<p dir="ltr">Methods of protein engineering and mutation to achieve selective and designed enzymatic function are often challenged by issues with foldamer stability. Molecular nanocages present an exciting new opportunity for biomimetic-defined cavities capable of biomolecule recognition and catalysis. While many different types of molecular cages exist, covalent organic molecular cages offer great flexibility and control over the design of the cage. Furthermore, the covalent linkages provide a robust framework resistant to degradation and stable in many chemical environments. Lastly, covalent organic cages may be designed for the precise placement of functional groups, including group placement inside the cage cavity for molecular recognition and binding. I report our recent advances in developing new synthetic methods for robust organic molecular cages with well-defined cavities and tunable functions for artificial enzyme catalysis and recognition. The basic design philosophy for such protein-mimetic structures will be introduced for the scalable synthesis of these macromolecules. Herein, we report two approaches to a [8+12] triazine-linked organic cage and a similar [8+12] triazine and boroxine-linked cage. While our first approach attempts a kinetically controlled tethered cage formation, our second method relies on the principles of dynamic covalent chemistry in the thermodynamically controlled self-assembly of the final cage structure.</p>

<p>  </p> <p>Ubiquitination is a post-translational modification that activates a variety of signaling pathways. The process of tagging ubiquitin (Ub) onto a substrate protein requires three proteins. First, the E1-activating protein primes Ub for attachment to the E2-conjugating enzymes. The E2-conjugating enzyme then brings Ub to E3 ligases, which also recruit the substrate proteins. The final step of this cascade is the transfer of Ub onto the substrate protein. More commonly, ubiquitinated proteins are then degraded via the proteasome. This cascade to downregulate proteins is employed as a cellular adaptation mechanism in response to various threats, including bacterial and viral pathogens. Although the Ub system exists exclusively in eukaryotes, in recent years many bacterial effector proteins and viral factors have been shown to hijack the system through highly regulated mechanisms. In my Ph.D. work, I characterized the hijacking mechanism of a protein produced by human papillomavirus (HPV) that causes downregulation of p53. Downregulation of p53 leads to the oncogenic effects of HPV infection. A strain of oncogenic HPV, HPV-16, produces the E6 protein, which forms a complex with the human ubiquitin E3 ligase, E6AP. This allows E6AP to recognize p53 for ubiquitination. Furthermore, the ability of E6 to act as an adaptor protein to target unnatural substrate proteins has been employed by medicinal chemists as the basis of <u>pro</u>teolysis <u>ta</u>rgeting <u>c</u>himeras (PROTACs). To this extent, my thesis covers three broad ideas that will add to our understanding of <strong>1) Cellular adaptor protein regulation, 2) viral adaptor protein hijacking, and 3) PROTAC ligand development.</strong></p>

No description available.

No description available.

<p dir="ltr">In life sciences, the determination of macromolecular structures and their functions, particularly proteins and protein complexes, is of paramount importance, as these molecules play critical roles within cells. The specific physical interactions of macromolecules govern molecular and cellular functions, making the 3D structure elucidation of these entities essential for comprehending the mechanisms underlying life processes, diseases, and drug discovery. Cryo-electron microscopy (cryo-EM) has emerged as a promising experimental technique for obtaining 3D macromolecular structures. In the course of my research, I proposed CryoREAD, an innovative AI-based method for <i>de nov</i>o DNA/RNA structure modeling. This novel approach represents the first fully automated solution for DNA/RNA structure modeling from cryo-EM maps at near-atomic resolution. However, as the resolution decreases, structure modeling becomes significantly more challenging. To address this challenge, I introduced Emap2sec+, a 3D deep convolutional neural network designed to identify protein secondary structures, RNA, and DNA information from cryo-EM maps at intermediate resolutions ranging from 5-10 Å. Additionally, I presented Alpha-EM-Multimer, a groundbreaking method for automatically building full protein complexes from cryo-EM maps at intermediate resolution. Alpha-EM-Multimer employs a diffusion model to trace the protein backbone and subsequently fits the AlphaFold predicted single-chain structure to construct the complete protein complex. Notably, this method stands as the first to enable the modeling of protein complexes with more than 10,000 residues for cryo-EM maps at intermediate resolution, achieving an average TM-Score of predicted protein complexes above 0.8, which closely approximates the native structure. Furthermore, I addressed the recognition of local structural errors in predicted and experimental protein structures by proposing DAQ, an evaluation approach for experimental protein structure quality that utilizes detection probabilities derived from cryo-EM maps via a pretrained multi-task neural network. In the pursuit of evaluating protein complexes generated through computational methods, I developed GNN-DOVE and DOVE, leveraging convolutional neural networks and graph neural networks to assess the accuracy of predicted protein complex structures. These advancements in cryo-EM-based structural modeling and evaluation methodologies hold significant promise for advancing our understanding of complex macromolecular systems and their biological implications.</p>

Mathematical models of regulatory networks become more difficult to construct and understand as they grow in size and complexity. Large regulatory network models can be built up from smaller models, representing subsets of reactions within the larger network. This dissertation focuses on novel model construction techniques that extend the ability of biological modelers to construct larger models by supplying them with tools for decomposing models and using the resulting components to construct larger models. Over the last 20 years, molecular biologists have amassed a great deal of information about the genes and proteins that carry out fundamental biological processes within living cells --- processes such as growth and reproduction, movement, signal reception and response, and programmed cell death. The full complexity of these macromolecular regulatory networks is too great to tackle mathematically at the present time. Nonetheless, modelers have had success building dynamical models of restricted parts of the network. Systems biologists need tools now to support composing "submodels" into more comprehensive models of integrated regulatory networks. We have identified and developed four novel processes (fusion, composition, flattening, and aggregation) whose purpose is to support the construction of larger models. Model Fusion combines two or more models in an irreversible manner. In fusion, the identities of the original (sub)models are lost. Beyond some size, fused models will become too complex to grasp and manage as single entities. In this case, it may be more useful to represent large models as compositions of distinct components. In Model Composition one thinks of models not as monolithic entities but rather as collections of smaller components (submodels) joined together. A composed model is built from two or more submodels by describing their redundancies and interactions. While it is appealing in the short term to build larger models from pre-existing models, each developed independently for their own purposes, we believe that ultimately it will become necessary to build large models from components that have been designed for the purpose of combining them. We define Model Aggregation as a restricted form of composition that represents a collection of model elements as a single entity (a "module"). A module contains a definition of pre-determined input and output ports. The process of aggregation (connecting modules via their interface ports) allows modelers to create larger models in a controlled manner. Model Flattening converts a composed or aggregated model with some hierarchy or connections to one without such connections. The relationships used to describe the interactions among the submodels are lost, as the composed or aggregated model is converted into a single large (flat) model. Flattening allows us to use existing simulation tools, which have no support for composition or aggregation. / Ph. D.

Macromolecular assemblies such as the ribosome, spliceosome, polymerases are imperative for cellular functions. The current understanding of these important machineries and many other assemblies at the molecular level is poor. The lack of structural data for many macromolecular assemblies further causes a bottleneck in understanding the cellular processes and the various disease manifestations. Hence, it is essential to characterize the structures and molecular architectures of these macromolecular assemblies. Though the number of 3-D structures for individual proteins structures or domains in the Protein Data Bank (PDB) is growing, the number of structures deposited for macromolecular assemblies is relatively poor. Hence, apart from the use of experimental techniques for characterizing macromolecular assembly structures, the use of computational techniques would help in supplementing the growth of macromolecular assembly structures. This thesis deals with the use of integrative approaches where computational methods are combined with experimental data to model and understand the mechanistic features of macromolecular assemblies with a special focus on a sub-complex of the spliceosome machinery. Chapter 1 of this thesis provides an introduction to protein-protein interactions and macromolecular assemblies. Further, the modelling of macromolecular assemblies using integrative methods are discussed, with a subsequent introduction to the spliceosome machinery. In chapter 2, modelling studies were performed on the proteins involved in the general amino acid control mechanism, which is triggered in yeast under amino acid starvation conditions. The proteins involved in the study were Gcn1, a ribosome binding protein and the RWD-domain containing proteins Gcn2, Yih1, Gir2 and Mtc5. From laboratory experiments it is known that in order for Gcn2 activation, an eIF2α kinase, its RWD-domain has to bind to Gcn1 and the residue Arg-2259 is important for this interaction. As the 3-D structure for the Gcn1 region containing Arg-2259 is not currently available, its 3-D structure was inferred using fold recognition and comparative modelling techniques. Further, in order to understand the Gcn2 RWD domain-Gcn1 molecular interaction, a complex structure was inferred by using a restrained protein-protein docking procedure. As the proteins, Yih1 and Gir2 are known to bind to Gcn1 using their RWD-domains, first the structures of the RWD-domain containing proteins including Mtc5 were inferred using a Gcn2 RWD domain NMR structure. Additionally, the Gcn1-Gcn2 complex was used to build a set of complexes to explain the binding of other RWD domain containing proteins Yih1, Gir2 and Mtc5. The important molecular interactions were obtained on analysing the interacting residues in these complexes. Thus, the Gcn1-Gcn2 interaction at the molecular level has been proposed for the first time. Future experiments guided by the protein-protein complex models and the proposed set of mutations should provide an understanding about the critical molecular interactions involved in the general amino acid control mechanism. Chapter 3 describes an integrative approach that was used to decipher a pseudo-atomic model of the closed form of human SF3b complex. SF3b is a multi-protein complex containing seven components – p14, SF3b49, SF3b155, SF3b145, SF3b130, SF3b14b and SF3b10. It recognizes the branch point adenosine in the pre-mRNA as part of U2 snRNP or U11/U12 di-snRNP in the spliceosome. Although, the cryo-EM map for human SF3b complex has been available for more than a decade, the structure and relative spatial arrangement of all components in the complex are not yet known. The integrative modelling approach used here involved utilizing structural data in the form of available X-ray and NMR structures, fold recognition and comparative modelling as well as currently available experimental datasets, along with the available cryo-EM density map to provide a model with high structural coverage. Hence, the molecular architecture of closed form human SF3b complex was derived that can now provide insights into the functioning of SF3b in splicing. This might also help the future high resolution structure determination efforts of the entire human spliceosome machinery In chapter 4, the molecular architecture of the closed form of SF3b complex obtained from the use of integrative modelling approach (Chapter 3) is extensively discussed. The structure-function relationships for some of the SF3b components based on the pseudo-atomic model has also been provided. In addition, the extreme flexibility associated with some of the SF3b components based on dynamics analysis has also been examined. Further, using an existing U11/U12 di-snRNP cryo-EM map and the closed form SF3b complex pseudo-atomic model, an open form of the SF3b complex was modelled and the component structures were fit into it. Hence, it was found that the transition between closed and open forms is primarily caused by a flap containing the HEAT repeat protein, SF3b155. This Protein is also known to harbour cancer causing mutations and has the potential to affect the Closed to open transition as well as SF3b complex structure and stability. Thus, this provides a framework for the future understanding of the closed to open transition in SF3b functioning within the spliceosome. Chapter 5 builds upon the integrative modelling approach (Chapter 3) that proposed the molecular architecture of the closed form of human SF3b complex and an open form of SF3b that was derived due to a flap opening of the closed form and which might help in accommodating RNA and other trans-acting factors within the U11/U12 di-snRNP (Chapter 4). In the current chapter, the SF3b open form and its interaction with the RNA elements is studied. The 5' end of U12 snRNA and its interaction with pre-mRNA in branch point duplex was modelled guided by the open form of SF3b that provided the necessary structural constraints and the RNA model is topologically consistent with the existing biochemical data. Further, utilizing the SF3b opens form-RNA model and the existing experimental knowledge, an extensive discussion has been provided on how the architecture of SF3b acts as a scaffold for U12 snRNA: pre-mRNA branch point duplex formation as well as its potential implications for branch point adenosine recognition fidelity. Moreover, the reasons for SF3b to be defined as a “fuzzy” complex - a complex with highly flexible folded regions along with intrinsically disordered regions is also discussed. Hence, the current work adds to the excellent developments made previously and deepens the understanding of the structure-function relationship of the human SF3b complex in the context of the spliceosome machinery. In chapter 6, a methodology has been proposed for the use of evolutionary conservation of protein-protein interfacial residues in multiple protein cryo-EM density based fitting of the protein components in the low-resolution density maps of multi-protein assemblies. First, the methodology was tested on a dataset of simulated density maps generated at four different resolutions -10, 15, 20 and 25 Å. On utilizing the evolutionary conservation scores obtained from multiple sequence alignments to score the fitted complexes, it was found that there was a decrease in the conservation scores when compared to that of the crystal structures, which were used to generate the simulated density maps. Further, the assessment of the multiple protein density fitting technique to align the actual protein-protein interface residues correctly using a performance metric called F-measure showed there was a decrease in performance as the resolutions became poorer. Hence, based on evolutionary conservations scores as well as F-measure the decrease in conservation scores or performance was found to be mainly due to the errors associated with the fitting process. Subsequently, a refinement methodology was designed involving the use of conservation scores, which improved the accuracy of the fitted models and the same, was observed in an experimental cryo-EM density test case of RyR1-FKBP12 complex. Hence, the conservation information acts as an effective filter to distinguish the incorrectly fitted structures and improves the accuracy of the fitting of the protein structures in the density maps. Thus, one can incorporate the conserved surface residues information in the current density fitting tools to reduce ambiguity and improve the accuracy of the macromolecular assembly structures determined using cryo-EM. In the concluding chapter 7, the learnings on the structural and mechanistic features of protein assemblies obtained from the use of computational techniques and integration of experimental datasets is discussed. In chapter 2, the modelling of a binary macromolecular complex such as the Gcn1-Gcn2 complex was performed using computational structure prediction strategies to understand the molecular basis of its interaction. Due to the potential inaccuracies which can exist in computational modelling, the chapters 3 to 5 dealt with the use of integrative approaches, primarily guided by the cryo-EM map, in order to decipher the molecular architecture of the human SF3b complex in the closed and open forms as well as its contribution for branch point adenosine recognition. Based on the extensive experience gained in modelling of assemblies using cryo-EM data in the previous chapters, a new method has been proposed on the use of evolutionary conservation information to improve the accuracy of cryo-EM density based fitting. Hence, these studies have provided strategies for modelling macromolecular assemblies as well as a deeper understanding of its mechanistic features.

My thesis is divided into five chapters, starting with a general introduction in first chapter and sample preparation and protein-NMR assignment techniques in second chapter. The remaining three chapters focus on three different areas/projects that I have worked on. Chapter 1: Introduction to nanomaterials and all the experimental techniques This chapter reviews different kinds of nanomaterials and their application utilized for protein-nanomaterial interaction in our study, along with the introduction to different spectroscopy and microscopy techniques used for the interaction studies. Starting with introduction of nanomaterials and all the experimental techniques, which constitute the arsenal for structural studies of the protein-nanomaterial interaction, different steps enroute to structural and dynamic interaction are outlined in detail. Chapter 2: Preparation and Characterization of Proteins used for nanomaterial interaction studies Proteins are generally of three kinds- globular (structured), intrinsically disordered and membrane bound. These proteins have different functions in living organisms and play a major role to maintain metabolism and other important factors. To probe protein-nanomaterial interactions, we have chosen different protein/peptides. This chapter describes the protocol/procedure used for purifying the proteins. For studying a globular protein, ubiquitin was chosen. Nanomaterial-IDP interaction was investigated using the intrinsically disordered central linker domain of human insulin like growth factor binding protein-2 (L-hIGFBP2). The hydrophobic membrane interacting part of the prion protein was chosen as a representative membrane protein. The characterization of the proteins by NMR spectroscopy is also described. Chapter 3: A nanomaterial based novel macromolecular crowding agent Carbon quantum dots (CQD) are nanomaterials with size less than 10 nm, first obtained in 2004 during purification of single-walled carbon-nanotubes. Since then CQDs have been used in a wide range of applications due to their low cost of preparation and favorable properties such as chemical inertness, biocompatibility, non-toxicity and solubility in aqueous medium. One of the applications of CQDs has been their use for imaging and tracking proteins inside cells, based on their intrinsic fluorescence. Further, quantum dots exhibit concentration dependent aggregation while retaining their solubility. Fluorescent carbon quantum dots (CQD) induce macromolecular crowding making them suitable for probing the structure, function and dynamics of both hydrophilic and hydrophobic peptides/ proteins under near in-cell conditions. We have prepared hydrophilic and hydrophobic quantum dots to see the crowding effect. After characterization of CQD, we tested the property of proteins with CQD and found that CQD behaves as a macromolecular crowding agent by mimicking near in-cell conditions. In our study, we have chosen a globular protein, an intrinsically disordered protein (IDP) and one hydrophobic membrane peptide. We have also compared the crowding property of CQD with ficoll which is widely used commercial crowding agent. The overall study tells that the CQD acts like crowding agent and can be used for the study of macromolecular crowding effect. This makes them suitable for structural and functional studies of proteins in near in-cell conditions. Chapter 4: Ubiquitin-Graphene oxide interactions Described here is the interaction of human ubiquitin with GO using NMR spectroscopy and other techniques such as Fluorescence spectroscopy, isothermal titration calorimetry (ITC), UV-Visible spectroscopy, dynamic light scattering (DLS), zeta potential measurements and transmission electron microscopy (TEM). The globular protein ubiquitin interacts with GO and undergoes a dynamic and reversible association-dissociation in a fast exchange regimen as revealed by NMR spectroscopy. The conformation of the protein is not affected and the primary interaction is seen to be electrostatic in nature due to the polar functional groups present on the protein and GO sheet surface. For the first time we have shown that the interaction between ubiquitin and GO is dynamic in nature with fast and reversible adsorption/desorption of protein from the surface of GO. This insight will help in understanding the mechanistic aspects of interaction of GO with cellular proteins and will help in designing appropriate functionalized graphene oxide for its biological application. Chapter 5: Section A: Interaction of an intrinsically disordered protein (L-HIGFBP2) with graphene oxide The interaction between intrinsically disordered linker domain of human insulin-like growth factor binding protein-2 (L-hIGFBP2) with GO was studied using NMR spectroscopy and other techniques such as isothermal titration calorimetry (ITC), dynamic light scattering (DLS), zeta-potential measurements. The study revealed that the disordered protein L-hIGFBP2 interacts with GO through electrostatic interaction and undergoes a dynamic and reversible association-dissociation in a fast exchange regime. The conformation of the protein is not affected. Section B: Stability of an Intrinsically disordered protein through weak interaction with Silver nanoparticles Using NMR spectroscopy and other techniques we probed the mechanism of L-hIGFBP2–AgNP interactions which render the IDP stable. The study reveals a mechanism which involves a relatively fast and reversible association–dissociation of L-hIGFBP2 (dynamic exchange) from the surface of AgNP. The AgNP–L-hIGFBP2 complex remains stable for more than a month. The techniques employed in addition to NMR include UV-Visible spectroscopy, dynamic light scattering (DLS), zeta potential measurements and transmission electron microscopy (TEM) to probe the protein-AgNP interaction here in this section.

No description available.

Thesis (PhD (Chemistry and Polymer Science))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The effects of the macromolecular composition and content of different wood species on the properties of wood-polymer composites (WPCs) achieved when using poly(vinyl alcohol-co-ethylene) (EVOH) as a compatibiliser and linear low density polyethylene (LLDPE) as a matrix, were investigated. Four wood different species (A. cyclops (acacia), E. grandis (eucalyptus), P. radiata (pine) and Q. alba (oak)) with different macromolecular composition and contents and average particle lengths were used. WPCs filled with these species and WPCs filled with the same species but without extractives were prepared using 10% wood content and different amounts (0, 2, 5, 7 and 10%) of EVOH. An EVOH content of 7% was found to be optimum. Unextracted woods produced WPCs with higher mechanical properties and better resistance to ultraviolet (UV) degradation, while the extracted woods produced WPCs with lower water absorption (WA) rates and better thermal stability. Use of unextracted A. cyclops resulted in composites with superior mechanical and thermal properties compared with the other unextracted species, most probably due to its higher cellulose and lignin contents and a favourable average wood particle length (0.225 mm). A. cyclops composites also had higher WA and thickness swelling (TS) rates most likely due to the greater number of free hydroxyl groups present in these composites because of higher cellulose content. Composites containing wood species with a high lignin and extractive content, such as A. cyclops and Q. alba, exhibited higher resistance to UV degradation. Poly(vinyl alcohol-co-ethylenes) (EVOHs) with different ethylene content (27, 32, 38 and 44%) and A. cyclops with different particle sizes (180, 250 and 450 ìm) were used to prepare WPCs with 10% A. cyclops content. The effect of the contact area between the A. cyclops particles and LLDPE achieved when using EVOHs as compatibilisers on the properties of WPCs was also investigated. The greatest improvements in the mechanical and thermal properties of composites made with A. cyclops with particle size 180 ìm were obtained when EVOH with 44% ethylene content was used. The greatest improvements in the composites made with A. cyclops with particle size 250 ìm were achieved when EVOH with 38% ethylene content was used. Composites made with A. cyclops with particle size 450 ìm exhibited better properties when EVOH with 27% ethylene content was used. All the composites that had better mechanical and thermal properties, also exhibited better compatibility and interface adhesion. Two successful approaches were used to impart more attractive ecological and economical advantages to WPCs. In the first approach, (0, 2, 5 and 7%) degraded LLDPE was used as a compatibiliser in WPCs at levels of 10, 30 and 50% wood content. The resulting mechanical properties, such as tensile strength and hardness, thermal and morphological properties of the compatibilised composites were slightly higher than those of noncompatibilised composites and virgin LLDPE. Elongation at break and impact properties of the compatibilised composites were lower than in virgin LLDPE, but higher than in noncompatibilised composites. In the second approach, polyethylene (PE) and various functionalised polyethylenes (PEs) were synthesised by copolymerising ethylene and 10-undecen-1-ol using a soluble metallocene/methylaluminoxane catalyst at room temperature. The incorporation of functional groups increased with increasing comonomer content. WPCs with 10 and 30% wood content were prepared. The composites prepared with functionalised PEs had better mechanical, thermal and morphogical properties than the composites prepared with PE. Composites made with functionalised PE with higher hydroxyl groups content exhibited better properties than composites made with functionalised PE with lower hydroxyl groups content. Composites with 10% wood content exhibited better properties and performance than composites with 30% wood content. / AFRIKAANSE OPSOMMING: Die gevolg van die makromolekulere samestelling van verskillende houtspesies op die eienskappe van hout-polimeer saamgestelde materiale (HPS) wanneer poli(viniel alcohol-ko-etileen) (EVOH) as versoeningsmiddel gebruik word saam met linieere lae digtheid poli(etileen) (LLDPE) as matriks is ondersoek. Vier houtspesies (A. cyclops (acacia), E. grandis (eucalyptus), P. radiata (pine) and Q. alba (oak)) met verskillende makromolekulere samestelling and partikelgrootte-verspreiding is gebruik in die studie. HPS materiale is berei met hierdie vesels, beide voor en na ekstraksie van die houtpartikels met onderskeidelik warm water en oplosmiddels (alleen en in kombinasie). In hierdie HPS materiale is 10% hout gebruik en 0, 2, 5, 7 en 10% EVOH. 'n EVOH inhoud van 7% is as optimum bepaal. Houtpartikels voor ekstraksie het HPS materiale met beter meganiese eienskappe en beter weerstand teen UV bestraling, terwyle partikels wat ekstraksie ondergaan het HPS materiale met laer water-absorpsie en beter hitte-stabiliteit to gevolg gehad het. Die gebruik van ongeekstraheerde A. cyclops het samegestelde materiale met die beste meganiese en termiese eienskappe tot gevolg gehad in vergelyking met die ander houtspesies (voor ekstraksie), as gevolg van die hoer sellulose en lignien inhoud van die spesie, sowel as 'n voordelige partikelgrootte-verspreiding. A. Cyclops saamgestelde materiale he took hoer waterabsorpsie (WA) en dikte-swelling (DS) tempos gehad, weens die groter hoeveelheid vrye hidroksielgroepe teenwoording in die materiale, direk in verwantskap met die sellulose-inhoud. Saamgestelde materiale met 'n hoe hoevellheid lignien en ekstraheerbare materiale (A. cyclops and Q. alba) het beter weerstand teen UV-degradasie geopenbaar. Verskillende poli(viniel alkohol-ko-etileen) polimere (EVOHs) met wisselende etileen-inhoud (27, 32, 38 en 44%) en A. Cyclops met verskillende partikel-groottes (180, 250 en 250 µm) is gebruik om HPS materiale met 10% hout te vervaardig. Die gevolg van die kontak-area tussen die houtpartikels en die LLDPE wanneer EVOHs as versoeningsmiddel gebruik is, is ook ondersoek. Die beste verbetering in die meganiese en termiese eienskappe van die saamgestelde materiale met A. cyclops met partikel-grootte 180 µm is gekry met EVOH met 44% etileen-inhoud, terwyl die beste resultate met 250 µm partikels verkry is met 'n EVOH met 38% etileen, en met 27% etileen in die geval van die 450 µm partikels. Twee benaderinge om meer aantreklike ekologiese en ekonomiese eienskappe by die HPS materiale te bewerkstellig was suksesvol. In die eerste geval is gedegradeerde LLDPE as versoeningsmiddel gebruik. Die resulterende meganiese eienskappe van die HPS materiale met LLDPE as versoeningsmiddel was beter as die HPS mateirale daarsonder. Samegestelde materiale met 10, 30 en 50% hout is vervaardig. Die trekverlenging by die breekpunt sowel as die impaksterkte van die HPS materiale was laer as LLDPE alleen, maar beter as die nie-versoende HPS materiale. In die tweede benadering is polietileen (PE) en gefunksionaliseerde PE gesintetiseer deur etileen en 10-undekeen-1-ol te koplimeriseer met ‘n oplosbare metalloseen/metiel alumoksaan katalis. Die hoeveelheid funskionele (OH) groepe is verhoog deur toenemend ekomonommer-inhoud. HPS materiale met 10 en 30% hout is vervaardig. Die saamgestelde materiale met funksionele PE het beter maganiese eienskappe gehad as die met gewone PE. Hoe hoër die hidroksielgroep-inhoud, hoe beter die eienskappe van die HPS materiale, terwyl die materiale met 10% hout beter eienskappe openbaar het as materiale met 30% hout.