of the specific binding of effector proteins to PA is not known.The PA binding domains thus far identified (for review, see Ref. 10) and more recently (7)) are diverse and share no apparent sequence homology, in contrast to other lipid binding domains, such as e.g. the PH, PX, FYVE, and C2 domains (11-15). One general feature that the PA binding domains do have in common is the presence of basic amino acids (7). Where examined in detail, these basic amino acids were shown to be essential for the interaction with PA, which underscores the importance of electrostatic interactions. Indeed, the negatively charged phosphomonoester head group of PA would be expected to interact electrostatically with basic amino acids in a lipid binding domain. However, such a simple electrostatic interaction cannot explain the strong preference of PA-binding proteins for PA over other, often more abundant, negatively charged phospholipids.In a recent study we showed that the phosphomonoester head group of PA has remarkable properties (8). The phosphomonoester head group is able to form an intramolecular hydrogen bond upon initial deprotonation (when its charge is Ϫ1), which stabilizes the second proton against dissociation. We provided evidence that competing hydrogen bonds, e.g. from the primary amine of the head group of phosphatidylet...

In this review, the synthesis and application of biomedical and pharmaceutical polymers synthesized via the copper(I)-catalyzed alkyne-azide cycloaddition, the thiol-ene reaction, or a combination of both click reactions are discussed. Since the introduction of both "click" methods, numerous articles have disclosed new approaches for the synthesis of polymers with different architectures, e.g., block and graft copolymers, dendrimers, and hydrogels, for pharmaceutical and biomedical applications. By describing selected examples, an overview is given of the possibilities and limitations that these two "click" methods may offer.

Amyloid deposits in the pancreatic islets of Langerhans are thought to be a main factor responsible for death of the insulin-producing islet b-cells in type 2 diabetes. It is hypothesized that b-cell death is related to interaction of the 37 amino acid residue human islet amyloid polypeptide (hIAPP), the major constituent of islet amyloid, with cellular membranes. However, the mechanism of hIAPP-membrane interactions is largely unknown. Here, we study the nature and the molecular details of the initial step of hIAPPmembrane interactions by using the monolayer technique. It is shown that both freshly dissolved hIAPP and the non-amyloidogenic mouse IAPP (mIAPP) have a pronounced ability to insert into phospholipid monolayers, even at lipid packing conditions that exceed the conditions that occur in biological membranes. In contrast, the fibrillar form of hIAPP has lost the ability to insert. These results, combined with the observations that both the insertion kinetics and the dependence of insertion on the initial surface pressure are similar for freshly dissolved hIAPP and mIAPP, indicate that hIAPP inserts into phospholipid monolayers most likely as a monomer. In addition, our results suggest that the N-terminal part of hIAPP, which is nearly identical with that of mIAPP, is largely responsible for insertion. This is supported by experiments with hIAPP fragments, which show that a peptide consisting of the 19 N-terminal residues of hIAPP efficiently inserts into phospholipid monolayers, whereas an amyloidogenic decapeptide, consisting of residues 20-29 of hIAPP, inserts much less efficiently. The results obtained here suggest that hIAPP monomers might insert with high efficiency in biological membranes in vivo. This process could play an important role as a first step in hIAPP-induced membrane damage in type 2 diabetes.

No abstract

To gain insight into the parameters that determine the arrangement of proteins in membranes, 2 H NMR experiments were performed to analyze tilt and rotation angles of membrane-spanning R-helical model peptides upon incorporation in diacylphosphatidylcholine bilayers with varying thickness. The peptides consisted of the sequence acetyl-GW 2 (LA) 8 LW 2 A-NH 2 (WALP23) and analogues thereof, in which the interfacial Trp residues were replaced by Lys (KALP23) and/or the hydrophobic sequence was replaced by Leu (WLP23 and KLP23). The peptides were synthesized with a single deuterium-labeled alanine at four different positions along the hydrophobic segment. For all peptides a small but systematic increase in tilt angle was observed upon decreasing the bilayer thickness. However, significantly larger tilt angles were obtained for the Lys-flanked KALP23 than for the Trp-flanked WALP23, suggesting that interfacial anchoring interactions of Trp may inhibit tilting. Increasing the hydrophobicity resulted in an increase in tilt angle for the Trp-flanked analogue only. For all peptides the maximum tilt angle obtained was remarkably small (less than 12°), suggesting that further tilting is inhibited, most likely due to unfavorable packing of lipids around a tilted helix. The results furthermore showed that the direction of tilt is determined almost exclusively by the flanking residues: Trp-and Lys-flanked peptides were found to have very different rotation angles, which were influenced significantly neither by hydrophobicity of the peptides nor by the extent of hydrophobic mismatch. Finally, very small changes in the side chain angles of the deuterated alanine probes were observed in Trp-flanked peptides, suggesting that these peptides may decrease their hydrophobic length to help them to adapt to thin membranes.Membrane proteins carry out many essential functions in cells. In the specific case of integral proteins, the membrane spanning parts of these molecules are in direct contact with acyl chains and headgroups of the surrounding lipids. Consequently, the lipid environment can modulate structure and dynamics and, hence, activity of membrane proteins (1). For example, changes in hydrophobic thickness of a lipid bilayer can affect the structure of an integral membrane protein by causing a hydrophobic mismatch between the bilayer thickness and the length of the membrane spanning parts of that protein (reviewed in refs 2-4). Understanding the molecular basis of how lipids influence membrane proteins requires precise information on structural properties of transmembrane segments of proteins and knowledge of how these properties are sensitive to protein and lipid composition. It is most suitable to perform such experiments on model membranes of synthetic lipids and transmembrane peptides, since the composition of both the lipids and the peptides can be systematically varied. Model membranes, made of phosphatidylcholine derivatives (PC) 1 and model transmembrane peptides, have already been extensively studied to inves...

This report describes the design and synthesis of a series of a V b 3 integrin-directed monomeric, dimeric and tetrameric cyclo [Arg-Gly-Asp-D-Phe-Lys] dendrimers using "click chemistry". It was found that the unprotected N-e-azido derivative of cyclo[Arg-Gly-Asp-D-Phe-Lys] underwent a highly chemoselective conjugation to amino acid-based dendrimers bearing terminal alkynes using a microwave-assisted Cu(I)-catalyzed 1,3-dipolar cycloaddition. The a V b 3 binding characteristics of the dendrimers were determined in vitro and their in vivo a V b 3 targeting properties were assessed in nude mice with subcutaneously growing human SK-RC-52 tumors. The multivalent RGD-dendrimers were found to have enhanced affinity toward the a V b 3 integrin receptor as compared to the monomeric derivative as determined in an in vitro binding assay. In case of the DOTA-conjugated 111 In-labeled RGD-dendrimers, it was found that the radiolabeled multimeric dendrimers showed specifically enhanced uptake in a V b 3 integrin expressing tumors in vivo. These studies showed that the tetrameric RGD-dendrimer had better tumor targeting properties than its dimeric and monomeric congeners.