@phdthesis{LopezGarcia2019,
  author    = {L{\´o}pez Garc{\´i}a, Patricia},
  title     = {Coiled coils as mechanical building blocks},
  doi       = {10.25932/publishup-42956},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-429568},
  school      = {Universit{\"a}t Potsdam},
  pages     = {xi, 130},
  year      = {2019},
  abstract  = {The natural abundance of Coiled Coil (CC) motifs in cytoskeleton and extracellular matrix proteins suggests that CCs play an important role as passive (structural) and active (regulatory) mechanical building blocks. CCs are self-assembled superhelical structures consisting of 2-7 α-helices. Self-assembly is driven by hydrophobic and ionic interactions, while the helix propensity of the individual helices contributes additional stability to the structure. As a direct result of this simple sequence-structure relationship, CCs serve as templates for protein design and sequences with a pre-defined thermodynamic stability have been synthesized de novo. Despite this quickly increasing knowledge and the vast number of possible CC applications, the mechanical function of CCs has been largely overlooked and little is known about how different CC design parameters determine the mechanical stability of CCs. Once available, this knowledge will open up new applications for CCs as nanomechanical building blocks, e.g. in biomaterials and nanobiotechnology. With the goal of shedding light on the sequence-structure-mechanics relationship of CCs, a well-characterized heterodimeric CC was utilized as a model system. The sequence of this model system was systematically modified to investigate how different design parameters affect the CC response when the force is applied to opposing termini in a shear geometry or separated in a zipper-like fashion from the same termini (unzip geometry). The force was applied using an atomic force microscope set-up and dynamic single-molecule force spectroscopy was performed to determine the rupture forces and energy landscape properties of the CC heterodimers under study. Using force as a denaturant, CC chain separation is initiated by helix uncoiling from the force application points. In the shear geometry, this allows uncoiling-assisted sliding parallel to the force vector or dissociation perpendicular to the force vector. Both competing processes involve the opening of stabilizing hydrophobic (and ionic) interactions. Also in the unzip geometry, helix uncoiling precedes the rupture of hydrophobic contacts. In a first series of experiments, the focus was placed on canonical modifications in the hydrophobic core and the helix propensity. Using the shear geometry, it was shown that both a reduced core packing and helix propensity lower the thermodynamic and mechanical stability of the CC; however, with different effects on the energy landscape of the system. A less tightly packed hydrophobic core increases the distance to the transition state, with only a small effect on the barrier height. This originates from a more dynamic and less tightly packed core, which provides more degrees of freedom to respond to the applied force in the direction of the force vector. In contrast, a reduced helix propensity decreases both the distance to the transition state and the barrier height. The helices are 'easier' to unfold and the remaining structure is less thermodynamically stable so that dissociation perpendicular to the force axis can occur at smaller deformations. Having elucidated how canonical sequence modifications influence CC mechanics, the pulling geometry was investigated in the next step. Using one and the same sequence, the force application points were exchanged and two different shear and one unzipping geometry were compared. It was shown that the pulling geometry determines the mechanical stability of the CC. Different rupture forces were observed in the different shear as well as in the unzipping geometries, suggesting that chain separation follows different pathways on the energy landscape. Whereas the difference between CC shearing and unzipping was anticipated and has also been observed for other biological structures, the observed difference for the two shear geometries was less expected. It can be explained with the structural asymmetry of the CC heterodimer. It is proposed that the direction of the α-helices, the different local helix propensities and the position of a polar asparagine in the hydrophobic core are responsible for the observed difference in the chain separation pathways. In combination, these factors are considered to influence the interplay between processes parallel and perpendicular to the force axis. To obtain more detailed insights into the role of helix stability, helical turns were reinforced locally using artificial constraints in the form of covalent and dynamic 'staples'. A covalent staple bridges to adjacent helical turns, thus protecting them against uncoiling. The staple was inserted directly at the point of force application in one helix or in the same terminus of the other helix, which did not experience the force directly. It was shown that preventing helix uncoiling at the point of force application reduces the distance to the transition state while slightly increasing the barrier height. This confirms that helix uncoiling is critically important for CC chain separation. When inserted into the second helix, this stabilizing effect is transferred across the hydrophobic core and protects the force-loaded turns against uncoiling. If both helices were stapled, no additional increase in mechanical stability was observed. When replacing the covalent staple with a dynamic metal-coordination bond, a smaller decrease in the distance to the transition was observed, suggesting that the staple opens up while the CC is under load. Using fluorinated amino acids as another type of non-natural modification, it was investigated how the enhanced hydrophobicity and the altered packing at the interface influences CC mechanics. The fluorinated amino acid was inserted into one central heptad of one or both α-helices. It was shown that this substitution destabilized the CC thermodynamically and mechanically. Specifically, the barrier height was decreased and the distance to the transition state increased. This suggests that a possible stabilizing effect of the increased hydrophobicity is overruled by a disturbed packing, which originates from a bad fit of the fluorinated amino acid into the local environment. This in turn increases the flexibility at the interface, as also observed for the hydrophobic core substitution described above. In combination, this confirms that the arrangement of the hydrophobic side chains is an additional crucial factor determining the mechanical stability of CCs. In conclusion, this work shows that knowledge of the thermodynamic stability alone is not sufficient to predict the mechanical stability of CCs. It is the interplay between helix propensity and hydrophobic core packing that defines the sequence-structure-mechanics relationship. In combination, both parameters determine the relative contribution of processes parallel and perpendicular to the force axis, i.e. helix uncoiling and uncoiling-assisted sliding as well as dissociation. This new mechanistic knowledge provides insight into the mechanical function of CCs in tissues and opens up the road for designing CCs with pre-defined mechanical properties. The library of mechanically characterized CCs developed in this work is a powerful starting point for a wide spectrum of applications, ranging from molecular force sensors to mechanosensitive crosslinks in protein nanostructures and synthetic extracellular matrix mimics.},
  language  = {en}
}
@phdthesis{Federico2011,
  author    = {Federico, Stefania},
  title     = {Synthetic peptides derived from decorin as building blocks for biomaterials based on supramolecular interactions},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-59661},
  school      = {Universit{\"a}t Potsdam},
  year      = {2011},
  abstract  = {In this work, the development of a new molecular building block, based on synthetic peptides derived from decorin, is presented. These peptides represent a promising basis for the design of polymer-based biomaterials that mimic the ECM on a molecular level and exploit specific biological recognition for technical applications. Multiple sequence alignments of the internal repeats of decorin that formed the inner and outer surface of the arch-shaped protein were used to develop consensus sequences. These sequences contained conserved sequence motifs that are likely to be related to structural and functional features of the protein. Peptides representative for the consensus sequences were synthesized by microwave-assisted solid phase peptide synthesis and purified by RP-HPLC, with purities higher than 95 mol\%. After confirming the desired masses by MALDI-TOF-MS, the primary structure of each peptide was investigated by 1H and 2D NMR, from which a full assignment of the chemical shifts was obtained. The characterization of the peptides conformation in solution was performed by CD spectroscopy, which demonstrated that using TFE, the peptides from the outer surface of decorin show a high propensity to fold into helical structures as observed in the original protein. To the contrary, the peptides from the inner surface did not show propensity to form stable secondary structure. The investigation of the binding capability of the peptides to Collagen I was performed by surface plasmon resonance analyses, from which all but one of the peptides representing the inner surface of decorin showed binding affinity to collagen with values of dissociation constant between 2•10-7 M and 2.3•10-4 M. On the other hand, the peptides representative for the outer surface of decorin did not show any significant interaction to collagen. This information was then used to develop experimental demonstration for the binding capabilities of the peptides from the inner surface of decorin to collagen even when used in more complicated situations close to possible appications. With this purpose, the peptide (LRELHLNNN) which showed the highest binding affinity to collagen (2•10-7 M) was functionalized with an N-terminal triple bond in order to obtain a peptide dimer via copper(I)-catalyzed cycloaddition reaction with 4,4'-diazidostilbene-2,2'-disulfonic acid. Rheological measurements showed that the presence of the peptide dimer was able to enhance the elastic modulus (G') of a collagen gel from ~ 600 Pa (collagen alone) to ~ 2700 Pa (collagen and peptide dimer). Moreover, it was shown that the mechanical properties of a collagen gel can be tailored by using different molar ratios of peptide dimer respect to collagen. The same peptide, functionalized with the triple bond, was used to obtain a peptide-dye conjugate by coupling it with N-(5'-azidopentanoyl)-5-aminofluorescein. An aqueous solution (5 vol\% methanol) of the peptide dye conjugate was injected into a collagen and a hyaluronic acid (HA) gel and images of fluorescence detection showed that the diffusion of the peptide was slower in the collagen gel compared to the HA gel. The third experimental demonstration was gained using the peptide (LSELRLHNN) which showed the lower binding affinity (2.3•10-4 M) to collagen. This peptide was grafted to hyaluronic acid via EDC-chemistry, with a degree of functionalization of 7 ± 2 mol\% as calculated by 1H-NMR. The grafting was further confirmed by FTIR and TGA measurements, which showed that the onset of decomposition for the HA-g-peptide decreased by 10 °C compared to the native HA. Rheological measurements showed that the elastic modulus of a system based on collagen and HA-g-peptide increased by almost two order of magnitude (G' = 200 Pa) compared to a system based on collagen and HA (G' = 0.9 Pa). Overall, this study showed that the synthetic peptides, which were identified from decorin, can be applied as potential building blocks for biomimetic materials that function via biological recognition.},
  language  = {en}
}
@phdthesis{Hahn2009,
  author    = {Hahn, Harald},
  title     = {Modularer Ansatz zu multifunktionellen Polymer-Peptid-Fasern},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-33016},
  school      = {Universit{\"a}t Potsdam},
  year      = {2009},
  abstract  = {Die Kombination von Polymeren mit Peptiden vereint die Eigenschaften beider Stoffklassen miteinander. Dabei k{\"o}nnen die strukturbildenden Eigenschaften der Peptide genutzt werden, um Polymere zu organisieren. In der vorliegenden Arbeit wurde ein Polymer-Peptid-Konjugat verwendet, das sich in Wasser zu B{\"a}ndern anordnet. Die treibende Kraft f{\"u}r diesen Prozess ist die Anordnung des Peptidteils zu β-Faltblattstrukturen. Das Polymer-Peptid-Aggregat besitzt einen Peptidkern mit funktionalen Oberfl{\"a}chen, der lateral von einer Polyethylenoxidschale umgeben ist. Durch {\"A}nderung der Peptidsequenz war es bisher m{\"o}glich, die Eigenschaften dieser Fasern zu variieren. In der Arbeit wird ein modularer Ansatz zur vielf{\"a}ltigen Modifizierung einer Polymer-Peptid-Faser entwickelt. So ist es m{\"o}glich, die Eigenschaften der Fasern einzustellen, ohne die strukturbildende β-Faltblattsequenz ver{\"a}ndern zu m{\"u}ssen. Um weitere Funktionen an den Fasern anzubringen, wurde die 1,3-dipolaren Addition verwendet. Diese Reaktion beschreibt die konzertierte Umlagerung eines Azides mit einem Alkin. Sie ist in den meisten L{\"o}sungsmitteln unter hohen Ausbeuten durchf{\"u}hrbar. Im Rahmen der Arbeit wird die Erzeugung von Aziden untersucht und auf die Polymer-Peptid-Fasern {\"u}bertragen. Der Diazotransfer stellte dabei die Methode der Wahl dar, so k{\"o}nnen Azidgruppen aus Aminen gewonnen werden. Unter Verwendung der 1,3-dipolaren Addition konnten verschiedene alkinfunktionale Molek{\"u}le kovalent an die azidfunktionalisierten Polymer-Peptid-Fasern gebunden werden. So wurde ein Fluoreszenzfarbstoff an die Fasern gebunden, der eine Abbildung der Fasern mittels konfokaler Mikroskopie erlaubte. Weiterhin wurden die Eigenschaften der Fasern durch Addition dreier carboxylfunktionaler Molek{\"u}le modifiziert. Diese Fasern konnten weiter genutzt werden, um Kalzium zu binden. Dabei variierte die Anzahl der gebundenen Kalziumionen in Abh{\"a}ngigkeit der jeweiligen Fasermodifikation erheblich. Weitere Untersuchungen, die Morphologie von Kalziumcarbonatkristallen betreffend, werden aktuell durchgef{\"u}hrt. Die kovalente Anbringung eines reduzierenden Zuckers an die Polymer-Peptid-Fasern erlaubt die Abscheidung von Silber aus Tollens Reagenz. Durch eine Entwicklung analog zur Schwarz-Weiss-Photographie k{\"o}nnen in nachfolgenden Arbeiten so Silberdr{\"a}hte in Nanogr{\"o}ße erzeugt werden. An die azidfunktionalen Fasern k{\"o}nnen weitere funktionale Molek{\"u}le angebracht werden, um die Eigenschaften und das Anwendungsspektrum der Polymer-Peptid-Fasern zu erweitern.},
  language  = {de}
}
@phdthesis{Maltseva2005,
  author    = {Maltseva, Elena},
  title     = {Model membrane interactions with ions and peptides at the air/water interface},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-5670},
  school      = {Universit{\"a}t Potsdam},
  year      = {2005},
  abstract  = {The interactions between peptides and lipids are of fundamental importance in the functioning of numerous membrane-mediated biochemical processes including antimicrobial peptide action, hormone-receptor interactions, drug bioavailability across the blood-brain barrier and viral fusion processes. Alteration of peptide structure could be a cause of many diseases. Biological membranes are complex systems, therefore simplified models may be introduced in order to understand processes occurring in nature. The lipid monolayers at the air/water interface are suitable model systems to mimic biological membranes since many parameters can be easily controlled. In the present work the lipid monolayers were used as a model membrane and their interactions with two different peptides B18 and Amyloid beta (1-40) peptide were investigated. B18 is a synthetic peptide that binds to lipid membranes that leads to the membrane fusion. It was demonstrated that it adopts different structures in the aqueous solutions and in the membrane interior. It is unstructured in solutions and forms alpha-helix at the air/water interface or in the membrane bound state. The peptide has affinity to the negatively charged lipids and even can fold into beta-sheet structure in the vicinity of charged membranes at high peptide to lipid ratio. It was elucidated that in the absence of electrostatic interactions B18 does not influence on the lipid structure, whereas it provides partial liquidization of the negatively charged lipids. The understanding of mechanism of the peptide action in model system may help to develop the new type of antimicrobial peptides as well as it can shed light on the general mechanisms of peptide/membrane binding. The other studied peptide - Amyloid beta (1-40) peptide, which is the major component of amyloid plaques found in the brain of patients with Alzheimer's disease. Normally the peptide is soluble and is not toxic. During aging or as a result of the disease it aggregates and shows a pronounced neurotoxicity. The peptide aggregation involves the conformational transition from a random coil or alpha-helix to beta-sheets. Recently it was demonstrated that the membrane can play a crucial role for the peptide aggregation and even more the peptide can cause the change in the cell membranes that leads to a neuron death. In the present studies the structure of the membrane bound Amyloid beta peptide was elucidated. It was found that the peptide adopts the beta-sheet structure at the air/water interface or being adsorbed on lipid monolayers, while it can form alpha-helical structure in the presence of the negatively charged vesicles. The difference between the monolayer system and the bulk system with vesicles is the peptide to lipid ratio. The peptide adopts the helical structure at low peptide to lipid ratio and folds into beta-sheet at high ratio. Apparently, Abeta peptide accumulation in the brain is concentration driven. Increasing concentration leads to a change in the lipid to peptide ratio that induces the beta-sheet formation. The negatively charged lipids can act as seeds in the plaque formation, the peptide accumulates on the membrane and when the peptide to lipid ratio increases it the peptide forms toxic beta-sheet containing aggregates.},
  subject      = {Lipide},
  language  = {en}
}