Synthesis, self-assembly and (absence of) protein interactions of poly(glycerol methacrylate)-silicone macro-amphiphiles

The present study focuses on the synthesis of amphiphilic block copolymers containing poly(glycerol monomethacrylate) (PGMMA), showing the advantages of a protection/deprotection strategy based on silyl groups. PGMMA blocks were synthesized via ATRP started by a double functional poly(dimethyl siloxane) (PDMS) macroinitiator of molecular weight ≈7000 g mol-1. The resulting triblock copolymers were characterized by low polydispersity (generally ≤1.1) and their aggregation concentration in water was essentially dominated by the PDMS block length (critical aggregation concentration substantially invariant for GMMA degree of polymerization ≥30). For GMMA blocks with DP > 50, the self-assembly in water produced 35-50 nm spherical micelles, while shorter hydrophilic chains produced larger aggregates apparently displaying worm-like morphologies. Block copolymers with long GMMA chains (DP ≈ 200) produced particularly stable micellar aggregates, which were then selected for a preliminary assessment of the possibility of adsorption of plasma proteins (albumin and fibrinogen); using diffusion NMR as an analytical technique, no significant adsorption was recorded both on micelles and on soluble PGMMA employed as a control, indicating the possibility of a "stealth" behaviour. This journal is © 2013 The Royal Society of Chemistry.

Self-assembly of trehalose molecules on a lysozyme surface: the broken glass hypothesis

To help understand how sugar interactions with proteins stabilise biomolecular structures, we compare the three main hypotheses for the phenomenon with the results of long molecular dynamics simulations on lysozyme in aqueous trehalose solution (0.75 M). We show that the water replacement and water entrapment hypotheses need not be mutually exclusive, because the trehalose molecules assemble in distinctive clusters on the surface of the protein. The flexibility of the protein backbone is reduced under the sugar patches supporting earlier findings that link reduced flexibility of the protein with its higher stability. The results explain the apparent contradiction between different experimental and theoretical results for trehalose effects on proteins.

Identifying a novel functional domain within the p115 tethering factor required for Golgi ribbon assembly and membrane trafficking

The tethering factor p115 has been shown to facilitate Golgi biogenesis and membrane traffic in cells in culture. However, the role of p115 within an intact animal is largely unknown. Here, we document that RNAi-mediated depletion of p115 in C. elegans causes accumulation of the yolk protein (YP170) in body cavity and the retention of the yolk receptor RME-2 in the ER and the Golgi within oocytes.Structure-function analyses of p115 have identified two homology (H1-2) regions within the N-terminal globular head and the coiled-coil 1 (CC1) domain as essential for p115 function. We identify a novel C-terminal domain of p115 as necessary for Golgi ribbon formation and cargo trafficking. We show that p115 mutants lacking the fourth CC domain (CC4) act in a dominant negative manner to disrupt Golgi and prevent cargo trafficking in cells containing endogenous p115. Furthermore, using RNAi-mediated "replacement" strategy we show that CC4 is necessary for Golgi ribbon formation and membrane trafficking in cells depleted of endogenous p115.p115 has been shown to bind a subset of ER-Golgi SNAREs through CC1 and CC4 domains (Shorter et al., 2002). Our findings show that CC4 is required for p115 function and suggest that both the CC1 and the CC4 SNARE-binding motifs may participate in p115-mediated membrane tethering.

A proteo-liposome system for the analysis of the intracellular interactome of membrane proteins using amyloid precursor protein as a model

Transmembrane proteins play crucial roles in many important physiological processes. The intracellular domain of membrane proteins is key for their function by interacting with a wide variety of cytosolic proteins. It is therefore important to examine this interaction. A recently developed method to study these interactions, based on the use of liposomes as a model membrane, involves the covalent coupling of the cytoplasmic domains of membrane proteins to the liposome membrane. This allows for the analysis of interaction partners requiring both protein and membrane lipid binding. This thesis further establishes the liposome recruitment system and utilises it to examine the intracellular interactome of the amyloid precursor protein (APP), most well-known for its proteolytic cleavage that results in the production and accumulation of amyloid beta fragments, the main constituent of amyloid plaques in Alzheimer’s disease pathology. Despite this, the physiological function of APP remains largely unclear. Through the use of the proteo-liposome recruitment system two novel interactions of APP’s intracellular domain (AICD) are examined with a view to gaining a greater insight into APP’s physiological function. One of these novel interactions is between AICD and the mTOR complex, a serine/threonine protein kinase that integrates signals from nutrients and growth factors. The kinase domain of mTOR directly binds to AICD and the N-terminal amino acids of AICD are crucial for this interaction. The second novel interaction is between AICD and the endosomal PIKfyve complex, a lipid kinase involved in the production of phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) from phosphatidylinositol-3-phosphate, which has a role in controlling ensdosome dynamics. The scaffold protein Vac14 of the PIKfyve complex binds directly to AICD and the C-terminus of AICD is important for its interaction with the PIKfyve complex. Using a recently developed intracellular PI(3,5)P2 probe it is shown that APP controls the formation of PI(3,5)P2 positive vesicular structures and that the PIKfyve complex is involved in the trafficking and degradation of APP. Both of these novel APP interactors have important implications of both APP function and Alzheimer’s disease. The proteo-liposome recruitment method is further validated through its use to examine the recruitment and assembly of the AP-2/clathrin coat from purified components to two membrane proteins containing different sorting motifs. Taken together this thesis highlights the proteo-liposome recruitment system as a valuable tool for the study of membrane proteins intracellular interactome. It allows for the mimicking of the protein in its native configuration therefore identifying weaker interactions that are not detected by more conventional methods and also detecting interactions that are mediated by membrane phospholipids.

The automation of protein crystal presentation for x ray diffraction experiments using standing acoustic waves in a microfluidic chip environment

As the pressure continues to grow on Diamond and the world's synchrotrons for higher throughput of diffraction experiments, new and novel techniques are required for presenting micron dimension crystals to the X ray beam. Currently this task is both labour intensive and primarily a serial process. Diffraction measurements typically take milliseconds but sample preparation and presentation can reduce throughput down to 4 measurements an hour. With beamline waiting times as long as two years it is of key importance for researchers to capitalize on available beam time, generating as much data as possible. Other approaches detailed in the literature [1] [2] [3] are very much skewed towards automating, with robotics, the actions of a human protocols. The work detailed here is the development and discussion of a bottom up approach relying on SSAW self assembly, including material selection, microfluidic integration and tuning of the acoustic cavity to order the protein crystals.

The automation of protein crystal presentation for X ray diffraction experiments using standing acoustic waves in a microfluidic chip environment

As the pressure continues to grow on Diamond and the world's synchrotrons for higher throughput of diffraction experiments, new and novel techniques are required for presenting micron dimension crystals to the X ray beam. Currently this task is both labour intensive and primarily a serial process. Diffraction measurements typically take milliseconds but sample preparation and presentation can reduce throughput down to 4 measurements an hour. With beamline waiting times as long as two years it is of key importance for researchers to capitalize on available beam time, generating as much data as possible. Other approaches detailed in the literature [1] [2] [3] are very much skewed towards automating, with robotics, the actions of a human protocols. The work detailed here is the development and discussion of a bottom up approach relying on SSAW self assembly, including material selection, microfluidic integration and tuning of the acoustic cavity to order the protein crystals.