TY - JOUR A1 - Hornemann, Andrea A1 - Eichert, Diane Madeleine A1 - Hoehl, Arne A1 - Tiersch, Brigitte A1 - Ulm, Gerhard A1 - Ryadnov, Maxim G. A1 - Beckhoff, Burkhard T1 - Investigating Membrane-Mediated Antimicrobial Peptide Interactions with Synchrotron Radiation Far-Infrared Spectroscopy JF - ChemPhysChem : a European journal of chemical physics and physical chemistry N2 - Synchrotron radiation-based Fourier transform infrared spectroscopy enables access to vibrational information from mid over far infrared to even terahertz domains. This information may prove critical for the elucidation of fundamental bio-molecular phenomena including folding-mediated innate host defence mechanisms. Antimicrobial peptides (AMPs) represent one of such phenomena. These are major effector molecules of the innate immune system, which favour attack on microbial membranes. AMPs recognise and bind to the membranes whereupon they assemble into pores or channels destabilising the membranes leading to cell death. However, specific molecular interactions responsible for antimicrobial activities have yet to be fully understood. Herein we probe such interactions by assessing molecular specific variations in the near-THz 400-40 cm(-1) range for defined helical AMP templates in reconstituted phospholipid membranes. In particular, we show that a temperature-dependent spectroscopic analysis, supported by 2D correlative tools, provides direct evidence for the membrane-induced and folding-mediated activity of AMPs. The far-FTIR study offers a direct and information-rich probe of membrane-related antimicrobial interactions. KW - antimicrobial peptides KW - electrostatic interactions KW - IR spectroscopy KW - phospholipid membranes KW - protein folding Y1 - 2022 U6 - https://doi.org/10.1002/cphc.202100815 SN - 1439-4235 SN - 1439-7641 VL - 23 IS - 4 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Bremer, Anne A1 - Wolff, Martin A1 - Thalhammer, Anja A1 - Hincha, Dirk K. T1 - Folding of intrinsically disordered plant LEA proteins is driven by glycerol-induced crowding and the presence of membranes JF - The FEBS journal N2 - Late embryogenesis abundant (LEA) proteins are related to cellular dehydration tolerance. Most LEA proteins are predicted to have no stable secondary structure in solution, i.e., to be intrinsically disordered proteins (IDPs), but they may acquire alpha-helical structure upon drying. In the model plant Arabidopsis thaliana, the LEA proteins COR15A and COR15B are highly induced upon cold treatment and are necessary for the plants to attain full freezing tolerance. Freezing leads to increased intracellular crowding due to dehydration by extracellular ice crystals. In vitro, crowding by high glycerol concentrations induced partial folding of COR15 proteins. Here, we have extended these investigations to two related proteins, LEA11 and LEA25. LEA25 is much longer than LEA11 and COR15A, but shares a conserved central sequence domain with the other two proteins. We have created two truncated versions of LEA25 (2H and 4H) to elucidate the structural and functional significance of this domain. Light scattering and CD spectroscopy showed that all five proteins were largely unstructured and monomeric in dilute solution. They folded in the presence of increasing concentrations of trifluoroethanol and glycerol. Additional folding was observed in the presence of glycerol and membranes. Fourier transform infra red spectroscopy revealed an interaction of the LEA proteins with membranes in the dry state leading to a depression in the gel to liquid-crystalline phase transition temperature. Liposome stability assays revealed a cryoprotective function of the proteins. The C- and N-terminal extensions of LEA25 were important in cryoprotection, as the central domain itself (2H, 4H) only provided a low level of protection. KW - intrinsically disordered proteins KW - late embryogenesis abundant proteins KW - osmolytes KW - protein folding KW - protein-membrane interaction Y1 - 2017 U6 - https://doi.org/10.1111/febs.14023 SN - 1742-464X SN - 1742-4658 VL - 284 SP - 919 EP - 936 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Hofmann, Hagen A1 - Soranno, Andrea A1 - Borgia, Alessandro A1 - Gast, Klaus A1 - Nettels, Daniel A1 - Schuler, Benjamin T1 - Polymer scaling laws of unfolded and intrinsically disordered proteins quantified with single-molecule spectroscopy JF - Proceedings of the National Academy of Sciences of the United States of America N2 - The dimensions of unfolded and intrinsically disordered proteins are highly dependent on their amino acid composition and solution conditions, especially salt and denaturant concentration. However, the quantitative implications of this behavior have remained unclear, largely because the effective theta-state, the central reference point for the underlying polymer collapse transition, has eluded experimental determination. Here, we used single-molecule fluorescence spectroscopy and two-focus correlation spectroscopy to determine the theta points for six different proteins. While the scaling exponents of all proteins converge to 0.62 +/- 0.03 at high denaturant concentrations, as expected for a polymer in good solvent, the scaling regime in water strongly depends on sequence composition. The resulting average scaling exponent of 0.46 +/- 0.05 for the four foldable protein sequences in our study suggests that the aqueous cellular milieu is close to effective theta conditions for unfolded proteins. In contrast, two intrinsically disordered proteins do not reach the T-point under any of our solvent conditions, which may reflect the optimization of their expanded state for the interactions with cellular partners. Sequence analyses based on our results imply that foldable sequences with more compact unfolded states are a more recent result of protein evolution. KW - protein folding KW - single-molecule FRET KW - coil-globule transition KW - polymer theory Y1 - 2012 U6 - https://doi.org/10.1073/pnas.1207719109 SN - 0027-8424 VL - 109 IS - 40 SP - 16155 EP - 16160 PB - National Acad. of Sciences CY - Washington ER - TY - THES A1 - Gomez, David T1 - Mechanisms of biochemical reactions within crowded environments T1 - Mechanismus der Biochemische Reaktionen im vollgestopfte Umgebungen N2 - The cell interior is a highly packed environment in which biological macromolecules evolve and function. This crowded media has effects in many biological processes such as protein-protein binding, gene regulation, and protein folding. Thus, biochemical reactions that take place in such crowded conditions differ from diluted test tube conditions, and a considerable effort has been invested in order to understand such differences. In this work, we combine different computationally tools to disentangle the effects of molecular crowding on biochemical processes. First, we propose a lattice model to study the implications of molecular crowding on enzymatic reactions. We provide a detailed picture of how crowding affects binding and unbinding events and how the separate effects of crowding on binding equilibrium act together. Then, we implement a lattice model to study the effects of molecular crowding on facilitated diffusion. We find that obstacles on the DNA impair facilitated diffusion. However, the extent of this effect depends on how dynamic obstacles are on the DNA. For the scenario in which crowders are only present in the bulk solution, we find that at some conditions presence of crowding agents can enhance specific-DNA binding. Finally, we make use of structure-based techniques to look at the impact of the presence of crowders on the folding a protein. We find that polymeric crowders have stronger effects on protein stability than spherical crowders. The strength of this effect increases as the polymeric crowders become longer. The methods we propose here are general and can also be applied to more complicated systems. N2 - Innerhalb einer Zelle, im Zytosol, entstehen und arbeiten sehr viele biologische Makromoleküle. Die Dichte dieser Moleküle ist sehr hoch und dieses ‘vollgestopfte’ Zytosol hat vielfältige Auswirkungen auf viele biologische Prozessen wie zum Beispiel Protein-Protein Interaktionen, Genregulation oder die Faltung von Proteinen. Der Ablauf von vielen biochemische Reaktionen in dieser Umgebung weicht von denen unter verdünnte Laborbedingungen ab. Um die Effekte dieses ‘makromolekularen Crowdings’ zu verstehen, wurde in den letzten Jahren bereits viel Mühe investiert. In dieser Arbeit kombinieren wir verschiede Computermethoden, um die Wirkungen des ‘makromolekularen Crowdings’ auf biologische Prozesse besser zu verstehen. Zuerst schlagen wir ein Gittermodell vor, um damit die Effekte des ‘makromolekularen Crowdings’ auf enzymatische Reaktionen zu studieren. Damit stellen wir ein detailliertes Bild zusammen, wie Crowding die Assoziations- und Dissozotationsraten beeinflusst und wie verschiedene crowding-Effekte zusammen auf die Gleichgewichtskonstante wirken. Weiterhin implementieren wir ein Gittermodell der ‘erleichterte Diffusion’. Unsere Ergebnisse zeigen, dass Hindernisse an der DNA die vereinfachte Diffusion beeinträchtigen. Das Ausmass dieser Wirkung hängt dabei von der Dynamik der Hindernisse an der DNA ab. Im dem Fall dass Crowder ausschließlich in der Lösung vorhanden sind, erhöhen sich unter bestimmten Bedingungen DNA-spezifische Bindungen. Schließlich nutzten wir strukturbasierte Techniken um damit die Auswirkungen von Crowding auf die Faltung von Proteinen zu untersuchen. Wir fanden dabei, dass Polymer Crowder stärkere Wirkungen auf die Proteinstabilität haben als kugelförmige Crowder. Dieser Effekt verstärkte sich mit der Länge der untersuchten Polymere. Die Methoden die hier vorgeschlagen werden, sind generell anwendbar und können auch an deutlich komplexeren Systemen angewandt werden. KW - molecular crowding KW - gene expression KW - enzymatic activity KW - protein folding KW - Molecular crowding KW - enzymatische Reaktionen KW - Genregulation KW - Faltung von Proteinen Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-94593 ER - TY - THES A1 - Weikl, Thomas R. T1 - Transition states and loop-closure principles in protein folding T1 - Übergangszustände und Schleifenschließungsprinzipien bei der Proteinfaltung N2 - Proteins are chain molecules built from amino acids. The precise sequence of the 20 different types of amino acids in a protein chain defines into which structure a protein folds, and the three-dimensional structure in turn specifies the biological function of the protein. The reliable folding of proteins is a prerequisite for their robust function. Misfolding can lead to protein aggregates that cause severe diseases, such as Alzheimer's, Parkinson's, or the variant Creutzfeldt-Jakob disease. Small single-domain proteins often fold without experimentally detectable metastable intermediate states. The folding dynamics of these proteins is thought to be governed by a single transition-state barrier between the unfolded and the folded state. The transition state is highly instable and cannot be observed directly. However, mutations in which a single amino acid of the protein is substituted by another one can provide indirect access. The mutations slightly change the transition-state barrier and, thus, the folding and unfolding times of the protein. The central question is how to reconstruct the transition state from the observed changes in folding times. In this habilitation thesis, a novel method to extract structural information on transition states from mutational data is presented. The method is based on (i) the cooperativity of structural elements such as alpha-helices and beta-hairpins, and (ii) on splitting up mutation-induced free-energy changes into components for these elements. By fitting few parameters, the method reveals the degree of structure formation of alpha-helices and beta-hairpins in the transition state. In addition, it is shown in this thesis that the folding routes of small single-domain proteins are dominated by loop-closure dependencies between the structural elements. N2 - Proteine sind Kettenmoleküle, die aus einzelnen Aminosäuren aufgebaut sind. Die genaue Sequenz der 20 verschiedenartigen Aminosäuren innerhalb der Proteinkette bestimmt dabei, in welche spezielle Struktur sich ein Protein faltet. Die dreidimensionale Struktur bestimmt wiederum die Funktion der Proteine. Doch nur korrekt gefaltet kann ein Protein seine Funktion erfüllen. Fehler bei der Faltung können zu Proteinaggregaten führen, die schwere Krankheiten wie Alzheimer, Parkinson oder das Creutzfeldt-Jakob-Syndrom hervorrufen. Viele kleine Proteine falten ohne experimentell beobachtbare metastabile Zwischenzustände. Entscheidend für die Faltungsdynamik dieser Proteine ist der Übergangszustand zwischen dem ungefalteten und gefalteten Zustand. Der Übergangszustand ist instabil und kann nicht direkt beobachtet werden. Einen indirekten Zugang ermöglichen jedoch Mutationen eines Proteins, bei denen einzelne Aminosäuren ausgetauscht werden. Die Mutationen verändern geringfügig die Übergangszustandsbarriere, und damit die Faltungs- und Entfaltungszeiten des Proteins. Die zentrale Frage ist, wie sich der Übergangszustand aus den beobachteten Änderungen der Faltungszeit rekonstruieren lässt. In dieser Habilitationsschrift wird eine neuartige Methode zur Rekonstruktion von Übergangszuständen aus Mutationsdaten vorgestellt. Die Methode beruht auf (i) der Kooperativität von Strukturelementen wie alpha-Helizes und beta-Haarnadeln, und (ii) der Aufspaltung von mutationsinduzierten Veränderungen der freien Energie in Komponenten für diese Strukturelemente. Die Modellierung der experimentellen Daten verrät, in welchem Grad alpha-Helizes and beta-Haarnadeln im Übergangszustand strukturiert sind. Zudem wird in dieser Habilitationsschrift gezeigt, dass die Faltungswege vieler kleiner Proteine durch Schleifenschließungsbeziehungen zwischen den Strukturelementen dominiert werden. KW - Proteinfaltung KW - Faltungsdynamik KW - Übergangszustand KW - Stochastische Prozesse KW - Schleifenschließung KW - protein folding KW - folding dynamics KW - transition state KW - stochastic processes KW - loop closure Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-26975 ER -