Session S50: Physics of proteins IV: Intrinsically Disordered and Aggregated States of Proteins

The Importance of Disorder in the Highly Ordered Circadian Clock

The circadian clock is a tightly-regulated molecular circuit that allows an organism to anticipate the time of day in order to better regulate its physiology and behavior. It is believed that the clock enhances fitness by ensuring that many organismal functions are timed to coincide with the proper phase of the day. As clock regulation is extensive, disruptions of the clock can have a negative impact on human health, for example increased risks for ischemic, metabolic, oncological and mental health diseases. The mechanism that underlies circadian timing is a highly-conserved molecular transcription-translation negative feedback loop. This molecular clock consists of two sets of protein complexes, the positive arm and the negative arm. The positive arm drives the expression of the negative arm, which then regulates its own transcription by inhibiting the activity of the positive arm. The negative arm is then targeted for degradation, allowing for the reactivation of the positive arm, restarting the cycle. Our work has demonstrated that intrinsic disorder is essential for the timing of this circadian circuit. We have established in Neurospora crassa, a fungi and clock model organism, that proteins in the positive arm as well as in the negative arm are Intrinsically Disordered Proteins (IDPs) and that their IDP nature is important to clock function. Moreover, this IDP feature of clock proteins is conserved in higher eukaryotes. Our lab is using biochemical and biophysical methods to identify the underlying purpose for this high level of conservation of protein disorder in the circadian clock. We hypothesize that the conservation of protein disorder in clock proteins allows for conformational flexibility and that this flexibility is key in regulating the timing and interactions of clock proteins over the circadian day.   

Multi-State Coarse Grained Modeling for Intrinsically Disordered Peptides

Many proteins display a marginally stable tertiary structure, which can be altered via external stimuli. Since majority of coarse grained (CG) models are aimed at structure prediction, their success for an intrinsically disordered peptide's conformational space with marginal stability and sensitivity to external stimuli cannot be taken for granted. In this study, by using the LKα14 peptide as a test system, we demonstrate a bottom-up approach for constructing a multi-state CG model, which can capture the conformational behavior of this peptide in three distinct environments with a unique set of interaction parameters. LKα14 is disordered in dilute solutions, however it strictly adopts the α-helix conformation upon aggregation or at a hydrophobic/hydrophilic interface. Our bottom-up approach combines a generic base model, unbiased for any particular secondary structure, with nonbonded interactions which represent hydrogen bonds, electrostatics and hydrophobic forces. We demonstrate that by using carefully designed all atom potential of mean force calculations from all three states, one can obtain a CG model, which behaves intrinsically disordered in bulk water, folds into an α-helix at an interface or a neighboring peptide, and is stable as a tetramer.   

Self-assembly and soluble aggregate behavior of computationally designed coiled-coil peptide bundles

Peptides are excellent candidates for nanomaterials design and controlled assembly due to their atomistic precision in designed structure formation as in biological systems. Unlike natural proteins and natural structural motifs, this effort is completely de novo in order to build arbitrary structures with desired size, shape and display of functional groups. We have successfully prepared soluble, coiled-coil, peptide tetramer bundles that are robust and stable. Using circular dichroism we demonstrated bundle thermal stability and confirmed their alpha helical and coiled-coil nature. The coiled-coil tetramer was confirmed by analytical ultra-centrifugation sedimentation studies. We also established how these bundles behave in solution using small angle neutron scattering. The form factor of the bundles is well represented by a cylinder model with dimensions consistent with the size of the designed bundles. At high concentrations, the behavior of the bundles is modeled using a structure factor for soluble aggregates with an average inter bundle separation. These experiments support our claim that the designed coiled-coil bundles were achieved in solution and interact in a way similar to natural proteins in concentrated solution.   

Study of Amyloidogenic Peptide Aggregation via Dielectric Relalxation Spectroscopy

Recent studies suggest that Alzheimer's disease (AD) and Type II Diabetic Disease are interlinked. The Aβ amyloid, associated with the AD, and insulin modulate each other's functions in the brain. However, the underlying mechanism of Aβ-insulin interaction is not well understood.
Here, we present our results on the Aβ1-42 and Aβ42-1 scrambled and their interactions with a bovine serum albumin - glycerol solution and insulin to mimic in vivo conditions. We used dielectric relaxation spectroscopy to measure the dielectric permittivity as a function of frequency (10-3 Hz to 10-7 Hz), temperature (133K to 223K), and aggregation time (0h-24h). Multiple Havriliak-Negami dispersions plus an additional conductivity and electrode polarization term were used to fit the relaxation processes. We observed that the dielectric response of Aβ1-42 and Aβ42-1 scrambled can be detected and differentiated via the evolution of the relaxation peak over time.   

Photoinduced Suppression of Alzheimer’s Aβ42 Amyloidogenesis by Highly Hydrophobic and Photoactive ZnO/AAO Composite Membranes

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by symptoms of memory loss and cognitive impairment and caused by amyloidogenesis of amyloid beta 42 (Aβ42). Photosensitized hydrophobic nanomaterials can retard the such amyloidogenic aggregation via reactive oxygen species (ROS) and surface interactions. Here, we report a novel type of highly hydrophobic and photoactive ZnO/AAO membranes for efficient prevention of Aβ42 amyloidogenesis. The membranes were fabricated using the facile hydrothermal and mild anodization approaches. The resulting membranes were characterized and found highly hydrophobic with a contact angle ~120^o. The inhibitory activity of the membranes in vitro was investigated as a function of incubation time (0-120 h) under visible light illumination and probed by tunneling electron microscope (TEM), dielectric relaxation spectroscopy (DRS), circular dichroism spectroscopy (CD), UV-visible spectroscopy and thioflavin-T (ThT) fluorescence assay. The fabricated ZnO/AAO membranes showed a great deal of suppression of Aβ42 amyloidogenesis after 120 h of incubation. Our results support a novel biological application of fabricated ZnO/AAO porous membranes and provide new insights into design of new multifunctional nanomaterials for treatment of AD.
  

Targeting the Prion-like Aggregation of α-synuclein in Parkinson's Disease and Mutant p53 in Cancer

Protein misfolding results in devastating neurodegenerative diseases and cancer. Key proteins involved in these diseases, such as Aβ, tau, α-synuclein, SOD1, and TDP43 can show a prion-like behavior. In the case of the intrinsically disordered protein (IDP) α-synuclein, we used high hydrostatic pressure to identify the mechanism through which α-syn amyloid fibrils are dissociated into monomers. We provide molecular evidence of how hydrophobic interaction and the formation of water-excluded cavities jointly contribute to the assembly and stabilization of the fibrils. We investigated the structural and dynamic properties of these monomers dissociated from HHP-disturbed fibrils and the remaining fibrillar species at the atomic level, and examined how these species might seed amyloid fibril formation. We are now examining the disassembly profile of disease-related mutants of α-syn to evaluate the potential use of intermediates as targets for drug development. In the case of p53, the function of this tumor suppressor protein is lost in more than 50% of human cancers. Studies from our laboratory and others have demonstrated that the formation of prion-like aggregates of mutant p53 is associated with loss-of-function, dominant-negative and gain-of-function (GoF) effects. p53 aggregates in a mixture of oligomers and fibrils that sequestrates the native protein into an inactive conformation. These aggregates are present in tissue biopsies of breast cancer especially in more aggressive ones. We will present data on (i) the loss-of-function (LoF), dominant negative activity (DN) and gain-of-function (GoF) effects of prion-like mutant p53 aggregation in cancer; (ii) discuss current challenges in preventing p53 aggregation, including the use of small molecules with high anticancer potential; and (iii) describe our methodology for trapping aggregation precursor states in solution.   

Single-molecule force-spectroscopy reveals aggregation dynamics of intrinsically disordered proteins

Intrisically disordered proteins (IDPs) are flexible biopolymers that lack a fixed, stable structure. Instead, their inherent flexibility expands their interaction radius, thereby increasing the rate and prevalence of molecular interactions. Hence, many IDPs are involved in aggregation-related diseases, such as amyotrophic lateral sclerosis (ALS). Determining the solution conditions and dynamics under which IDP aggregates form is crucial for better understanding these diseases. We use a single-molecule magnetic stretching technique, magnetic tweezers, to measure nanometer-scale structural changes of neurofilament aggregates in real time. We probe single protein chains to determine the interaction energetics and timescales that lead to aggregation. By varying pH and ionic content of the solution, we show that the formation of these aggregates is driven by electrostatic complexation of this polyampholyte. Further, we narrow down the regions responsible for these interactions by probing a truncated version of the protein.   

Aggregation of Tau-Fragments in Osmolytic Environment

Dysfunction of the intrinsically disordered Tau protein in the forms of oligomerization and fibril formation can cause numerous neurodegenerative diseases including the Alzheimer's disease. Cellular osmolytic agents such as urea and trimethylamine N-oxide (TMAO) are known for regulating the aggregation propensity of the Tau protein. Interestingly, urea inhibits Tau aggregation while TMAO promotes it. In the present work, we study the effects of mixed urea-TMAO solutions on the aggregation propensities of two fragments of Tau, R2 (²⁷³GKVQIINKKLDL²⁸⁴) and R3 (³⁰⁶VQIVYKPVDLSK³¹⁷), which contain the nucleating segments PHF6* and PHF6 respectively. Using replica-exchange molecular dynamics simulations we find that in the mixed urea-TMAO solution TMAO counteracts the aggregation-inhibiting effects of urea and promotes oligomerization of the peptides. The molecular mechanism behind the counteraction process has been studied by means of the peptide-osmolyte preferential interactions along with the morphological changes in the peptide dimers and oligomers in pure and mixed urea-TMAO solutions.   

Structural insights into amyloid-β(1-42) fibril elongation

The aggregation of the intrinsically disordered amyloid-β peptides into higher order structures called amyloid fibrils is associated with the onset and development of Alzheimer’s disease.
Here, we use molecular dynamics simulations to explore the structural rearrangement of amyloid-β(1-42) monomers during fibril elongation. Starting from the recent solid-state NMR structure of the double horseshoe amyloid-β(1-42) fibril [1,2] we employ both conventional and enhanced sampling techniques to access metastable states that are outside of the resolution of experimental techniques.
We identify the growing end of the double filament amyloid-β fibril polymorph and hypothesize on the monomer attachment/detachment mechanisms. Additionally, we inform on the fibril twist and correlate results with experimental findings.

[1] Wälti MA et al. PNAS 2016;113:E4976-E84
[3] Colvin MT et al. JACS 2016;138:9663-74
  

Fluorescent lifetime of amino acid tryptophan in the protein xylanase

We employ fluorescence lifetime microscopy (FLIM) to quantify the emission lifetime of a model biopolymer, xylanase. We excite tryptophan using a femtosecond pulsed laser and quantify the emission lifetime by Time Correlated Single Photon Counting (TCSPC). We observe two major fluorescence decay rates associated with tryptophan. In addition to the intrinsic decay rate of tryptophan in this protein (~2.4ns), we observed a fast decay that indicates quenching between tryptophan and its neighbors. We also observed an increase in emission intensity after higher power laser exposure, as opposed to the expected decrease in the intensity due to photobleaching. We characterize the relation between the lifetime decay rates of tryptophan emission and the increase in its intensity after high power laser pulses.