TY - JOUR A1 - Knox-Brown, Patrick A1 - Rindfleisch, Tobias A1 - Günther, Anne A1 - Balow, Kim A1 - Bremer, Anne A1 - Walther, Dirk A1 - Miettinen, Markus S. A1 - Hincha, Dirk K. A1 - Thalhammer, Anja T1 - Similar Yet Different BT - Structural and Functional Diversity among Arabidopsis thaliana LEA_4 Proteins JF - International Journal of Molecular Sciences N2 - The importance of intrinsically disordered late embryogenesis abundant (LEA) proteins in the tolerance to abiotic stresses involving cellular dehydration is undisputed. While structural transitions of LEA proteins in response to changes in water availability are commonly observed and several molecular functions have been suggested, a systematic, comprehensive and comparative study of possible underlying sequence-structure-function relationships is still lacking. We performed molecular dynamics (MD) simulations as well as spectroscopic and light scattering experiments to characterize six members of two distinct, lowly homologous clades of LEA_4 family proteins from Arabidopsis thaliana. We compared structural and functional characteristics to elucidate to what degree structure and function are encoded in LEA protein sequences and complemented these findings with physicochemical properties identified in a systematic bioinformatics study of the entire Arabidopsis thaliana LEA_4 family. Our results demonstrate that although the six experimentally characterized LEA_4 proteins have similar structural and functional characteristics, differences concerning their folding propensity and membrane stabilization capacity during a freeze/thaw cycle are obvious. These differences cannot be easily attributed to sequence conservation, simple physicochemical characteristics or the abundance of sequence motifs. Moreover, the folding propensity does not appear to be correlated with membrane stabilization capacity. Therefore, the refinement of LEA_4 structural and functional properties is likely encoded in specific patterns of their physicochemical characteristics. KW - IDP KW - LEA protein KW - abiotic stress KW - dehydration KW - conformational rearrangement KW - membrane stabilization KW - sequence-structure-function relationship Y1 - 2020 U6 - https://doi.org/10.3390/ijms21082794 SN - 1422-0067 VL - 21 IS - 8 PB - Molecular Diversity Preservation International CY - Basel ER - TY - GEN A1 - Knox-Brown, Patrick A1 - Rindfleisch, Tobias A1 - Günther, Anne A1 - Balow, Kim A1 - Bremer, Anne A1 - Walther, Dirk A1 - Miettinen, Markus S. A1 - Hincha, Dirk K. A1 - Thalhammer, Anja T1 - Similar Yet Different BT - Structural and Functional Diversity among Arabidopsis thaliana LEA_4 Proteins T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The importance of intrinsically disordered late embryogenesis abundant (LEA) proteins in the tolerance to abiotic stresses involving cellular dehydration is undisputed. While structural transitions of LEA proteins in response to changes in water availability are commonly observed and several molecular functions have been suggested, a systematic, comprehensive and comparative study of possible underlying sequence-structure-function relationships is still lacking. We performed molecular dynamics (MD) simulations as well as spectroscopic and light scattering experiments to characterize six members of two distinct, lowly homologous clades of LEA_4 family proteins from Arabidopsis thaliana. We compared structural and functional characteristics to elucidate to what degree structure and function are encoded in LEA protein sequences and complemented these findings with physicochemical properties identified in a systematic bioinformatics study of the entire Arabidopsis thaliana LEA_4 family. Our results demonstrate that although the six experimentally characterized LEA_4 proteins have similar structural and functional characteristics, differences concerning their folding propensity and membrane stabilization capacity during a freeze/thaw cycle are obvious. These differences cannot be easily attributed to sequence conservation, simple physicochemical characteristics or the abundance of sequence motifs. Moreover, the folding propensity does not appear to be correlated with membrane stabilization capacity. Therefore, the refinement of LEA_4 structural and functional properties is likely encoded in specific patterns of their physicochemical characteristics. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 901 KW - IDP KW - LEA protein KW - abiotic stress KW - dehydration KW - conformational rearrangement KW - membrane stabilization KW - sequence-structure-function relationship Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-469419 SN - 1866-8372 IS - 901 ER - TY - GEN A1 - Roethlein, Christoph A1 - Miettinen, Markus S. A1 - Ignatova, Zoya T1 - A flexible approach to assess fluorescence decay functions in complex energy transfer systems T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe 819 N2 - Background: Time-correlated Forster resonance energy transfer (FRET) probes molecular distances with greater accuracy than intensity-based calculation of FRET efficiency and provides a powerful tool to study biomolecular structure and dynamics. Moreover, time-correlated photon count measurements bear additional information on the variety of donor surroundings allowing more detailed differentiation between distinct structural geometries which are typically inaccessible to general fitting solutions. Results: Here we develop a new approach based on Monte Carlo simulations of time-correlated FRET events to estimate the time-correlated single photon counts (TCSPC) histograms in complex systems. This simulation solution assesses the full statistics of time-correlated photon counts and distance distributions of fluorescently labeled biomolecules. The simulations are consistent with the theoretical predictions of the dye behavior in FRET systems with defined dye distances and measurements of randomly distributed dye solutions. We validate the simulation results using a highly heterogeneous aggregation system and explore the conditions to use this tool in complex systems. Conclusion: This approach is powerful in distinguishing distance distributions in a wide variety of experimental setups, thus providing a versatile tool to accurately distinguish between different structural assemblies in highly complex systems. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 819 KW - time resolved FRET KW - Monte-Carlo simulations KW - complex heterogeneous systems KW - protein aggregation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-427557 SN - 1866-8372 IS - 819 ER - TY - JOUR A1 - Wong-Ekkabut, Jirasak A1 - Miettinen, Markus S. A1 - Dias, Christiano A1 - Karttunen, Mikko T1 - Static charges cannot drive a continuous flow of water molecules through a carbon nanotube Y1 - 2010 UR - http://www.nature.com/nnano U6 - https://doi.org/10.1038/nnano.2010.152 SN - 1748-3387 ER - TY - JOUR A1 - Roethlein, Christoph A1 - Miettinen, Markus S. A1 - Borwankar, Tejas A1 - Buerger, Joerg A1 - Mielke, Thorsten A1 - Kumke, Michael Uwe A1 - Ignatova, Zoya T1 - Architecture of polyglutamine-containing fibrils from time-resolved fluorescence decay JF - The journal of biological chemistry N2 - The disease risk and age of onset of Huntington disease (HD) and nine other repeat disorders strongly depend on the expansion of CAG repeats encoding consecutive polyglutamines (polyQ) in the corresponding disease protein. PolyQ length-dependent misfolding and aggregation are the hallmarks of CAG pathologies. Despite intense effort, the overall structure of these aggregates remains poorly understood. Here, we used sensitive time-dependent fluorescent decay measurements to assess the architecture of mature fibrils of huntingtin (Htt) exon 1 implicated in HD pathology. Varying the position of the fluorescent labels in the Htt monomer with expanded 51Q (Htt51Q) and using structural models of putative fibril structures, we generated distance distributions between donors and acceptors covering all possible distances between the monomers or monomer dimensions within the polyQ amyloid fibril. Using Monte Carlo simulations, we systematically scanned all possible monomer conformations that fit the experimentally measured decay times. Monomers with four-stranded 51Q stretches organized into five-layered beta-sheets with alternating N termini of the monomers perpendicular to the fibril axis gave the best fit to our data. Alternatively, the core structure of the polyQ fibrils might also be a zipper layer with antiparallel four-stranded stretches as this structure showed the next best fit. All other remaining arrangements are clearly excluded by the data. Furthermore, the assessed dimensions of the polyQ stretch of each monomer provide structural evidence for the observed polyQ length threshold in HD pathology. Our approach can be used to validate the effect of pharmacological substances that inhibit or alter amyloid growth and structure. Y1 - 2014 U6 - https://doi.org/10.1074/jbc.M114.581991 SN - 0021-9258 SN - 1083-351X VL - 289 IS - 39 SP - 26817 EP - 26828 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - JOUR A1 - Röthlein, Christoph A1 - Miettinen, Markus S. A1 - Ignatova, Zoya T1 - A flexible approach to assess fluorescence decay functions in complex energy transfer systems JF - BMC biophysics N2 - Background: Time-correlated Forster resonance energy transfer (FRET) probes molecular distances with greater accuracy than intensity-based calculation of FRET efficiency and provides a powerful tool to study biomolecular structure and dynamics. Moreover, time-correlated photon count measurements bear additional information on the variety of donor surroundings allowing more detailed differentiation between distinct structural geometries which are typically inaccessible to general fitting solutions. Results: Here we develop a new approach based on Monte Carlo simulations of time-correlated FRET events to estimate the time-correlated single photon counts (TCSPC) histograms in complex systems. This simulation solution assesses the full statistics of time-correlated photon counts and distance distributions of fluorescently labeled biomolecules. The simulations are consistent with the theoretical predictions of the dye behavior in FRET systems with defined dye distances and measurements of randomly distributed dye solutions. We validate the simulation results using a highly heterogeneous aggregation system and explore the conditions to use this tool in complex systems. Conclusion: This approach is powerful in distinguishing distance distributions in a wide variety of experimental setups, thus providing a versatile tool to accurately distinguish between different structural assemblies in highly complex systems. KW - Time resolved FRET KW - Monte-Carlo simulations KW - Complex heterogeneous systems KW - Protein aggregation Y1 - 2015 U6 - https://doi.org/10.1186/s13628-015-0020-z SN - 2046-1682 VL - 8 PB - BioMed Central CY - London ER - TY - JOUR A1 - Javanainen, Matti A1 - Hammaren, Henrik A1 - Monticelli, Luca A1 - Jeon, Jae-Hyung A1 - Miettinen, Markus S. A1 - Martinez-Seara, Hector A1 - Metzler, Ralf A1 - Vattulainen, Ilpo T1 - Anomalous and normal diffusion of proteins and lipids in crowded lipid membranes JF - Faraday discussions N2 - Lateral diffusion plays a crucial role in numerous processes that take place in cell membranes, yet it is quite poorly understood in native membranes characterized by, e.g., domain formation and large concentration of proteins. In this article, we use atomistic and coarse-grained simulations to consider how packing of membranes and crowding with proteins affect the lateral dynamics of lipids and membrane proteins. We find that both packing and protein crowding have a profound effect on lateral diffusion, slowing it down. Anomalous diffusion is observed to be an inherent property in both protein-free and protein-rich membranes, and the time scales of anomalous diffusion and the exponent associated with anomalous diffusion are found to strongly depend on packing and crowding. Crowding with proteins also has a striking effect on the decay rate of dynamical correlations associated with lateral single-particle motion, as the transition from anomalous to normal diffusion is found to take place at macroscopic time scales: while in protein-poor conditions normal diffusion is typically observed in hundreds of nanoseconds, in protein-rich conditions the onset of normal diffusion is tens of microseconds, and in the most crowded systems as large as milliseconds. The computational challenge which results from these time scales is not easy to deal with, not even in coarse-grained simulations. We also briefly discuss the physical limits of protein motion. Our results suggest that protein concentration is anything but constant in the plane of cell membranes. Instead, it is strongly dependent on proteins' preference for aggregation. Y1 - 2013 U6 - https://doi.org/10.1039/c2fd20085f SN - 1359-6640 VL - 161 IS - 1 SP - 397 EP - 417 PB - Royal Society of Chemistry CY - Cambridge ER - TY - JOUR A1 - Miettinen, Markus S. A1 - Knecht, Volker A1 - Monticelli, Luca A1 - Ignatova, Zoya T1 - Assessing polyglutamine conformation in the nucleating event by molecular dynamics simulations JF - The journal of physical chemistry : B, Condensed matter, materials, surfaces, interfaces & biophysical chemistry N2 - Polyglutamine (polyQ) diseases comprise a group of dominantly inherited pathology caused by an expansion of an unstable polyQ stretch which is presumed to form beta-sheets. Similar to other amyloid pathologies, polyQ amyloidogenesis occurs via a nucleated polymerization mechanism, and proceeds through energetically unfavorable nucleus whose existence and structure are difficult to detect. Here, we use atomistic molecular dynamics simulations in explicit solvent to assess the conformation of the polyQ stretch in the nucleus that initiates polyQ fibrillization. Comparison of the kinetic stability of various structures of polyQ peptide with a Q-length in the pathological range (Q(40)) revealed that steric zipper or nanotube-like structures (beta-nanotube or beta-pseudohelix) are not kinetically stable enough to serve as a template to initiate polyQ fibrillization as opposed to beta-hairpin-based (beta-sheet and beta-sheetstack) or alpha-helical conformations. The selection of different structures of the polyQ stretch in the aggregation-initiating event may provide an alternative explanation for polyQ aggregate polymorphism. Y1 - 2012 U6 - https://doi.org/10.1021/jp305065c SN - 1520-6106 VL - 116 IS - 34 SP - 10259 EP - 10265 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Miettinen, Markus S. A1 - Monticelli, Luca A1 - Nedumpully-Govindan, Praveen A1 - Knecht, Volker A1 - Ignatova, Zoya T1 - Stable polyglutamine dimers can contain beta-hairpins with interdigitated side chains but not a-helices, alpha-nanotubes, beta-pseudohelices, or steric zippers JF - Biophysical journal N2 - A common thread connecting nine fatal neurodegenerative protein aggregation diseases is an abnormally expanded polyglutamine tract found in the respective proteins. Although the structure of this tract in the large mature aggregates is increasingly well described, its structure in the small early aggregates remains largely unknown. As experimental evidence suggests that the most toxic species along the aggregation pathway are the small early ones, developing strategies to alleviate disease pathology calls for understanding the structure of polyglutamine peptides in the early stages of aggregation. Here, we present a criterion, grounded in available experimental data, that allows for using kinetic stability of dimers to assess whether a given polyglutamine conformer can be on the aggregation path. We then demonstrate that this criterion can be assessed using present-day molecular dynamics simulations. We find that although the a-helical conformer of polyglutamine is very stable, dimers of a-helices lack the kinetic stability necessary to support further oligomerization. Dimers of steric zipper, beta-nanotube, and beta-pseudohelix conformers are also too short-lived to initiate aggregation. The beta-hairpin-containing conformers, instead, invariably form very stable dimers when their side chains are interdigitated. Combining these findings with the implications of recent solid-state NMR data on mature fibrils, we propose a possible pathway for the initial stages of polyglutamine aggregation, in which beta-hairpin-containing conformers act as templates for fibril formation. Y1 - 2014 U6 - https://doi.org/10.1016/j.bpj.2014.02.027 SN - 0006-3495 SN - 1542-0086 VL - 106 IS - 8 SP - 1721 EP - 1728 PB - Cell Press CY - Cambridge ER - TY - CHAP A1 - Miettinen, Markus S. A1 - Monticelli, Luca A1 - Nedumpully-Govindan, Praveen A1 - Knecht, Volker A1 - Ignatova, Zoya T1 - Initiating polyglutamine aggregation - computational clarification of the structural details T2 - Biophysical journal Y1 - 2015 SN - 0006-3495 SN - 1542-0086 VL - 108 IS - 2 SP - 386A EP - 386A PB - Cell Press CY - Cambridge ER -