Browsing by Author "Linder, Markus, Prof., Aalto University, Department of Bioproducts and Biosystems, Finland"

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  • Analytical ultracentrifugation as a tool to understand interactions in biomolecular materials
    (2024) Fedorov, Dmitrii
     School of Chemical Technology | Doctoral dissertation (article-based)
    The modern world dictates new rules and requirements for materials used in various applications. The development of human society has reached a point where, in addition to the requirements for good functional properties, the requirements for safety and sustainability of the materials are also increasing. Bio-based materials satisfy all these criteria. They are functional, sustainable, renewable and have a good mechanical properties. One of the most promising representatives is spider silk, the fibers of which are formed on the basis of triblock proteins. Spider silk stands out for its excellent mechanical properties. However, to achieve the properties similar to native spider silk using artificial analogues, a deep understanding of processes and interactions at the molecular level is required. In overall this is the true for any materials. The lack of the knowledge about molecular interactions is critical and significantly complicates the material development. It can be compared to forging a sword when one knows nothing about metal processing. Theoretically, it is possible to forge a sword that looks like a sword. Applying a lot of efforts, one even can make it beautiful. But most likely it will be either fragile or too soft, since the person does not know how to process it correctly. Applying even more efforts, after many attempts, one can find the conditions under which the sword would have the desired properties, but what if after sword it is needed to forge a horseshoe, for example. There could be different requirements for properties and all the process of searching the best conditions will be repeated all over again. However, knowing the rules of hardening and alloying of iron, everything would be much simpler. Absolutely the same situation occurs with biomaterials and intermolecular interactions. This work devoted to certain gaps in the understanding of protein interactions at the molecular level, how they affect intermediate states such as liquid-liquid phase separation (LLPS), shows the importance of understanding the interactions, and also demonstrates the capabilities of Analytical Ultracentrifugation (AUC) in combination with other techniques for material science application. Publication 1 demonstrates the effect of terminal domains on LLPS of engineered three-block spider silk proteins. The discovered patterns will serve for creation of coacervates with controlled properties. Publication 2 is devoted to the stability of NT-2Rep-CT, studying the component composition of NT-2Rep-CT solution and demonstrating the influence of the history of the sample on its properties and the observed state. Publication 3 investigated the dimerization of the CBM molecule and tested various methods for AUC data analysis. In publication 4, the shape of molecule of the CBM-AQ12-CBM protein was investigated using a combination of AUC and MD simulation. A similar approach was used in publication 5, where the polypeptides PLL and PGA were the objects of study. Solution composition, molecular weights and shape were determined.
  • Assembly of silk-like proteins towards functional bio-inspired materials
    (2024) Yin, Yin
     School of Chemical Technology | Doctoral dissertation (article-based)
    Protein assembly plays a crucial role in the development of functional materials, leveraging the natural properties of proteins to create sophisticated, sustainable, and high-performance materials that meet the demands of diverse applications. Nature offers numerous examples of protein assembly into functional materials, such as silk and collagen. As a prevalent phenomenon in nature, liquid-liquid phase separation (LLPS) has been found involved in the organization and function of living cells and the formation of functional materials outside cells. LLPS is a process where certain mixtures undergo demixing, resulting in a solute-rich dense phase and a solute-poor dilute phase. The dense phase is often referred to as coacervates or condensates. LLPS has gained increasing attention for its significant implications for the design of biological materials. Understanding and exploiting the principles underlying protein assembly through LLPS are therefore important for the design and engineering of bioinspired materials in synthetic biosystems. This thesis work utilized recombinant silk-like proteins as the main building blocks and incorporated other components using the powerful SpyCatcher-SpyTag protein pair to develop strategies and provide insights for functional materials development through LLPS. Publication I investigated the role of coacervates and 3,4-dihydroxyphenylalanine (DOPA), two key features of marine mussel adhesion, on adhesion. This was achieved by incorporating silk-like proteins with mussel foot proteins, resulting in the development of a strong and tough adhesive. Publication II provided strategies for selective condensates that utilize the intrinsically disordered sequence derived from spider silk as a tag for recruiting client proteins into silk-like protein-based condensates. Publication III explored the effect of phosphate, an important element involved in spider silk formation, on silk-like proteins. This study revealed that phosphate induced β-sheet formation in silk-like proteins, stiffening the protein layers and reducing their ability to form new interactions, thereby impeding fiber formation for phosphate-induced coacervates. Publication IV further investigated the assemblies involved in LLPS for longer silk-like proteins and provided insights into the assembly pathway of these proteins. The results of this thesis provided strategies and insights for creating functional silk-like protein-based materials with specific properties, such as strong adhesives, selective condensates, and fibers. This research paves the way for future developments in biomaterials inspired by nature, highlighting the potential of protein-based materials in various applications.
  • Biologically Inspired High Performance Material - Coacervation of genetically engineered silk-like fusion proteins as an intermediate step toward fabrication of next generation fiber, adhesive and composite
    (2018) Mohammadi, Pezhman
     School of Chemical Technology | Doctoral dissertation (article-based)
    Nature can serve as a source of inspiration for the design of the next generation high performance materials. Proteins can play a major role in structuring the novel sustainable and advanced functional materials. Given the precise design of proteins at molecular level together with expanding knowledge of new protein sequences, the ease of gene synthesis, cloning strategies and optimized biological production, various potential designs and applications can be anticipated. However, one of the main challenge toward this goal is the lack of understanding of the processes in which such materials could be assembled and form their functional molecular interactions. Inspired by the natural structural material, this thesis highlights solutions to some of the fundamental challenges related to the design strategies and processing routes with the extends the scopes toward potential applications. In publication 1, the general problem of how to directly assemble genetically engineered and recombinantly produced fusion proteins toward functional states was touched from a biological structural materials perspective. This was approached by exploring how the overall protein architecture and modularity affect liquid-liquid demixing and coacervate formation as the functioning intermediate entities toward assemblies of protein based fiber and also adhesive fiber. In publication 2, the nature of phase separated liquid-like coacervate assemblies was characterized in detail using various state-of-the-art techniques. Overall, the assemblies showed a range of properties including low surface tension, low viscosity, fast molecular diffusion, coalescence, cohesiveness and difformability under shear flow. It was further demonstrated how these could be used as an intermediate state for strong water based assemblies between various cellulosic surfaces. In publication 3, cellulose nanofibril (CNF) was used to fabricate high performance fibers with exceptional mechanical properties. This was carried out by exploring how CNF could be aligned under shear forces while being extruded through rela-tively long and thin capillaries.In publication 4, the central challenge of biocomposite mimicry as to how to minimize stress singularity at interfaces of dissimilar components for achieving high toughness was approached. This was carried out by effective infiltration and crosslinking of oriented CNF network (publication 3) through the use of low surface energy, adhesive and energy dissipating phase separated coacervates (publication 1 and 2). The resulting material showed exceptional strength, stiffness and overall toughness.
  • Engineering Principles of Hydrophobin Fusion Proteins
    (2017) Kurppa, Katri
     School of Chemical Technology | Doctoral dissertation (article-based)
    The research presented in this thesis focuses on the design and use of hydrophobin fusion proteins for technological applications. Hydrophobins are small fungal proteins with interfacial function. This characteristic arises from a unique, bipolar structure. Hydrophobins also partition effectively in liquid two-phase systems. The aim of the work presented in this thesis was to connect the molecular function of the hydrophobin HFBI to other operational functionalities by methods of protein engineering.  Proteins have become a central focus of reseach in the fields of biotechnology and material development. The vast interest is due to the inherently detailed structure of proteins, forming complex functionalities that build up to great application potential. Nature has created detailed and precise function to these molecules, which can be harnessed to build new materials. The art of protein engineering may be used to join and modify elements in new combinations.  A central theme throughout this research was to evaluate aspects such as protein component stoichiometry, material geometry and charge effects, as well as holistic factors influencing application desing. Firstly, suitable model hydrophobin fusion proteins were designed and produced, and their functionality at liquid-liquid and solid-liquid interfaces was studied. In the following segment of this study, the functionality of the fusion proteins was assessed in model applicaions as a hybrid material with carbon nanoparticles. The results presented in this thesis demonstrate the design and use of protein functionalities for creation of biomolecular assemblies based on the self-assembly of hydrophobin HFBI.  The solution equilibrium of class II hydrophobins plays a crucial role in the usability of its fusion derivatives, alongside with the mechanistic details of the interfacial assembly. The results were evaluated in the frame of the design process of hydrophobin fusion proteins. This process consists of an engineering step, a formulation step and a final application step. Thereby, this thesis highlights the importance of considering protein architecture and stoichiometry throughout the process.
  • Exploring silk protein assembly mechanisms for high-performance materials
    (2024) Välisalmi, Teemu
     School of Chemical Technology | Doctoral dissertation (article-based)
    Silk proteins present remarkable potential as the building blocks for high-performance materials. Due to their unique protein structure, they exhibit a variety of properties, such as a combination very high tensile strength and elasticity, biodegradability, biocompatibility, and ability to be spun in natural conditions — qualities uncommon in synthetic fibres. While challenging to acquire through natural means, the application of gene engineering allows to produce substantial quantities of recombinant silk proteins. However, the assembly pathways leading to the formation of functional materials remain largely unknown, with the evidence suggesting that crucial insights lie within the intricate spinning mechanism of silk-producing animals. Publication 1 delves into an investigation of the native spinning system of Bombyx mori silkworm. Amino acid mapping throughout the middle silk gland revealed a gradual change in various amino acids, attributed to the increase in different sericin proteins. This change correlated with the extensional behaviour of the silk dope, which was further enhanced by a straightforward pH modification inspired by the native spinning system. Publication 2 introduces a more sophisticated silk pulling device designed for controlled pulling and tensile testing, with a focus in automation. The device was employed to pull fibers from regenerated silk solution. Although immune to the pH induced dimerization due to irreversible changes by the regeneration, the fibroin fibers displayed a considerable median tensile strength of 147 MPa. In addition, a recombinant silk protein was observed to preassemble at an air-water interface, allowing pulling of thin fibers from droplets of silk. Liquid-liquid phase separation was found to be a significant factor in creating stronger fibers, demonstrating the necessity of controlling this intricate aspect of molecular assembly. Not limited to fibers, silk proteins find application as building blocks for films. Publication 3 details the development of a simple deposition method to create highly hydrophobic silk films. These films, characterized by the arrangement of intrinsically hydrophobic regions of the silk protein, formed in the presence of a specific salt concentration and high relative humidity during drying. Notably, the phenomenon was not strictly dependent on the silk protein sequence or the type of salt, although certain variations displayed superior performance. The films were altered by wetting, disrupting the interaction between the silk and salt. Although the lack of durability limits applicability of the silk films, this work demonstrates the versatile nature of the amphiphilic silk proteins.
  • Molecular assembly routes for biological materials
    (2022) Lemetti, Laura
     School of Chemical Technology | Doctoral dissertation (article-based)
    Biological materials present a wide variety of functional properties that provide inspiration for the development of new synthetic biomaterials. For example, spider silk has been extensively studied due to its exceptional toughness and adhesion properties. The exact pathway leading to the correct molecular assembly, which is the foundation of its extraordinary mechanical properties, is a complex process and not fully understood. This is one of the main reasons why the properties of synthetic silk materials are inferior to natural ones. This thesis attempts to answer the question of how recombinant spider silk proteins can be brought together in soluble form in a way that enables the interactions and microstructures that yield strong materials. The possible role of liquid-liquid phase separation, also known as coacervation, in the correct molecular assembly is of special interest. It has been suggested that many other natural organisms utilize coacervation as an intermediate step to produce functional materials, so gaining more information about its role on molecular assembly would be of great value to materials science. In publication I the coacervation of a recombinant spider silk-like protein, CBM-eADF3-CBM, was studied as an intermediate step for fiber assembly. Depending on the conditions, two kinds of coacervates were obtained: liquid-like and solid-like coacervates, but only the liquid-like ones enabled fiber formation and thus were more interesting for further studies. Publication II focused on the effect of molecular crowding on coacervation of CBM-eADF3-CBM. Dextran was used as an inert crowding agent, which enabled studying phase separation in lower protein concentrations, and in a controlled set-up. Furthermore, this set-up enabled thermodynamic studies of the system, which provided insight into the coacervation mechanism. Publication III presents the adhesion properties of CBM-eADF3-CBM in a system with delignified cellulose. Delignified cellulose proved to be a well-functioning model system to study silk as an adhesive, and CBM-eADF3-CBM formed a strong adhesive system with it. A method to produce native-sized recombinant silk proteins was developed in publication IV, and the effect of protein length on coacervation was studied. Post-translational in vitro ligation with the SpyCatcher2-SpyTag protein-peptide pair proved to be a robust way to produce native-sized silk proteins with high yield and solubility. Lengthening the protein lowered the critical concentration needed for coacervation and provided insight into the molecular interactions that lead to phase separation. To conclude, the results show that engineered recombinant silk proteins can be used to gain a deeper understanding of the molecular assembly behind the material formation process. Coacervation proved to be an important step in material formation and the methods to study it developed in this thesis increased the knowledge of molecular assembly processes.
  • Nanocellulose for bio-inspired nanocomposites - surface modification with recombinant proteins
    (2018) Fang, Wenwen
     School of Chemical Technology | Doctoral dissertation (article-based)
    The increased concern about the environment and legislation pressure, such as plastic bag ban, has generated the fast-grown demand for renewable and sustainable materials to replace the petroleum-based products. Cellulose is the most abundant natural polymer on earth, which can be found in many different organisms ranging from microbes to plants and animals. Nanocellulose, including cellulose nanofibrils and cellulose nanocrystals, can be extracted from plant cell wall by mechanical treatment or acid hydrolysis. Due to its extraordinary mechanical properties, low density and good biocompatibility, nanocellulose attracts increasing interests for materials scientist. In this work, we used genetically engineered recombinant proteins containing multiple functional domains to functionalize nanocellulose and make nanocomposites together with other building components. First, we needed to understand how the proteins interact with nanocellulose. The cellulose binding domains were coupled with gold nanoparticles through EDC-NHS chemistry and their binding to nanocellulose was visualized with electron microscopy. Further, the in situ interaction of recombinant protein and nanocellulose surface was investigated with QCM-D. It showed that the recombinant proteins containing resilin like polypeptide and cellulose binding module could bind to cellulose surface and form a pH responsive layer. From the rheology study, we also found that the recombinant protein could cross-link the cellulose nanofibrils. This finding led to our next study, improve the mechanical properties of nanocellulose film by cross linking the fibrils with cellulose binding proteins. The stiffness of the cellulose film was clearly increased due to the cross-linking of fibrils. In order to make conductive cellulose film, carbon nanotubes were added to the cellulose matrix. A bifunctional protein containing a hydrophobic patch and a glycosylated domain was used to disperse carbon nanotube into the cellulose matrix. In addition, the protein-protein interaction was also investigated in this work. The recombinant protein contains resilin like polypeptides could form salt induced coacervates. The effect of ionic strength, pH, protein concentration and different termimal domains were studied with dynamic light scattering and electron microscopies. In conclusion, we demonstrated that genetically engineered proteins provide a new toolbox for the functionalization of nanocellulose and modify the interface between different building components in nanocomposites.
  • On the Nanoscale Interactions and the Self-Assembly of Recombinant Proteins and Hybrid Nanostructures: an AFM Study
    (2019) Griffo, Alessandra
     School of Chemical Technology | Doctoral dissertation (article-based)
    The study presented in this Thesis is focussed on the characterization and the design of new polymeric materials, taking inspiration from the Nature. Here, new hybrid architectures in which adhesive and elastic proteins coexist with inorganic or cellulosic surfaces, or where ligand capped metal nanoclusters self-assemble in monolayer films, are investigated. Genetic engineering is used to produce new synthetic fusion proteins having specific functionalities starting from microbes. The particle self-assembly is indeed inspired on the symmetrical and directional arrangement of natural architectures such as globular proteins and viral capsids. The study is fundamental and performed at nanoscale level. Single molecular interactions on surfaces are analysed as well as the structure and the conformation of individual fusion proteins. The self-assembly process of protein films is deeply studied as well as the stiffness and elastic modulus of self-assembled silver nanocluster composite films. The candidate proteins for making biohybrids are hydrophobins, cellulose binding modules and resilins. Hydrophobins (HFB), with their unique assembly mechanism, are well known for their hydrophobic patch, that strongly bind to hydrophobic surfaces. Cellulose binding modules (CBMs), turned out to be highly interesting domains for their binding affinity to their primary substrate, the cellulose. On the other hand, resilin, for its ability to dissipate energy upon tensile stress, could find use as a sacrificial bond in high strength materials. Atomic force microscope (AFM) is here used for detecting the binding and interaction forces between proteins and surfaces. For the resilin, this such powerful tool is also used to characterize the length of the biopolymer under different environments, upon stretching. AFM was also employed for determining the elastic modulus of the nanocluster monolayers. According to the results achieved, HFBI ranged a quite high adhesion force value near 100 pN on the chosen hydrophobic surfaces, whereas the CBMs reported a binding affinity for different kind of cellulosic surfaces between 40-50 pN. The silver nanoclusters ligand-capped films revealed an elastic modulus value around 20 GPa. The Thesis sheds light on the importance of replacing plastic materials with new bio hybrids for a more sustainable approach, in an age where the ecosystem risks to be compromised by pollution and not biodegradable waste.
  • Silica-drug delivery systems: From prolonged drug release to wound dressings and orthopedic applications
    (2019) Ahmed Mosselhy Abdelrehiem, Dina
     School of Chemical Technology | Doctoral dissertation (article-based)
    Wounds and orthopedic implants afflicted with methicillin resistant Staphylococcus aureus (MRSA) infections and their biofilms are recalcitrant to treatment. Silica (SiO2) could be used as a carrier in delivery systems for targeting drugs (silver and gentamicin) to potentially prevent the growth of bacteria and treat bacterial infections in wound dressings and orthopedic applications. Publication [1] presents the prolonged rapid initial release of silver (Ag) from a SiO2-Ag nanocomposite delivery system over the first 24 h (~38.2%) from the stock suspension of the nanocomposite, followed by a slower sustained release after 48 (~46.9%) and 72 h (~49.1%), favorable for wound dressing applications. The nanocomposite was impregnated in gauze and compared with a commercial silver-containing (CSD) dressing, and demonstrated to have better prolonged antibacterial effects than the CSD. The underlying mechanisms of action of the nanocomposite in MRSA were putatively attributed to silver ions released from the nanocomposite, resulting at the end in the loss of bacterial membranes. Publication [2] demonstrates a prolonged rapid initial release of gentamicin from the SiO2-gentamicin nanohybrids delivery system over the first 24 h (~21.4%) from the stock suspension of the nanohybrids, followed by a slower sustained release after 120 h (~43.9%), which is essential in orthopedic surgery. Publication [3] examines the in vitro toxicities of SiO2-gentamicin nanohybrids to human osteoblast-like SaOS-2 cells, and demonstrates a significant decrease of the viability of SaOS-2 cells treated with SiO2-gentamicin nanohybrids (250 μg/mL) in a time-dependent manner (68 ± 0% after 24 h; 25 ± 5% after 5 days), demonstrating severe cytotoxicity, and a significant reduction of the expression of alkaline phoshphatase (i.e., reaching < 1/3 of the control group). Publication [4] shows the potential of SiO2-gentamicin nanohybrids to kill planktonic MRSA cells that are commonly encountered in orthopedic infections, and to eradicate Escherichia coli cells in biofilms (i.e., minimum biofilm eradication concentration of 250 μg/mL) via a complete deformation of the shape, wrinkling of the cell walls, and the reduction of the size of E. coli cells. The in vivo toxicities of SiO2-gentamicin delivery systems were also assessed, in Publication [4], in zebrafish embryos, demonstrating non-significant mortality rates and the biocompatibility of the nanohybrids at concentrations as high as 500 and 1000 μg/mL. The thesis illustrates the complexities of safety issues associated with future orthopedic applications of SiO2-gentamicin nanohybrids, resulting from discrepancies in the toxicities recorded in vitro and in vivo.