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 et.al., 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)

Characterization of Chimaeras of a Thermoresponsive Polymer and the Parathyroid Hormone

Bruno Voigt, Zhanna Evgrafova, Monika Baumann, Madlen Stephani, Marvin Umlandt, Wolfgang H. Binder, Jochen Balbach

Faculty of Natural Sciences II, Martin-Luther-Universität Halle-Wittenberg, D-06120 Halle, Germany

The parathyroid hormone (PTH) is an 84 residue peptide from the parathyroid glands which controls the calcium and phosphate level in human blood. The peptide adopts an α-helical conformation at the N-terminus and is intrinsically disordered at the C-terminus. Amyloidogenic properties of PTH have been reported [1]. Here we show the in vitro formation of amyloid fibrils of PTH 1-84 and of the pharmaceutically relevant N-terminal fragment PTH 1-34 under physiological conditions. To get further insights into the mechanism of amyloid fibrillation we investigated the effect of thermoresponsive polymers [2] on PTH. We covalently attached polyacrylate based polymers to 15N isotope labelled PTH 1-84 and used two dimensional NMR techniques for the characterization of the resulting chimaeras. This allows the observation of amino acid sequence specific changes of the cross peaks corresponding to the peptide backbone according to the polymer state. The studies revealed strong dependencies of chemical shifts on the temperature, the peptide attachment site and the polymer molecular weight.

[1] Gopalswamy, M.; Kumar, A.; Adler, J.; Baumann, M.; Henze, M.; Kumar, S.T.; Fändrich, M.; Scheidt, H.A.; Huster, D.; Balbach, J., Biochim Biophys Acta 2015, 1854, 249-257. (link)
[2] Funtan, S.; Evgrafova, Z.; Adler, J.; Huster, D.; Binder, W.H., Polymers 2016, 8, 178. (link)

Amyloid Protein Aggregation in the Presence of Temperature-Sensitive Polymers

Zhanna Evgrafova1, Sonu Kumar1, Bruno Voigt1,  Juliane Adler2, Daniel Huster2, Jochen Balbach1 and Wolfgang H. Binder1

1Faculty of Natural Science II, Martin Luther University Halle-Wittenberg, Von-Danckelmann-Platz 4, D-06120 Halle (Saale), Germany
2Institute for Medical Physics and Biophysics, Leipzig University, Härtelstraße 16-18, D-04107 Leipzig, Germany

The formation of amyloid fibrils is considered to be one of the main causes for many neurodegenerative diseases, such as Alzheimer’s, Parkinson’s or Huntington’s disease [1, 2]. Current knowledge suggests that amyloid-aggregation represents a nucleation-dependent aggregation process in vitro, where a sigmoidal growth phase follows an induction period. Here, we studied the fibrillation of amyloid β 1-40 (Aβ40) and Parathyroid hormone (PTH) in the presence of thermoresponsive polymers, expected to alter their fibrillation kinetics due to their specific hydrophobic and hydrophilic interactions with proteins [3]. Mixtures in varying concentrations and the conjugates of PTH or Aβ40 with poly(ethylene glycol) methyl ether acrylate were studied via time-dependent measurements of the thioflavin T (ThT) fluorescence and transition electron microscopy (TEM). The studies revealed that amyloid fibrillation was altered, accompanied by either reduction or elongation of the lag phase of PTH or Aβ40 fibrillation in the presence of studied polymers [4].

[1] Chiti, F.; Dobson, C.M., Annu. Rev. Biochem. 2006, 75, 333–366. (link)
[2] Hamley, I.W., Angew. Chem. Int. Ed. 2007, 46, 8128–8147. (link)
[3] Adler, J.; Huster, D., Phys. Chem. Chem. Phys. 2017, 19, 1839–1846. (link)
[4] Funtan, S.; Evgrafova, Z.; Adler, J.; Huster, D.; Binder, W.H., Polymers 2016, 8, 178. (link)

Nucleation processes in protein aggregation

Tuomas P. J. Knowles

University of Cambridge, Department of Chemistry, UK

Filamentous protein aggregation underlies a number of functional and pathological processes in nature. This talk focuses on the formation of amyloid fibrils, a class of beta-sheet rich protein filament. Such structures were initially discovered in the context of disease states where their uncontrolled formation impedes normal cellular function, but are now known to also possess numerous beneficial roles in organisms ranging from bacteria to humans. The formation of these structures commonly occurs through supra-molecular polymerisation following an initial primary nucleation step. In recent years it has become apparent that in addition to primary nucleation, secondary nucleation events which are catalysed in the presence of existing aggregates can play a significant role in the dynamics of such systems. This talk describes our efforts to understand the nature of the nucleation processes in protein aggregation as well as the dynamics of such systems and how these features connect to the biological roles that these structures can have in both health and disease.

Thermal Behavior of Silk Protein

P. Cebe1, D. Thomas1, J. Merfeld1, B. P. Parlow2, D. L. Kaplan2, R. Alamo3, A. Wurm4, E. Zhuravlev4, and C. Schick4

1Physics and Astronomy Dept., Tufts University, Medford, MA 02155 USA
2Biomedical Engineering Dept., Tufts University, Medford, MA 02155 USA
3Chemical and Biomedical Engineering Dept., Florida State University-FAMU, Tallahassee, FL 32310 USA
4 Institute for Physics and Competence Center Calorimetry of Interdisciplinary Faculty, University of Rostock, Rostock, Germany

Silk is a naturally occurring biopolymer which has been used in textiles for over 5000 years. Silk stands as an exemplar of the class of fibrous proteins. The properties of silk protein are related to its semicrystalline nature, imparted by the secondary structures, such as the non-crystalline helices and random coils, and the crystalline beta pleated sheets. Using techniques of condensed matter polymer physics, we investigate the structure and thermal behavior of silk fibroin [1-3]. Continue reading Thermal Behavior of Silk Protein

Amyloids from the origin to the end of life

Roland Riek1, Marielle Wälti1, Cedric Eichmann, and Jason Greenwald1

1Vladimir Prelog Weg 2, ETH, ETH Hönggerberg, CH-8093 Zurich

Protein aggregation is observed in many diseases including Alzheimer’s disease. These protein aggregates are termed amyloids. Amyloids are composed of pairs of tightly interacting, many-stranded, repetitive, inter-molecular beta-sheets termed the cross-beta-sheet structure. Because of this structure, amyloids can grow by recruitment of the same protein while their repeat can transform a weak activity into a potent one through cooperativity and avidity. Continue reading Amyloids from the origin to the end of life