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Lazzara T.D.,Institute of Organic and Biomolecular Chemistry | Aaron Lau K.H.,Northwestern University | Knoll W.,AIT Austrian Institute of Technology | Janshoff A.,Institute of Physical Chemistry | Steinem C.,Institute of Organic and Biomolecular Chemistry
Beilstein Journal of Nanotechnology | Year: 2012

Layer-by-layer (LbL) deposition of polyelectrolytes and proteins within the cylindrical nanopores of anodic aluminum oxide (AAO) membranes was studied by optical waveguide spectroscopy (OWS). AAO has aligned cylindrical, nonintersecting pores with a defined pore diameter d 0 and functions as a planar optical waveguide so as to monitor, in situ, the LbL process by OWS. The LbL deposition of globular proteins, i.e., avidin and biotinylated bovine serum albumin was compared with that of linear polyelectrolytes (linear-PEs), both species being of similar molecular weight. LbL deposition within the cylindrical AAO geometry for different pore diameters (d 0 = 25-80 nm) for the various macromolecular species, showed that the multilayer film growth was inhibited at different maximum numbers of LbL steps (n max). The value of n max was greatest for linear-PEs, while proteins had a lower value. The cylindrical pore geometry imposes a physical limit to LbL growth such that n max is strongly dependent on the overall internal structure of the LbL film. For all macromolecular species, deposition was inhibited in native AAO, having pores of d 0 = 25-30 nm. Both, OWS and scanning electron microscopy showed that LbL growth in larger AAO pores (d 0 > 25-30 nm) became inhibited when approaching a pore diameter of d eff,n_max = 25-35 nm, a similar size to that of native AAO pores, with d 0 = 25-30 nm. For a reasonable estimation of d eff,n_max, the actual volume occupied by a macromolecular assembly must be taken into consideration. The results clearly show that electrostatic LbL allowed for compact macromolecular layers, whereas proteins formed loosely packed multilayers. © 2012 Lazzara et al.


News Article | December 14, 2016
Site: www.eurekalert.org

The latest recipients of Germany's most prestigious research funding prize have been announced. In Bonn today, the Joint Committee of the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) chose ten researchers, three women and seven men, to receive the 2017 Leibniz Prize. The recipients of the prize were selected by the Nominations Committee from 134 nominees. Of the ten new prizewinners, three are from the natural sciences, three from the humanities and social sciences, two from the life sciences and two from the engineering sciences. Each of the ten winners will receive €2.5 million in prize money. They can use these funds for their research work in any way they wish, without bureaucratic obstacles, for up to seven years. The awards ceremony for the 2017 Leibniz Prizes will be held on 15 March in Berlin. The following researchers will receive the 2017 "Funding Prize in the Gottfried Wilhelm Leibniz Programme" awarded by the DFG: The Gottfried Wilhelm Leibniz Prize has been awarded by the DFG annually since 1986. Each year a maximum of ten prizes can be awarded, each with prize money of €2.5 million. With the ten prizes for 2017, a total of 348 Leibniz Prizes have been awarded to date. Of these, 115 were bestowed on researchers in the natural sciences, 101 in the life sciences, 79 in the humanities and social sciences, and 53 in the engineering sciences. The number of award recipients is higher than the number of awarded prizes because, in exceptional cases, the prizes and money can be shared. Accordingly, a total of 374 nominees have received the prize, including 326 men and 48 women. The Leibniz Prize is the most significant research prize in Germany. Seven past prizewinners have subsequently received the Nobel Prize: 1988 Professor Dr. Hartmut Michel (Chemistry), 1991 Professor Dr. Erwin Neher and Professor Dr. Bert Sakmann (Medicine), 1995 Professor Dr. Christiane Nüsslein-Volhard (Medicine), 2005 Professor Dr. Theodor W. Hänsch (Physics), 2007 Professor Dr. Gerhard Ertl (Chemistry) and most recently in 2014 Professor Dr. Stefan W. Hell (Chemistry). Professor Dr. Lutz Ackermann (43), Organic Molecular Chemistry, Institute of Organic and Biomolecular Chemistry, University of Göttingen Lutz Ackermann has been selected for the 2017 Leibniz Prize for his outstanding work in the field of organic chemistry. His international reputation is based especially on his research into the catalytic activation of carbon-hydrogen bonds. These bonds, which occur in all organic substances, are usually extremely inert and permit only very poor and frequently non-selective transformation. The methods developed by Ackermann and his colleagues have paved the way for fundamentally new and low-impact manufacturing methods for important chemical products including active substances, agrochemicals and fine chemicals. Through his other work, Ackermann has also created new concepts for environmentally friendly chemical synthesis. Lutz Ackermann studied chemistry in Kiel, and, after further studies in Rennes and Mülheim an der Ruhr, he obtained his doctorate from the University of Dortmund. He did postdoctoral research at Berkeley before going to Munich in 2003 to work as the leader of a DFG-funded Emmy Noether independent junior research group. Ackermann has held his current chair in Göttingen since 2007 and has headed the Institute of Organic and Biomolecular Chemistry there since 2015. He is one of the most frequently cited researchers in his field in the world. Professor Dr. Beatrice Gründler (52), Arabic Studies, Seminar for Semitic and Arabic Studies, Free University of Berlin Beatrice Gründler will receive the Leibniz Prize for her studies on the diversity of voices in Arabic poetry and culture. She has been interested in the medium of script and its fundamental importance to Arabic traditions since an early stage in her career, as evidenced for example by her book "The Development of the Arabic Script" (1993). Through her research she has developed a complex media history of the Arab world, from the introduction of paper to book printing and beyond - indeed, she refers to an 'Arabic book revolution'. In a pilot project for a critical, annotated digital edition of the "Kalila wa-Dimna", begun in 2015, Gründler has unravelled the history of the text, development and impact of this collection of fables, considered one of the earliest Arabic prose works and a central text of Arabic wisdom literature. Gründler's own approach puts into practice in an exemplary way the encounters between Arabic and European knowledge traditions that she investigates in her work - another reason for the importance of her research. Beatrice Gründler studied at Strasbourg, Tübingen and Harvard, where she received her doctorate in 1995. After a period at Dartmouth College, she began teaching at Yale University in 1996, first as an assistant professor and from 2002 as Professor of Arabic Literature. In 2014 she returned to Germany, and has since been undertaking research at the Free University of Berlin. Ralph Hertwig will be recognised with the 2017 Leibniz Prize for his pioneering work in the psychology of human judgement and decision-making. His research has expanded our understanding of the possibilities and limitations of human rationality. Hertwig investigates the strategies which humans use, faced with limited knowledge, limited cognitive resources and often limited time, to nonetheless make good decisions and organise their actions. Central to his work is the question why a limitation also constitutes a strength, in other words how adaptive heuristics, as simple rules of thumb for problem-solving, can be as effective as complex optimisation models. Another of Hertwig's important contributions to decision research is the distinction between experience-based and description-based assessment of risk. This explains why the dramatic consequences of climate change, for example, are systematically underestimated by society, because although there is plenty of information available to describe the problem, there is little everyday experience - the main thing that people base their decisions on. Ralph Hertwig has been the director of the Max Planck Institute for Human Development since 2012 and heads the Center for Adaptive Rationality. Hertwig began his scientific career in 1995 at the Max Planck Institute for Psychological Research in Munich. In 1997 he moved to the Max Planck Institute in Berlin. Between 2000 and 2002 he was a Research Fellow at Columbia University. In 2003 he obtained his habilitation from the Free University of Berlin. In 2005 he was appointed Professor of Cognitive Science and Decision Psychology at the University of Basel, and moved from there to his current position. Karl-Peter Hopfner will receive the Leibniz Prize for his outstanding work in structural molecular biology and genome biology, with which he has made pioneering contributions to the field of DNA repair and the cellular detection of foreign nucleic acids. Hopfner's research focused on the molecular mechanisms of multiprotein complexes, which play an important role in the detection of damaged or viral nucleic acids. These detection processes are crucial to the protection of the genome; errors in detection and repair are among the main reasons for the development of cancer. Building on that work, Hopfner has carried out essential work on DNA double-strand break repair and in recent years has decoded the mechanism of the central MRN complex Mre11-Rad50-Nbs1, a DNA damage sensor. He also contributed substantially to answering the question of how cellular sensors of the innate immune system recognise viral or bacterial nucleic acids in the case of infection. Here, the sensors must distinguish between the body's own RNA and foreign RNA. Karl-Peter Hopfner studied biology in Regensburg and in St. Louis, USA. He completed his doctorate at the Max Planck Institute for Biochemistry in Martinsried as part of the Division led by Nobel Prize winner Robert Huber. Between 1999 and 2001 he carried out postdoctoral research at the Scripps Research Institute in La Jolla, before accepting a tenure track professorship at the Gene Center at LMU Munich. He has been a full professor at LMU since 2007. Professor Dr. Frank Jülicher (51), Theory of Biological Physics, Max Planck Institute for the Physics of Complex Systems, Dresden The award of the Leibniz Prize to Frank Jülicher recognises a world-leading researcher in biophysics with the ability to identify universal physical principles in the complex world of living matter. He had already attracted attention with his early work on the physics of hearing and cell mechanics. Through his investigation of active matter - the components of which exhibit autonomous activity, such as molecular motors, which play a key role in cell movement and division - Jülicher has established a new field of research. This raises many fundamental questions in non-equilibrium physics and has also inspired numerous new applications as well as biomimetic design. In collaboration with French researchers, the biophysicist laid the foundations for the dynamics of active matter by formulating a general hydrodynamic theory of active matter. Most recently, Jülicher has turned his attention to the control and organisation of cells in tissue. His seminal work is contributing to our understanding of cell self-organisation in tissue. This phenomenon, as yet poorly understood, is of enormous importance to developmental biology and medical applications. Frank Jülicher studied physics in Stuttgart and Aachen, received his doctorate in Cologne in 1994 and then spent two years researching in the USA and Canada. He subsequently worked with leading researchers in Paris in the field of soft matter and biophysics, before obtaining his habilitation in 2000 at Paris Diderot University (Paris 7). Since 2002, Jülicher has been the director of the Max Planck Institute for the Physics of Complex Systems in Dresden and Professor of Biophysics at the Technical University of Dresden. Professor Dr. Lutz Mädler (45), Mechanical Process Engineering, Stiftung Institut für Werkstofftechnik (IWT) and Department of Production Engineering, University of Bremen Lutz Mädler will receive the Leibniz Prize in recognition of his pioneering work in the targeted reactive formation of nanoparticles in the gas phase and their effect on living matter. He has developed an improved variant of flame spray pyrolysis for the cost-effective synthesis of nanoparticles, involving the thermochemical splitting of organic compounds. His work has made flame spray pyrolysis available for industrial applications. Mädler subsequently refined this pyrolysis technique when he discovered the droplet explosion phenomenon in flame sprays and its effects on material synthesis. However, as well as looking at the tailored synthesis of nanoparticles, Mädler has also investigated how toxic these particles are to the human body. This is important because many applications, for example paints, textiles and dental fillings, have direct impacts on humans. Mädler was able to demonstrate that interactions between synthetic nanoparticles and biological tissue produce reactive oxygen species which can trigger undesirable reactions. Lutz Mädler studied physics at the Technical University of Zwickau and then process engineering at Technische Universität Bergakademie Freiberg, where he obtained his doctorate in 1999. He completed his habilitation at ETH Zurich and then, with the support of a DFG fellowship, became a Senior Researcher at the University of California, Los Angeles. In 2008 he was appointed professor at the University of Bremen. Britta Nestler has been selected to receive the 2017 Leibniz Prize for her significant, internationally recognised research in computer-assisted materials research and the development of new material models with multiscale and multiphysical approaches. Nestler has developed extremely flexible and high-performing simulation environments to simulate the microstructure of materials for use on supercomputers. These are based on her own quantitative models for the description of multicomponent systems. She has thus achieved a new quality of microstructure representation in the thermomechanical simulation of materials and the simulation of solidification processes and thus depicted these processes through realistic 3D simulation for the first time. Through her creative application and further development of the phase field method, Nestler has achieved outstanding fundamental insights which are also of enormous practical relevance. For example, her simulation calculations are used to predict the spread of cracks in design materials such as brake discs and therefore help to extend their lifetime. Britta Nestler studied physics and mathematics in Aachen, where she also received her doctorate. Research visits took her to Southampton, UK and Paris. In 2001 Nestler accepted a professorship in the Faculty of Computer Science at Karlsruhe University of Applied Sciences and in 2009 her current chair at KIT. Professor Dr. Joachim P. Spatz (47), Biophysics, Max Planck Institute for Intelligent Systems, Stuttgart, and Institute of Physical Chemistry, University of Heidelberg Joachim Spatz will be recognised with the Leibniz Prize for his outstanding research at the boundaries of materials sciences and cell biophysics. His research is concerned with cell adhesion, that is, the adhesion and bonding of cells to one another and to surfaces. His exemplary experimental approach has garnered precise insights into the control of cell adhesion and indeed physiological processes. To achieve this, Spatz used artificial, molecularly structured boundary surfaces to reduce possible interactions to a minimum of molecular components. Joachim Spatz' scientific achievement lies in the fact that he can study the communication mechanisms between cells in a new way with the help of concepts from materials science and physics. Using these resources, he was able to explain how the molecular mechanism of collective cell migration works in wound healing. Joachim Spatz studied physics in Ulm and at Colorado State University. He obtained his doctorate in macromolecular chemistry in Ulm, and it was also there that he completed his habilitation with a topic on cell mechanics. Since 2000 he has been a professor of biophysical chemistry in Heidelberg. In 2004 he was appointed director of the Max Planck Institute for Metals Research, now the Max Planck Institute for Intelligent Systems, in Stuttgart. Since 2008 he has also held a visiting professorship in molecular cell biology at the Weizmann Institute in Rehovot, Israel. Professor Dr. Anne Storch (48), African Studies, Institute for African Studies and Egyptology, University of Cologne In awarding the 2017 Leibniz Prize to Anne Storch, the DFG is honouring an extremely innovative and world-renowned researcher in African Studies who has contributed to a far-reaching reorientation of her field through her pioneering work. Drawing on questions and methods from cultural anthropology and the social sciences, Storch has introduced new thematic and methodological dimensions, both theoretical and practical, to African Studies. Her exemplary studies have also shown how linguistically based analyses can be used in an interdisciplinary approach to develop a cultural-anthropological understanding of contemporary Africa. Of particular significance was her study of taboos and secret languages in central Africa, published in 2011, which describes linguistic observations in such a way that they lead to complex sociological descriptions of power practices and political mechanisms of effect. Storch's case studies, rooted in, yet transcending, linguistic speech description, have become internationally significant model studies for a modern, self-critical approach to African Studies. Anne Storch has been Professor of African Studies in Cologne since 2004. She trained in anthropology, African Studies, Oriental Studies and archaeology in Frankfurt am Main and Mainz. Between 2006 and 2009 she served as president of the Fachverband Afrikanistik, the specialist society for Africa-related scholarship in Germany. Since 2014 she has been the president of the International Association for Colonial and Postcolonial Linguistics. Awarding the Leibniz Prize to Jörg Vogel recognises one of the world's leading researchers in the field of ribonucleic acid biology. He was selected for his pioneering contributions to our understanding of regulatory RNA molecules in infection biology. Vogel recognised the importance of RNA biochemistry in prokaryotes very early on and has done pioneering work in the application and development of high-throughput sequencing for RNA analysis. Using this method, he has studied the influence of pathogens on the host cell. Vogel has also discovered how small regulatory RNA molecules control protein synthesis and the breakdown of RNA. This in turn has contributed to the development of new methods which can be used in gene therapy. Together with Emmanuelle Charpentier, who won the Leibniz Prize in 2016, Vogel was able to understand tracrRNA (trans-activating RNA) and its function, which made the application of the CRISPR/Cas9 system possible. Vogel thus uncovered general biological principles which play a major role in our understanding of pathogenic microorganisms and are resulting in new treatment approaches. Jörg Vogel studied biochemistry at the Humboldt University of Berlin, where he also obtained his doctorate on RNA splicing in plants. After doing postdoctoral research in Uppsala and Jerusalem, in 2004 he was appointed Head of Division at the Max Planck Institute for Infection Biology in Berlin. Since 2009 he has been a professor at the University of Würzburg, where he heads the Institute for Molecular Infection Biology. The Leibniz Prizes will be awarded on 15 March 2017 at 3.00 pm at the Berlin-Brandenburg Academy of Sciences and Humanities in Berlin. A separate invitation will be sent to members of the media. Additional information about the 2017 prizewinners can be requested at the start of the new year by contacting the DFG Press and Public Relations Office or at http://www. . Detailed information about the Gottfried Wilhelm Leibniz Programme is available at: http://www.


Lazzara T.D.,Institute of Organic and Biomolecular Chemistry | Kliesch T.-T.,Institute of Organic and Biomolecular Chemistry | Janshoff A.,Institute of Physical Chemistry | Steinem C.,Institute of Organic and Biomolecular Chemistry
ACS Applied Materials and Interfaces | Year: 2011

Anodic aluminum oxide (AAO) membranes with aligned, cylindrical, nonintersecting pores were selectively functionalized in order to create dual-functionality substrates with different pore-rim and pore-interior surface functionalities, using silane chemistry. We used a two-step process involving an evaporated thin gold film to protect the underlying surface functionality of the pore rims. Subsequent treatment with oxygen plasma of the modified AAO membrane removed the unprotected organic functional groups, i.e., the pore-interior surface. After gold removal, the substrate became optically transparent, and displayed two distinct surface functionalities, one at the pore-rim surface and another at the pore-interior surface. We achieved a selective hydrophobic functionalization with dodecyl-trichlorosilane of either the pore rims or the pore interiors. The deposition of planar lipid membranes on the functionalized areas by addition of small unilamellar vesicles occurred in a predetermined fashion. Small unilamellar vesicles only ruptured upon contact with the hydrophobic substrate regions forming solid supported hybrid bilayers. In addition, pore-rim functionalization with dodecyl-trichlorosilane allowed the formation of pore-spanning hybrid lipid membranes as a result of giant unilamellar vesicle rupture. Confocal laser scanning microscopy was employed to identify the selective spatial localization of the adsorbed fluorescently labeled lipids. The corresponding increase in the AAO refractive index due to lipid adsorption on the hydrophobic regions was monitored by optical waveguide spectroscopy. This simple orthogonal functionalization route is a promising method to control the three-dimensional surface functionality of nanoporous films. © 2011 American Chemical Society.


Rother J.,Institute of Physical Chemistry | Noding H.,Institute of Physical Chemistry | Mey I.,Institute of Organic and Biomolecular Chemistry | Janshoff A.,Institute of Physical Chemistry
Open Biology | Year: 2014

Mechanical phenotyping of cells by atomic force microscopy (AFM) was proposed as a novel tool in cancer cell research as cancer cells undergo massive structural changes, comprising remodelling of the cytoskeleton and changes of their adhesive properties. In this work, we focused on the mechanical properties of human breast cell lines with different metastatic potential by AFM-based microrheology experiments. Using this technique,we are not only able to quantify the mechanical properties of living cells in the context of malignancy, but we also obtain a descriptor, namely the loss tangent, which provides model-independent information about the metastatic potential of the cell line. Including also other cell lines from different organs shows that the loss tangent (G00/G0) increases generally with the metastatic potential from MCF-10A representing benign cells to highly malignant MDA-MB-231 cells. © 2014 The Authors.


Lazzara T.D.,Institute of Organic and Biomolecular Chemistry | Carnarius C.,Institute of Organic and Biomolecular Chemistry | Kocun M.,Institute of Physical Chemistry | Janshoff A.,Institute of Physical Chemistry | Steinem C.,Institute of Organic and Biomolecular Chemistry
ACS Nano | Year: 2011

Anodic aluminum oxide (AAO) is a porous material having aligned cylindrical compartments with 55-60 nm diameter pores, and being several micrometers deep. A protocol was developed to generate pore-spanning fluid lipid bilayers separating the attoliter-sized compartments of the nanoporous material from the bulk solution, while preserving the optical transparency of the AAO. The AAO was selectively functionalized by silane chemistry to spread giant unilamellar vesicles (GUVs) resulting in large continuous membrane patches covering the pores. Formation of fluid single lipid bilayers through GUV rupture could be readily observed by fluorescence microscopy and further supported by conservation of membrane surface area, before and after GUV rupture. Fluorescence recovery after photobleaching gave low immobile fractions (5-15%) and lipid diffusion coefficients similar to those found for bilayers on silica. The entrapment of molecules within the porous underlying cylindrical compartments, as well as the exclusion of macromolecules from the nanopores, demonstrate the barrier function of the pore-spanning membranes and could be investigated in three-dimensions using confocal laser scanning fluorescence imaging. © 2011 American Chemical Society.


Hofer I.,Institute of Organic and Biomolecular Chemistry | Steinem C.,Institute of Organic and Biomolecular Chemistry
Soft Matter | Year: 2011

Pore-spanning planar membranes on highly ordered porous silicon substrates were shown to be well suited to monitor the calcium ion mediated fusion of large unilamellar vesicles by means of confocal laser scanning fluorescence microscopy and scanning ion conductance microscopy in real-time. © 2011 The Royal Society of Chemistry.


Lazzara T.D.,Institute of Organic and Biomolecular Chemistry | Mey I.,Institute of Organic and Biomolecular Chemistry | Steinem C.,Institute of Organic and Biomolecular Chemistry | Janshoff A.,Institute of Physical Chemistry
Analytical Chemistry | Year: 2011

Porous substrates have gained widespread interest for biosensor applications based on molecular recognition. Thus, there is a great demand to systematically investigate the parameters that limit the transport of molecules toward and within the porous matrix as a function of pore geometry. Finite element simulations (FES) and time-resolved optical waveguide spectroscopy (OWS) experiments were used to systematically study the transport of molecules and their binding on the inner surface of a porous material. OWS allowed us to measure the kinetics of protein adsorption within porous anodic aluminum oxide membranes composed of parallel-aligned, cylindrical pores with pore radii of 10-40 nm and pore depths of 0.8-9.6 μm. FES showed that protein adsorption on the inner surface of a porous matrix is almost exclusively governed by the flux into the pores. The pore-interior surface nearly acts as a perfect sink for the macromolecules. Neither diffusion within the pores nor adsorption on the surface are rate limiting steps, except for very low rate constants of adsorption. While adsorption on the pore walls is mainly governed by the stationary flux into the pores, desorption from the inner pore walls involves the rate constants of desorption and adsorption, essentially representing the protein-surface interaction potential. FES captured the essential features of the OWS experiments such as the initial linear slopes of the adsorption kinetics, which are inversely proportional to the pore depth and linearly proportional to protein concentration. We show that protein adsorption kinetics allows for an accurate determination of protein concentration, while desorption kinetics could be used to capture the interaction potential of the macromolecules with the pore walls. © 2011 American Chemical Society.


Lazzara T.D.,Institute of Organic and Biomolecular Chemistry | Behn D.,Institute of Organic and Biomolecular Chemistry | Kliesch T.-T.,Institute of Organic and Biomolecular Chemistry | Janshoff A.,Institute of Physical Chemistry | Steinem C.,Institute of Organic and Biomolecular Chemistry
Journal of Colloid and Interface Science | Year: 2012

Anodic aluminum oxide (AAO) substrates with aligned, cylindrical, non-intersecting pores with diameters of 75. nm and depths of 3.5 or 10 μm were functionalized with lipid monolayers harboring different receptor lipids. AAO was first functionalized with dodecyl-trichlorosilane, followed by fusion of small unilamellar vesicles (SUVs) forming a lipid monolayer. The SUVs' lipid composition was transferred onto the AAO surface, allowing us to control the surface receptor density. Owing to the optical transparency of the AAO, the overall vesicle spreading process and subsequent protein binding to the receptor-doped lipid monolayers could be investigated in situ by optical waveguide spectroscopy (OWS). SUV spreading occurred at the pore-rim interface, followed by lateral diffusion of lipids within the pore-interior surface until homogeneous coverage was achieved with a lipid monolayer. The functionality of the system was demonstrated through streptavidin binding onto a biotin-DOPE containing POPC membrane, showing maximum protein coverage at 10. mol% of biotin-DOPE. The system enabled us to monitor in real-time the selective extraction of two histidine-tagged proteins, PIGEA14 (14. kDa) and ezrin (70. kDa), directly from cell lysate solutions using a DOGS-NTA(Ni)/DOPC (1:9) membrane. The purification process including protein binding and elution was monitored by OWS and confirmed by SDS-PAGE. © 2011 Elsevier Inc.


PubMed | Institute of Organic and Biomolecular Chemistry
Type: | Journal: Beilstein journal of nanotechnology | Year: 2012

Layer-by-layer (LbL) deposition of polyelectrolytes and proteins within the cylindrical nanopores of anodic aluminum oxide (AAO) membranes was studied by optical waveguide spectroscopy (OWS). AAO has aligned cylindrical, nonintersecting pores with a defined pore diameter d(0) and functions as a planar optical waveguide so as to monitor, in situ, the LbL process by OWS. The LbL deposition of globular proteins, i.e., avidin and biotinylated bovine serum albumin was compared with that of linear polyelectrolytes (linear-PEs), both species being of similar molecular weight. LbL deposition within the cylindrical AAO geometry for different pore diameters (d(0) = 25-80 nm) for the various macromolecular species, showed that the multilayer film growth was inhibited at different maximum numbers of LbL steps (n(max)). The value of n(max) was greatest for linear-PEs, while proteins had a lower value. The cylindrical pore geometry imposes a physical limit to LbL growth such that n(max) is strongly dependent on the overall internal structure of the LbL film. For all macromolecular species, deposition was inhibited in native AAO, having pores of d(0) = 25-30 nm. Both, OWS and scanning electron microscopy showed that LbL growth in larger AAO pores (d(0) > 25-30 nm) became inhibited when approaching a pore diameter of d(eff,n_max) = 25-35 nm, a similar size to that of native AAO pores, with d(0) = 25-30 nm. For a reasonable estimation of d(eff,n_max), the actual volume occupied by a macromolecular assembly must be taken into consideration. The results clearly show that electrostatic LbL allowed for compact macromolecular layers, whereas proteins formed loosely packed multilayers.


PubMed | Institute of Organic and Biomolecular Chemistry
Type: Journal Article | Journal: Biochimica et biophysica acta | Year: 2011

The mechanism of how full length Tat (aa 1-86) crosses artificial lipid membranes was elucidated by means of fluorescence spectroscopy and fluorescence microscopy. It was shown that full length Tat (aa 1-86) neither forms pores in large unilamellar vesicles (LUVs) nor in giant unilamellar vesicles (GUVs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). In contrast, an N-terminally truncated Tat protein (aa 35-86) that lacks the structurally defined proline- and cysteine-rich region as well as the highly conserved tryptophan residue at position 11 generates pores in artificial POPC-membranes, through which a water-soluble dye up to a size of 10kDa can pass. By means of fluorescence microscopy, the transfer of fluorescently labeled full length Tat across POPC-bilayers was unambiguously visualized with a concomitant accumulation of the protein in the membrane interface. However, if the dye was attached to the protein, also pore formation was induced. The size of the pores was, however smaller than the protein size, i.e. the labeled protein with a mass of 11.6kDa passed the membrane, while a fluorescent dye with a mass of 10kDa was excluded from the vesicles interior. The results demonstrate that pore formation is not the prime mechanism by which full length Tat crosses a membrane.

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