Growth factor release by vesicular phospholipid gels: in-vitro results and application for rotator cuff repair in a rat model

Background: Biological augmentation of rotator cuff repair is of growing interest to improve biomechanical properties and prevent re-tearing. But intraoperative single shot growth factor application appears not sufficient to provide healing support in the physiologic growth factor expression peaks. The purpose of this study was to establish a sustained release of granulocyte-colony stimulating factor (G-CSF) from injectable vesicular phospholipid gels (VPGs) in vitro and to examine biocompatibility and influence on histology and biomechanical behavior of G-CSF loaded VPGs in a chronic supraspinatus tear rat model. Methods: G-CSF loaded VPGs were produced by dual asymmetric centrifugation. In vitro the integrity, stability and release rate were analyzed. In vivo supraspinatus tendons of 60 rats were detached and after 3 weeks a transosseous refixation with G-CSF loaded VPGs augmentation (n = 15;control, placebo, 1 and 10 mu g G-CSF/d) was performed. 6 weeks postoperatively the healing site was analyzed histologically (n = 9;H&E by modified MOVIN score/Collagen I/III) and biomechanically (n = 6). Results: In vitro testing revealed stable proteins after centrifugation and a continuous G-CSF release of up to 4 weeks. Placebo VPGs showed histologically no negative side effects on the healing process. Histologically in vivo testing demonstrated significant advantages for G-CSF 1 mu g/d but not for G-CSF 10 mu g/d in Collagen III content (p = 0.035) and a higher Collagen I/III ratio compared to the other groups. Biomechanically G-CSF 1 mu g/d revealed a significant higher load to failure ratio (p = 0.020) compared to control but no significant differences in stiffness. Conclusions: By use of VPGs a continuous growth factor release could be obtained in vitro. The in vivo results demonstrate an improvement of immunohistology and biomechanical properties with a low dose G-CSF application via VPG. The VPG itself was well tolerated and had no negative influence on the healing behavior. Due to the favorable properties (highly adhesive, injectable, biocompatible) VPGs are a very interesting option for biologic augmentation. The study may serve as basis for further research in growth factor application models

Growth factor release by vesicular phospholipid gels: in-vitro results and application for rotator cuff repair in a rat model

Background: Biological augmentation of rotator cuff repair is of growing interest to improve biomechanical properties and prevent re-tearing. But intraoperative single shot growth factor application appears not sufficient to provide healing support in the physiologic growth factor expression peaks. The purpose of this study was to establish a sustained release of granulocyte-colony stimulating factor (G-CSF) from injectable vesicular phospholipid gels (VPGs) in vitro and to examine biocompatibility and influence on histology and biomechanical behavior of G-CSF loaded VPGs in a chronic supraspinatus tear rat model. Methods: G-CSF loaded VPGs were produced by dual asymmetric centrifugation. In vitro the integrity, stability and release rate were analyzed. In vivo supraspinatus tendons of 60 rats were detached and after 3 weeks a transosseous refixation with G-CSF loaded VPGs augmentation (n = 15;control, placebo, 1 and 10 mu g G-CSF/d) was performed. 6 weeks postoperatively the healing site was analyzed histologically (n = 9;H&E by modified MOVIN score/Collagen I/III) and biomechanically (n = 6). Results: In vitro testing revealed stable proteins after centrifugation and a continuous G-CSF release of up to 4 weeks. Placebo VPGs showed histologically no negative side effects on the healing process. Histologically in vivo testing demonstrated significant advantages for G-CSF 1 mu g/d but not for G-CSF 10 mu g/d in Collagen III content (p = 0.035) and a higher Collagen I/III ratio compared to the other groups. Biomechanically G-CSF 1 mu g/d revealed a significant higher load to failure ratio (p = 0.020) compared to control but no significant differences in stiffness. Conclusions: By use of VPGs a continuous growth factor release could be obtained in vitro. The in vivo results demonstrate an improvement of immunohistology and biomechanical properties with a low dose G-CSF application via VPG. The VPG itself was well tolerated and had no negative influence on the healing behavior. Due to the favorable properties (highly adhesive, injectable, biocompatible) VPGs are a very interesting option for biologic augmentation. The study may serve as basis for further research in growth factor application models

Investigation of local deformation of DNA in protein-DNA complexes by Molecular Dynamics Simulations

Die konformationelle Plastizität der DNA ist wichtig für essenzielle biologische Funktionen wie Transkription oder Replikation. Die Deformation von DNA-Doppelsträngen kann durch die DNA-Sequenz, durch Umgebungsfaktoren oder durch die Interaktion mit Proteinen begünstigt werden. In der vorliegenden Arbeit wurden zwei grundlegende Aspekte der Protein-DNA-Interaktion mit computergestützten Methoden untersucht: (1) Der Einfluss interkalierender Aminosäuren auf die Erzeugung von Knicken in DNA. (2) Die Rolle von DNA-Sequenzmerkmalen bei der Modulation der Proteinbindung ("shape readout"). Für die Untersuchung proteininduzierter Knicke wurde ein Workflow zur Identifizierung von Protein-DNA-Komplexstrukturen mit geknickter DNA etabliert. Damit wurden insgesamt 88 Strukturen für 15 verschiedene Systeme erhalten. Die Untersuchung der Komplexe hinsichtlich ihrer strukturellen Eigenschaften und der Art und Anzahl der interkalierenden Aminosäuren ergab, dass die Anwesenheit von interkalierenden Aminosäuren keine notwendige Voraussetzung für starke Knicke ist und dass ein bis drei hydrophobe Aminosäuren zur Interkalation beitragen. Um die Strukturprinzipien des proteininduzierten DNA-Knickens zu entschlüsseln, wurden Moleküldynamik (MD)-Simulationen für sechs Komplexe durchgeführt, die sich in ihrer Architektur, ihrer Funktion und der Identität der interkalierten Aminosäuren unterscheiden. Simulationen wurden für die DNA-Komplexe von Wildtyp-Proteinen (Sac7d, Cren7, Sox-4, CcpA, TFAM, TBP) und für Mutanten durchgeführt, bei denen die interkalierenden Aminosäuren einzeln oder in Kombination durch Alanin ersetzt wurden. Die Arbeit zeigte, dass bei Systemen mit mehreren interkalierten Aminosäuren nicht alle für die Knickbildung notwendig sind. In einigen Komplexen (Sox-4, TBP) erwies sich eine der Aminosäuren als wesentlich für die Knickbildung, während die zweite nur einen sehr geringen Einfluss auf die Größe des Knicks hatte. In anderen Systemen (z.B. Sac7d) erwies sich jede der interkalierenden Aminosäuren als unabhängig in der Lage einen starken Knick zu verursachen, was auf eine teilweise redundante Rolle der interkalierenden Aminosäuren hindeutet. Die Mutation der für das Knicken verantwortlichen Schlüsselreste führte entweder zu stabilen Komplexen mit reduzierten Knickwinkeln oder zu Konformationsinstabilitäten, wie z.B. einer Verschiebung des Knicks zu einer benachbarten Basenpaarstufe. Auf diese Weise können MD-Simulationen helfen, die Rolle der einzelnen interkalierten Aminosäuren bei der Erzeugung von Knicken zu identifizieren, was bei einer Analyse der statischen Strukturen nicht leicht möglich ist. Diese Informationen könnten hilfreich sein, um Protein-DNA-Interaktionen genauer zu verstehen und in der Zukunft Proteine mit veränderten DNA-Bindungseigenschaften zu entwerfen. Im zweiten Teil dieser Arbeit wurde die Rolle des shape readout für DNA-Protein-Erkennungsprozesse untersucht, wobei der Schwerpunkt auf den CcpA-DNA- und AmtR-DNA-Komplexen als Modellsysteme lag. Das dimere CcpA bindet bevorzugt palindromische DNA (cre)-Sequenzen mit einer zentralen CG- Basenpaarstufe, die im Komplex geknickt wird. Zur Untersuchung der zugrunde liegenden Strukturprinzipien wurden mutierte DNA-Sequenzen mit zentralen GC-, AT- und TA-Basenpaarstufen im Komplex mit CcpA modelliert und die Konformationsstabilität der Systeme durch MD-Simulationen bewertet. Die Erkennung der CG- und GC-Stufen wird durch spezifische Wasserstoffbrückenbindungen mit dem Protein begünstigt. Das Fehlen dieser Wasserstoffbrücken führt zu einer schwächeren Bindung oder sogar zu einer verminderten konformationellen Stabilität der Komplexe, wie sie für die TA-Basenpaarstufe beobachtet wird. Die Simulationen zeigen, dass CG- und GC-Stufen während der Simulationen ähnlich stark geknickt bleiben. Allerdings ist der Energiebedarf für das Knicken bei CG geringer als bei GC, was einer allgemein besseren Deformierbarkeit der Pyrimidin-Purin-Basenpaarstufen entspricht. Die obigen Ergebnisse zeigen, dass ein Zusammenspiel von base readout und shape readout für die spezifische Erkennung einer zentralen CG-Basenpaarstufe im CcpA-DNA-Komplex verantwortlich ist. Der letzte Teil der Arbeit befasste sich mit der Rolle der Formauslesung für die Erkennung von geraden DNA-Sequenzen mit einer fast regelmäßigen B-DNA-Geometrie. Dieser Aspekt wurde für das dimere AmtR-Protein untersucht, das die DNA an zwei palindromischen Stellen kontaktiert, die durch einen sechs Reste langen Spacer mit schlecht konservierter Sequenz verbunden sind. Um zu beurteilen, wie dieser zentrale Spacer die DNA-Struktur beeinflusst und ob die Deformierbarkeit des Spacers eine Rolle bei der differentiellen AmtR-Bindung spielt, wurden deformierbare und starre Spacer -Varianten entwickelt, die als 3CG bzw. 6G bezeichnet wurden. MD-Simulationen ergaben, dass der Wildtyp (amtB) und der 3CG-Spacer sehr ähnliche Eigenschaften in Bezug auf die DNA-Feinstruktur (Propeller Twist, Slide) und den gesamten Twistwinkel aufweisen. Im Gegensatz dazu unterschieden sich diese Parameter signifikant für den starren 6G Spacer, was zu einer schlechteren Anpassung des Proteins an die DNA führte, wie die Instabilität der Protein-DNA-Wasserstoffbrücken zeigt. Diese Ergebnisse deuten auf eine schwächere Bindung der DNA mit einem starren Spacer hin, was mit den jüngsten experimentellen Daten übereinstimmt. Zusammengenommen zeigt das Beispiel von AmtR, dass das Auslesen der Form nicht nur für geknickte DNA eine Rolle spielt, sondern auch die Bindung von geraden DNA-Sequenzen beeinflussen kann.The conformational plasticity of DNA is important for essential biological functions such as transcription or replication. The deformation of DNA double strands can be facilitated by DNA sequence, environmental factors or by the interaction with proteins. In the present thesis, two basic aspects of protein-DNA interaction were investigated using computer-assisted methods: (1) The influence of intercalating amino acids on the induction of kinks in DNA. (2) The role of DNA sequence features in the modulation of protein binding ("shape readout"). For the investigation of protein-induced kinks, a workflow for the identification of protein-DNA complex structures with kinked DNA was established. With this, a total of 88 structures for 15 different systems were obtained. The examination of the complexes with regard to their structural properties and the type and number of intercalating amino acids showed that the presence of intercalating amino acids is not a necessary condition for strong kinks and that one to three hydrophobic amino acids contribute to intercalation. In order to unravel the structural principles of protein-induced DNA kinking, molecular dynamics (MD) simulations were performed for six complexes that varied in their architecture, function and identity of the intercalated amino acids. Simulations were performed for the DNA complexes of wildtype proteins (Sac7d, Cren7, Sox-4, CcpA, TFAM, TBP) and for mutants in which the intercalating residues were replaced individually or in combination by alanine. The work revealed that for systems with multiple intercalated residues, not all of them are necessarily required for kink formation. In some complexes (Sox-4, TBP), one of the residues proved to be essential for kink formation, whereas the second residue has only a very small effect on the magnitude of the kink. In other systems (e.g. Sac7d) each of the intercalated residues proved to be individually capable of conferring a strong kink suggesting a partially redundant role of the intercalating amino acids. Mutation of the key residues responsible for kinking either resulted in stable complexes with reduced kink angles or caused conformational instability as evidenced by a shift of the kink to an adjacent base step. Thus, MD simulations can help to identify the role of individual inserted residues for kinking, which is not readily apparent from an inspection of the static structures. This information might be helpful for understanding protein-DNA interactions in more detail and for designing proteins with altered DNA binding properties in the future. In the second part of this thesis, the role of shape readout for DNA-protein recognition processes was investigated with a focus on the CcpA-DNA and AmtR-DNA complexes as model systems. The dimeric CcpA preferentially binds palindromic DNA (cre) sequences with a central CG base step that becomes kinked in the complex. To investigate the underlying structural principles, mutant DNA sequences with central GC, AT, and TA base steps were modelled in complex with CcpA and the conformational stability of the systems was assessed by MD-simulations. Recognition of the CG and GC steps is favoured by specific hydrogen bonds with the protein demonstrating the role of base readout in cre binding. The absence of this hydrogen bond results in weaker binding or even reduced conformational complex stability as observed for the TA base step. The simulations reveal that CG and GC steps remain kinked to a similar extent during the simulations. However, the energy required for kinking is lower for CG than for GC, which is in line with a generally enhanced deformability of pyrimidine-purine base steps. The findings above demonstrate that interplay of base readout and shape readout is responsible for the specific recognition of a central CG base step in the CcpA-DNA complex. The final part of the work addressed the role of shape readout for the recognition of straight DNA sequences with an almost regular B-DNA geometry. This aspect was investigated for the dimeric AmtR protein, which contacts the DNA at two palindromic sites that are connected by a six-residue Spacer of poor sequence conservation. To assess how this central Spacer influences the DNA structure and whether the deformability of the Spacer plays a role in differential AmtR binding, deformable and rigid Spacer variants were designed, which were termed 3CG and 6G, respectively. MD simulations revealed that the wildtype (amtB) and the 3CG Spacer exhibit very similar properties with respect to DNA fine structure (propeller twist, slide) and the overall twist angle. In contrast, these parameters differed significantly for the rigid 6G Spacer resulting in a poorer fit of the protein on the DNA as evident from the instability of protein-DNA hydrogen bonds. These results suggest a weaker binding of DNA with a rigid Spacer, which is in line with recent experimental data. Taken together, the example of AmtR demonstrates that shape readout does not only play a role for kinked DNA but may also affect the interaction of straight DNA sequences

Probing the role of intercalating protein sidechains for kink formation in DNA

<div><p>Protein binding can induce DNA kinks, which are for example important to enhance the specificity of the interaction and to facilitate the assembly of multi protein complexes. The respective proteins frequently exhibit amino acid sidechains that intercalate between the DNA base steps at the site of the kink. However, on a molecular level there is only little information available about the role of individual sidechains for kink formation. To unravel structural principles of protein-induced DNA kinking we have performed molecular dynamics (MD) simulations of five complexes that varied in their architecture, function, and identity of intercalated residues. Simulations were performed for the DNA complexes of wildtype proteins (Sac7d, Sox-4, CcpA, TFAM, TBP) and for mutants, in which the intercalating residues were individually or combined replaced by alanine. The work revealed that for systems with multiple intercalated residues, not all of them are necessarily required for kink formation. In some complexes (Sox-4, TBP), one of the residues proved to be essential for kink formation, whereas the second residue has only a very small effect on the magnitude of the kink. In other systems (e.g. Sac7d) each of the intercalated residues proved to be individually capable of conferring a strong kink suggesting a partially redundant role of the intercalating residues. Mutation of the key residues responsible for kinking either resulted in stable complexes with reduced kink angles or caused conformational instability as evidenced by a shift of the kink to an adjacent base step. Thus, MD simulations can help to identify the role of individual inserted residues for kinking, which is not readily apparent from an inspection of the static structures. This information might be helpful for understanding protein-DNA interactions in more detail and for designing proteins with altered DNA binding properties in the future.</p></div

Structural properties of the Sox-4-DNA complex.

<p>(A) Structure of Sox-4-DNA complex with intercalated ‘FM wedge’ residues highlighted in orange. (B) Enlargement showing the kinked DNA base pair step and intercalating residues Phe66 and Met67. The kinked TT base step is also indicated. (C) DNA roll angle profile of Sox-4 in the simulations of wildtype and mutant Sox-4 proteins (the wildtype crystal structure 3u2b is shown as reference). (D) Time course of roll angles for Sox-4 A67M mutant. Values are shown for all base steps over the MD simulation. The vertical black line denotes the boundary between the two independent 250-ns MD simulations performed, which are presented in a single panel.</p

Structural properties of the CcpA-DNA complex.

<p>(A) Structure of CcpA-DNA complex with intercalated residues highlighted in orange. The two subunits of CcpA are colored in blue and green, respectively, and the corepressor HPr is shown in gray. (B) Enlargement showing the kinked DNA base pair step and intercalating residues L56 and L56’. The kinked CG base step is also indicated. (C) DNA roll angle profile of CcpA in the simulations of wildtype and mutant CcpA proteins (the wildtype crystal structure 3oqm is shown as reference).</p

Distribution of kink angles for different systems.

<p>Multiple dots in a single line result from the analysis of multiple DNA-complexes available for the same protein. Proteins in green boxes have no intercalating side chains, whereas proteins in blue boxes do have intercalating side chains. Values of the kinks produced by TFAM HMG-Box-1 and HMG-Box-2 are depicted separately.</p

Structure of the TBP-DNA complex.

<p>(A) Structure of TBP-DNA complex with intercalated residue highlighted in orange. (B) Enlargement showing one of the kinked DNA base pair steps (TA) and the intercalating residues Phe284 (loop) and Phe301 (sheet).</p

DNA deformation by wildtype and mutant TBP.

<p>(A) DNA roll angle profile of TBP in the simulations of wildtype and mutant TBP proteins (the wildtype crystal structure 1cdw is shown as reference). The DNA sequence is shown as x-axis indicating that there are two kinks formed between a TA and an AG base step, respectively. (B) Time course of the roll angles for all base steps over the MD simulation. The two base steps exhibiting the largest kinks are highlighted by boxes at the y-axis. The vertical black line denotes the boundary between the two independent 250-ns MD simulations performed for each system, which are presented in a single panel. Colors of boxes match line colors of average roll angle plots of corresponding systems in (A). The type of mutation present in the individual systems is structurally depicted at the top of each panel.</p

Structural properties of the TFAM-DNA complex.

<p>(A) Structure of the TFAM-DNA complex. HMG-Box-2 is highlighted in color and HMG-Box-1 is shown in grey. (B) Enlargement showing the kinked CA base step (HMG-Box-2). The intercalating L140 as well as the partially inserting V124 and F128 are shown in stick presentation. (C) DNA roll angle profile of TFAM for the simulations of full-length and truncated systems containing only Box-2. The wildtype crystal structure (3tmm) is shown as reference. (D) DNA roll angle profiles for different TFAM Box-2 mutants.</p