@article{BoboneHilschStormetal.2017, author = {Bobone, Sara and Hilsch, Malte and Storm, Julian and Dunsing, Valentin and Herrmann, Andreas and Chiantia, Salvatore}, title = {Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes}, series = {Journal of virology}, volume = {91}, journal = {Journal of virology}, publisher = {American Society for Microbiology}, address = {Washington}, issn = {0022-538X}, doi = {10.1128/JVI.00267-17}, pages = {15}, year = {2017}, abstract = {Influenza A virus matrix protein 1 (M1) is an essential component involved in the structural stability of the virus and in the budding of new virions from infected cells. A deeper understanding of the molecular basis of virion formation and the budding process is required in order to devise new therapeutic approaches. We performed a detailed investigation of the interaction between M1 and phosphatidylserine (PS) (i.e., its main binding target at the plasma membrane [PM]), as well as the distribution of PS itself, both in model membranes and in living cells. To this end, we used a combination of techniques, including Forster resonance energy transfer (FRET), confocal microscopy imaging, raster image correlation spectroscopy, and number and brightness (N\&B) analysis. Our results show that PS can cluster in segregated regions in the plane of the lipid bilayer, both in model bilayers constituted of PS and phosphatidylcholine and in living cells. The viral protein M1 interacts specifically with PS-enriched domains, and such interaction in turn affects its oligomerization process. Furthermore, M1 can stabilize PS domains, as observed in model membranes. For living cells, the presence of PS clusters is suggested by N\&B experiments monitoring the clustering of the PS sensor lactadherin. Also, colocalization between M1 and a fluorescent PS probe suggest that, in infected cells, the matrix protein can specifically bind to the regions of PM in which PS is clustered. Taken together, our observations provide novel evidence regarding the role of PS-rich domains in tuning M1-lipid and M1-M1 interactions at the PM of infected cells. IMPORTANCE Influenza virus particles assemble at the plasma membranes (PM) of infected cells. This process is orchestrated by the matrix protein M1, which interacts with membrane lipids while binding to the other proteins and genetic material of the virus. Despite its importance, the initial step in virus assembly (i.e., M1-lipid interaction) is still not well understood. In this work, we show that phosphatidylserine can form lipid domains in physical models of the inner leaflet of the PM. Furthermore, the spatial organization of PS in the plane of the bilayer modulates M1-M1 interactions. Finally, we show that PS domains appear to be present in the PM of living cells and that M1 seems to display a high affinity for them.}, language = {en} } @article{DunsingLucknerZuehlkeetal.2018, author = {Dunsing, Valentin and Luckner, Madlen and Zuehlke, Boris and Petazzi, Roberto Arturo and Herrmann, Andreas and Chiantia, Salvatore}, title = {Optimal fluorescent protein tags for quantifying protein oligomerization in living cells}, series = {Scientific reports}, volume = {8}, journal = {Scientific reports}, publisher = {Nature Publ. Group}, address = {London}, issn = {2045-2322}, doi = {10.1038/s41598-018-28858-0}, pages = {12}, year = {2018}, abstract = {Fluorescence fluctuation spectroscopy has become a popular toolbox for non-disruptive analysis of molecular interactions in living cells. The quantification of protein oligomerization in the native cellular environment is highly relevant for a detailed understanding of complex biological processes. An important parameter in this context is the molecular brightness, which serves as a direct measure of oligomerization and can be easily extracted from temporal or spatial fluorescence fluctuations. However, fluorescent proteins (FPs) typically used in such studies suffer from complex photophysical transitions and limited maturation, inducing non-fluorescent states. Here, we show how these processes strongly affect molecular brightness measurements. We perform a systematic characterization of non-fluorescent states for commonly used FPs and provide a simple guideline for accurate, unbiased oligomerization measurements in living cells. Further, we focus on novel red FPs and demonstrate that mCherry2, an mCherry variant, possesses superior properties with regards to precise quantification of oligomerization.}, language = {en} } @article{SperberWelkePetazzietal.2019, author = {Sperber, Hannah Sabeth and Welke, Robert-William and Petazzi, Roberto Arturo and Bergmann, Ronny and Schade, Matthias and Shai, Yechiel and Chiantia, Salvatore and Herrmann, Andreas and Schwarzer, Roland}, title = {Self-association and subcellular localization of Puumala hantavirus envelope proteins}, series = {Scientific reports}, volume = {9}, journal = {Scientific reports}, publisher = {Nature Publ. Group}, address = {London}, issn = {2045-2322}, doi = {10.1038/s41598-018-36879-y}, pages = {15}, year = {2019}, abstract = {Hantavirus assembly and budding are governed by the surface glycoproteins Gn and Gc. In this study, we investigated the glycoproteins of Puumala, the most abundant Hantavirus species in Europe, using fluorescently labeled wild-type constructs and cytoplasmic tail (CT) mutants. We analyzed their intracellular distribution, co-localization and oligomerization, applying comprehensive live, single-cell fluorescence techniques, including confocal microscopy, imaging flow cytometry, anisotropy imaging and Number\&Brightness analysis. We demonstrate that Gc is significantly enriched in the Golgi apparatus in absence of other viral components, while Gn is mainly restricted to the endoplasmic reticulum (ER). Importantly, upon co-expression both glycoproteins were found in the Golgi apparatus. Furthermore, we show that an intact CT of Gc is necessary for efficient Golgi localization, while the CT of Gn influences protein stability. Finally, we found that Gn assembles into higher-order homo-oligomers, mainly dimers and tetramers, in the ER while Gc was present as mixture of monomers and dimers within the Golgi apparatus. Our findings suggest that PUUV Gc is the driving factor of the targeting of Gc and Gn to the Golgi region, while Gn possesses a significantly stronger self-association potential.}, language = {en} } @misc{LucknerDunsingChiantiaetal.2017, author = {Luckner, Madlen and Dunsing, Valentin and Chiantia, Salvatore and Herrmann, Andreas}, title = {Influenza virus vRNPs: quantitative investigations via fluorescence cross-correlation spectroscopy}, series = {European biophysics journal : with biophysics letters ; an international journal of biophysics}, volume = {46}, journal = {European biophysics journal : with biophysics letters ; an international journal of biophysics}, publisher = {Springer}, address = {New York}, issn = {0175-7571}, pages = {S368 -- S368}, year = {2017}, language = {en} } @article{MemczakLausterKaretal.2016, author = {Memczak, Henry and Lauster, Daniel and Kar, Parimal and Di Lella, Santiago and Volkmer, Rudolf and Knecht, Volker and Herrmann, Andreas and Ehrentreich-Foerster, Eva and Bier, Frank Fabian and Stoecklein, Walter F. M.}, title = {Anti-Hemagglutinin Antibody Derived Lead Peptides for Inhibitors of Influenza Virus Binding}, series = {PLoS one}, volume = {11}, journal = {PLoS one}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0159074}, pages = {82 -- 90}, year = {2016}, abstract = {Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza A spike glycoprotein. Their binding properties were studied experimentally, and by molecular dynamics simulations. Two peptide candidates showed binding to influenza A/Aichi/2/68 H3N2. One of them, termed PeB, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. PeB matches best the conserved receptor binding site of hemagglutinin. PeB bound also to other medical relevant influenza strains, such as human-pathogenic A/California/7/2009 H1N1, and avian-pathogenic A/MuteSwan/Rostock/R901/2006 H7N1. Strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. The peptides and their derivatives are of great potential for drug development as well as biosensing.}, language = {en} } @misc{SperberWelkePetazzietal.2019, author = {Sperber, Hannah Sabeth and Welke, Robert-William and Petazzi, Roberto Arturo and Bergmann, Ronny and Schade, Matthias and Shai, Yechiel and Chiantia, Salvatore and Herrmann, Andreas and Schwarzer, Roland}, title = {Self-association and subcellular localization of Puumala hantavirus envelope proteins}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {648}, issn = {1866-8372}, doi = {10.25932/publishup-42504}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-425040}, pages = {15}, year = {2019}, abstract = {Hantavirus assembly and budding are governed by the surface glycoproteins Gn and Gc. In this study, we investigated the glycoproteins of Puumala, the most abundant Hantavirus species in Europe, using fluorescently labeled wild-type constructs and cytoplasmic tail (CT) mutants. We analyzed their intracellular distribution, co-localization and oligomerization, applying comprehensive live, single-cell fluorescence techniques, including confocal microscopy, imaging flow cytometry, anisotropy imaging and Number\&Brightness analysis. We demonstrate that Gc is significantly enriched in the Golgi apparatus in absence of other viral components, while Gn is mainly restricted to the endoplasmic reticulum (ER). Importantly, upon co-expression both glycoproteins were found in the Golgi apparatus. Furthermore, we show that an intact CT of Gc is necessary for efficient Golgi localization, while the CT of Gn influences protein stability. Finally, we found that Gn assembles into higher-order homo-oligomers, mainly dimers and tetramers, in the ER while Gc was present as mixture of monomers and dimers within the Golgi apparatus. Our findings suggest that PUUV Gc is the driving factor of the targeting of Gc and Gn to the Golgi region, while Gn possesses a significantly stronger self-association potential.}, language = {en} } @misc{MemczakLausterKaretal.2016, author = {Memczak, Henry and Lauster, Daniel and Kar, Parimal and Di Lella, Santiago and Volkmer, Rudolf and Knecht, Volker and Herrmann, Andreas and Ehrentreich-F{\"o}rster, Eva and Bier, Frank Fabian and St{\"o}cklein, Walter F. M.}, title = {Anti-hemagglutinin antibody derived lead peptides for inhibitors of influenza virus binding}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {536}, issn = {1866-8372}, doi = {10.25932/publishup-41087}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-410872}, pages = {24}, year = {2016}, abstract = {Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza A spike glycoprotein. Their binding properties were studied experimentally, and by molecular dynamics simulations. Two peptide candidates showed binding to influenza A/Aichi/2/68 H3N2. One of them, termed PeB, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. PeB matches best the conserved receptor binding site of hemagglutinin. PeB bound also to other medical relevant influenza strains, such as human-pathogenic A/California/7/2009 H1N1, and avian-pathogenic A/MuteSwan/Rostock/R901/2006 H7N1. Strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. The peptides and their derivatives are of great potential for drug development as well as biosensing.}, language = {en} } @article{HaralampievMertensSchwarzeretal.2015, author = {Haralampiev, Ivan and Mertens, Monique and Schwarzer, Roland and Herrmann, Andreas and Volkmer, Rudolf and Wessig, Pablo and Mueller, Peter}, title = {Recruitment of SH-Containing peptides to lipid and biological membranes through the use of a palmitic acid functionalized with a Maleimide Group}, series = {Angewandte Chemie : a journal of the Gesellschaft Deutscher Chemiker ; International edition}, volume = {54}, journal = {Angewandte Chemie : a journal of the Gesellschaft Deutscher Chemiker ; International edition}, number = {1}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1433-7851}, doi = {10.1002/anie.201408089}, pages = {323 -- 326}, year = {2015}, abstract = {This study presents a novel and easily applicable approach to recruit sulfhydryl-containing biomolecules to membranes by using a palmitic acid which is functionalized with a maleimide group. Notably, this strategy can also be employed with preformed (biological) membranes. The applicability of the assay is demonstrated by characterizing the binding of a Rhodamine-labeled peptide to lipid and cellular membranes using methods of fluorescence spectroscopy, lifetime measurement, and microscopy. Our approach offers new possibilities for preparing biologically active liposomes and manipulating living cells.}, language = {en} } @inproceedings{HaralampievMertensSchwarzeretal.2015, author = {Haralampiev, Ivan and Mertens, Monique and Schwarzer, Roland and Herrmann, Andreas and Volkmer, Rudolf and Wessig, Pablo and M{\"u}ller, Peter}, title = {A palmitic acid functionalized with a maleimide group is used to recruit SH-containing peptides to lipid and biological membranes}, series = {The FEBS journal}, volume = {282}, booktitle = {The FEBS journal}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {1742-464X}, pages = {204 -- 204}, year = {2015}, language = {en} } @article{HeuvelingFrochauxZiomkowskaetal.2014, author = {Heuveling, Johanna and Frochaux, Violette and Ziomkowska, Joanna and Wawrzinek, Robert and Wessig, Pablo and Herrmann, Andreas and Schneider, Erwin}, title = {Conformational changes of the bacterial type I ATP-binding cassette importer HisQMP(2) at distinct steps of the catalytic cycle}, series = {Biochimica et biophysica acta : Biomembranes}, volume = {1838}, journal = {Biochimica et biophysica acta : Biomembranes}, number = {1}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0005-2736}, doi = {10.1016/j.bbamem.2013.08.024}, pages = {106 -- 116}, year = {2014}, abstract = {Prokaryotic solute binding protein-dependent ATP-binding cassette import systems are divided into type land type II and mechanistic differences in the transport process going along with this classification are under intensive investigation. Little is known about the conformational dynamics during the catalytic cycle especially concerning the transmembrane domains. The type I transporter for positively charged amino acids from Salmonella enterica serovar Typhimurium (1A0-Hi5QMP2) was studied by limited proteolysis in detergent solution in the absence and presence of co-factors including ATP, ADP, LAO/arginine, and Mg2+ ions. Stable peptide fragments could be obtained and differentially susceptible cleavage sites were determined by mass spectrometry as Lys-258 in the nucleotide-binding subunit, HisP, and Arg-217/Arg-218 in the transmembrane subunit, HisQ In contrast, transmembrane subunit HisM was gradually degraded but no stable fragment could be detected. HisP and HisQ were equally resistant under pre- and post-hydrolysis conditions in the presence of arginine-loaded solute-binding protein LAO and ATP/ADP. Some protection was also observed with LAO/arginine alone, thus reflecting binding to the transporter in the apo-state and transmembrane signaling. Comparable digestion patterns were obtained with the transporter reconstituted into proteoliposomes and nanodiscs. Fluorescence lifetime spectroscopy confirmed the change of HisQ(R218) to a more apolar microenvironment upon ATP binding and hydrolysis. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. Together, our data suggest similar conformational changes during the transport cycle as described for the maltose ABC transporter of Escherichia coli, despite distinct structural differences between both systems.}, language = {en} }