Towards Probing Structural Transition of Single-Polymer Chains with External Force

Sebastian Belau, Ralf Seidel

Peter Debye Institute for Soft Matter Physics, Leipzig University, Linnéstr. 5, 04013 Leipzig

The crystallization of polymers is typically investigated by cooling of a melt and characterizing the process or the finally formed structure with different methods (e.g. DSC, SAXS, NMR spectroscopy). Here we want to follow a different approach and investigate the crystallization at ambient conditions by using mechanical stress. Herby single polymer chains shall be stretched and the structure formation process will be induced by lowering the applied force. This way crystallization as well as structure disruption could be studied. To this end we focus on short polyethylene glycol (PEG) chains of approximately 5 kDa. The application of force to the PEG will be carried out in an optical tweezers setup as well as in a magnetic tweezer setup. For the convenient manipulation as well as for a precise length determination, we started to synthesize PEG-DNA hybrids consisting of a single polymer chain with dsDNA attached to both ends. Herby the reaction of thiol with maleimide and click chemistry with an azide-alkyne reaction is exploited. The successful coupling of one DNA oligomer to the polymer could be shown. Future experiments for binding DNA to two coupling sites of the polymer are planned.

Applying Principal-Components Analysis to Single DNA Molecules in a Thermophoretic Trap

Tobias Thalheim, Marco Braun, and Frank Cichos

Peter Debye Institute for Soft Matter Physics, Leipzig University, Linnéstr. 5, 04013 Leipzig

We report on single DNA molecules in liquids trapped over several minutes applying a feedback-driven dynamic temperature field. The thermophoretically induced drift velocities, which make the trapping of single nano-objects possible, are generated by spatially and temporally varying the temperature at a plasmonic nano-structure. The randomization of the positions and conformations by Brownian motion is prevented with the help of feedback-controlled switching of local temperature fields. A model-free statistical tool called principal-components analysis as introduced by Cohen & Moerner [1] is employed to assess the distortion of the DNA’s conformation and conformation dynamics.

[1] A. E. Cohen, and W. E. Moerner, PNAS 104 (31), 12622-12627 (2007). (link)

Serine substitution in Amyloid-β – a possible link between β-Methylamino-L-alanine and Alzheimer’s disease?

A. Korn1, M. Krüger2, S. Roßner3, D. Huster1

1Institute of Medical Physics and Biophysics, University of Leipzig, D-04107 Leipzig, Germany.
2Institute of Anatomy, University of Leipzig, D-04103 Leipzig, Germany
3Paul-Flechsig-Institut für Hirnforschung, Liebigstraße 19 D-04103 Leipzig, Germany

β-Methylamino-L-alanine (BMAA) was found as a possible reason for increased ALS-PDC (amyotrophic lateral sclerosis–parkinsonism/dementia complex) [1]. It is a non- proteinogenic amino acid produced by cyanobacteria that can be enriched via the food chain in plants, seafood, higher animals, and humans [2]. This is a critical factor because cyanobacteria are known for their excessive blooms not only in marine ecosystems but also in lakes that are used as fresh water source for millions of people supplying BMAA to human nutrition [3].
Although BMAA is known as a neurotoxin for several decades, its mode of action is still topic of controversial discussions. One of the more commonly accepted pathologic pathways is its function as a neurotransmitter mimetic where it can overstimulate glutamate receptors, deplete glutathione, increase free radical concentration and subsequently leads to neuronal damage [4]. Besides this, BMAA can also be misincorporated in proteins. Recent findings showed that serine tRNA synthetase accepts BMAA as substrate which may finally lead to a serine-BMAA substitution [5].
Assuming that BMAA can substitute Ser8 or Ser26 of Amyloid-β, the question arises if this may alter Amyloid-β fibrillation and structure leading to a higher risk for neurodegenerative pathogenesis.

[1] J. Pablo, S.A. Banack, PA Cox, T.E. Johnson, S. Papapetropoulos, W.G. Bradley, A. Buck, D.C. Mash, Acta Neurologica Scandinavica, 120, 216 (2009) (link)
[2] C.L. Garcia-Rodenas, M. Affolter, G. Vinyes-Pares, C.A. De Castro, L.G, Karagounis, Y.M. Zhang, P.Y Wang, S.K Thakkar, Nutrients, 8, 606, (2016) (link)
[3] M. Monteiro, M. Costa, C. Moreira, V.M. Vasconcelos, M.S. Baptista, Journal of Applied Phycology, 29, 879 (2017) (link)
[4] F. D’Mello, N. Braidy, H. Marcal, G. Guillemin, F. Rossi, M. Chinian, D. Laurent, C. Teo, B.A. Neilan, Neurotoxicity Research, 31, 245 (2017) (link)

Early oligomers and the process of oligomerization of the amyloid peptides Aβ40 and Aβ42

Jana Rüdel, Maria Ott

Institute of Physics, Martin-Luther-University Halle-Wittenberg

Similar to synthetic polymers like polyamides, amyloidogenic proteins as well as short peptide sequences display the inherent ability to form long and very stable fibers called fibrils. Along the pathway from a single peptide to the mature fibrils, various transient and long-lived intermediate states are formed spanning the whole range between small and mostly unstructured oligomers to well-ordered, β-sheet rich protofibrils. As early oligomeric states were found to be neurotoxic, they are a presumptive key to understand the development of neurodegenerative diseases [1].
In order to reveal the leading mechanisms of amyloid aggregation, we studied the appearance and development of early oligomeric states of the Aβ40- and the Aβ42-peptides using a combined approach of single-molecule fluorescence spectroscopy and imaging techniques, such as TEM and AFM. Additionally, thermodynamic stabilities of the detected amyloid aggregates were studied by the use of ultrafast-scanning calorimetry.
We could reveal and characterize soluble oligomers of the Aβ40- and the Aβ42-peptide and found distinct differences in terms of size distribution as well as the process of oligomerization. While the fibrillation of Aβ42-peptides includes small and large oligomers, the assembly of Aβ40-peptide display only small oligomers and an overall slower kinetic of fibril formation. We will discuss our results by the use of thermodynamic models of self-assembly.

[1] F. Bemporad and F. Chiti, Protein misfolded oligomers: Experimental approaches, mechanism of formation, and structure-toxicity relationships, Chem. Biol. 19 (2012), 315 (link)

Effects of Hofmeister Ion Series on Stability of a Salt Bridge

S. Pylaeva, H. Elgabarty, and D. Sebastiani

Theoretical Chemistry, Institute of Chemistry, Martin Luther University Halle-Wittenberg, Von-Danckelmann-Platz 4, 06120, Halle, Germany

Salt bridges are important components of protein structure stability. They can be defined as an interaction of two aminoacid side chains of opposite charge [1]. Such Coulomb attraction interaction is sensitive to presence of other charged species in the vicinity. Concentration of charged species – free ions can be significant in a crowded environment of a living cell. Additionally Hofmeister ion series have been shown to have a significant impact on structure and dynamics of water and solvated proteins [2, 3].

We have investigated effects of Hofmeister ion series on an arginine – aspartic acid salt bridge by means of computer simulations [4]. Changes in thermodynamic properties of a salt bridge and dynamic properties of their solvation shells will be discussed in a poster.

[1] J.E. Donald, D.W. Kulp, W.F. DeGrado, Proteins, 79(3), 898 (2011). (link)
[2] C. Allolio, N. Salas-Illanes, Y.S. Desmukh, M.R. Hansen, D. Sebastiani, JPCB 117(34), 9939 (2013) (link)
[3] M.D. Smith, L. Cruz, JPCB 117, 6614 (2013) (link)
[4] M. Fyta, R. Netz, JCP 136, 124103 (2012). (link)

Amyloid peptide aggregation near interfaces

T. John1,2,3, L.L. Martin3, H.J. Risselada1,4, and B. Abel1,2

1Leibniz Institute of Surface Modification, Leipzig (Germany)
2Wilhelm-Ostwald-Institute for Physical and Theoretical Chemistry, Leipzig University, Leipzig (Germany)
3School of Chemistry, Monash University, Clayton (Australia)
4Department of Theoretical Physics, Georg-August-University Göttingen, Göttingen (Germany)

Amyloid peptides aggregate into characteristic fibrils with cross-β-sheet structure, also known as amyloid plaque. They are associated with several diseases such as Alzheimer’s disease or type II diabetes. However, there is evidence that indicates the soluble transient oligomers, instead of mature fibrils, as the toxic species. Amyloid-forming peptides are natively soluble and only aggregate under certain circumstances. Comprehensive knowledge on the aggregation mechanism and a detailed characterisation of the transient species is essential to understand the physiological role of these peptides [1].

Interfaces, such as nanoparticles, can act to accelerate or inhibit peptide aggregation. Experimental studies and molecular dynamics simulations (MD) presented an accelerated fibril formation in the presence of citrate-stabilised gold nanoparticles [2,3]. The role of gold surfaces in oligomer formation and peptide aggregation is discussed in this study. Moreover, possible mechanisms for the observed acceleration of the peptide aggregation by a reduction of the conformational space that is sampled are presented.

[1] F. Chiti, and C.M. Dobson, Annu. Rev. Biochem., 75, 333 (2006). (link)
[2] A. Gladytz, B. Abel, and H.J. Risselada, Angew. Chem., Int. Ed., 55, 11242 
(2016). (link)
[3] A. Gladytz, M. Wagner, T. Häupl, C. Elsner, and B. Abel, Part. Part. Syst. 
Charact., 32, 573 (2015). (link)

Dynamic and Structural Properties of Polyglutamine

P. Enke, M. Schleeger, and T. Kiefhaber

Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 3, 06120 Halle

More than ten diseases are known that are based on the expansion of polyglutamine sequences within the disease-related proteins, which leads to the formation of amyloids. We tested, whether the propensity to form amyloids is due to distinct structural and/or dynamic properties of the monomeric state of polyglutamine (polyQ) chains. Using triplet-triplet energy transfer (TTET) and time-resolved fluorescence resonance energy transfer (trFRET) we were able to characterize the structure (end-to-end distribution) and the intrachain dynamics of monomeric polyQ chains of different length. The results were compared to the properties of the previously characterized model chains poly-(glycine-serine) and poly-serine [1-4] and of fragments from IDPs, which do not form amyloids. The results show that intrachain dynamics in polyQ chains are slower than the dynamics of all other investigated chains and that longer polyQ chains contain a fraction of pre-formed loop structures in the conformational ensemble, which may explain their tendency to form oligomers of β-hairpins.

[1] O. Bieri, T. Kiefhaber, Biol. Chem. 380, 923 (1999). (link)
[2] F. Krieger, B. Fierz, O. Bieri, M. Drewello, T. Kiefhaber, J. Mol. Biol. 332, 265 (2003). (link)
[3] A. Möglich, K. Joder, T. Kiefhaber, PNAS 103, 12394 (2006). (link)
[4] B. Fierz, H. Satzger, C. Root, P. Glich, W. Zinth, T. Kiefhaber, PNAS 104, 2163 (2007). (link)

Effects of thermal denaturation and UV-B irradiation on eye lens crystallin proteins

M. Camilles, S. Link, A. Krushelnitsky, J. Balbach, K. Saalwächter

Institute of Physics, Martin Luther University Halle-Wittenberg, Germany

Crystallins are the major vision-related (i.e. refractive) proteins found in the eye lens. The mammalian lens consist of three classes of proteins, α-, β- and γ-crystallins, the former also acts as chaperone [1]. Commonly, proteins are subject to a continuous degradation and replacement process, but the eye lens proteins have no turnover and hence have to remain stable and soluble for a lifetime. So far, most studies have focused on single eye lens proteins and their interactions at low concentrations [2]. Here we combine NMR and other biophysical techniques to monitor stress induced aggregation and changes of the interactions of crystallins at various concentrations [3]. This allows us to investigate molecular effects which might lead to cataract in a highly concentrated eye lens surrounding.

[1] H. Bloemendal et. al., Progress in Biophysics & Molecular Biology 86 407-485 (2004). (link)
[2] CN Kingsley, R.W. Martin, Structure 21, 2221 (2013). (link)
[3] M. Roos, S. Link et. al., Biophysical Journal 108, 98 (2015). (link)

Structure formation of peptides in the PRIME20 model

A. Böker and W. Paul

Martin-Luther-Universität Halle-Wittenberg, 06099 Halle

The relation between conformations of a polypeptide is governed by local minima in the free energy function. Coarse-grained models tend to simplify the free energy in such a way that these local minima are ignored. To circumvent this problem, the level of coarse graining needs to be chosen appropriately. PRIME20 [1] provides reasonable detail by mapping each amino acid to four beads, but keeps parameter space simple with the set of interactions reduced to 19 energy parameters.
Poly-Glutamines (polyQ) are associated with Huntington’s disease due to their ability to aggregate into an amyloid state. Single polyQ chains have been found to form a beta hairpin as a precursor to these aggregates. We will discuss the temperature dependent end-to-end distance of the chains in relation to TTET and FRET experiments performed on polyQ chains.
We perform thermodynamic simulations of single PRIME20 chains using the “SAMC” [2] variation of Wang-Landau Monte Carlo sampling which provides insight in different statistical ensembles at the expense of dynamic information. The aforementioned polyQ are compared to poly-Alanines with a lower tendency to form beta structure motifs.

[1] M. Cheon, I. Chang, C. K. Hall, Proteins, 78, 2950 (2010) (link)
[2] B. Werlich, T. Shakirov, M. P. Taylor, W. Paul, Comp. Phys. Comm., 186, 65 (2015) (link)

Characterization of the Self-Assembly Process of Hydrophobin SC3 at Interfaces and in Solution

M. Kordts; A. Kerth; D. Hinderberger

Institute of Chemistry; Martin Luther University Halle-Wittenberg; Von-Danckelmann-Platz 4, 06120 Halle (S.); Germany

Hydrophobins are small amphiphilic proteins (~ 7-10 kDa) produced by filamentous fungi that self-assemble at interfaces. They are divided into two classes based on hydropathy plots and solubility. By forming extremely stable amphipathic membranes, foams and emulsions hydrophobins are thought to fulfill a great variety of tasks during the fungal lifecycle such as coating of airborne spores and facilitating contact between fungus and host during infection. [1] Their ability to modify surfaces is of great interest in many fields such as medicine, material science and cosmetics. [2] To harness this ability to its full potential the exact mechanisms of the self-assembly process at interfaces and in solution must be better understood on a molecular level.
Class I hydrophobins are characterized as highly insoluble and known to form stable amyloid fibrils called rodlets at the air-water-interface. We investigated the self-assembled structures of class I hydrophobin SC3 at the air-water-interface using a Langmuir-filmbalance coupled with a fluorescence microscope. The use of two dyes of different hydropathy showed the potential to force the protein into different superstructures of several micrometers in dimension upon compression. To our knowledge, this difference in self-assembled superstructure has not been visualized before. Future investigations using Atomic Force Microscopy (AFM) are planned to investigate their underlying structures on a scale of several nanometers.
Furthermore, we attempted to covalently attach 3-Maleimido-PROXYL to the N-terminus of SC3. This stable radical can be investigated using electron paramagnetic resonance (EPR) spectroscopy in order to examine the self-assembly of the protein in solution. Again, to the best of our knowledge, no such experiments have been reported before. We are hoping to get a coherent picture of the aggregation that can then be compared to that of class II hydrophobins, which generally differ in their dominant interfacial structure.

[1] V. Aimanianda et al., Nature 460, 1117-1121 (2009) (link)
[2] H. J. Hektor and K. Scholtmeijer, Curr. Opin. Biotechnol. 16, 434-439 (2005) (link)