Chapter 2 REVIEW OF RELATED LITERATURE

History Cases of diseases in what appeared to be protein misfolding and aggregation disorders had been described during the 17th century (Kyle, 2001). Rudolf Virchow coined the term amyloid to describe the structural bodies of deposited tissue in the brain that reacted to the iodine solution in combination with hydrated sulfuric acid. These deposits, termed wax-like or lardaceaous, was believed to be cellulosic in nature. It was not until Fritz Friedrich and August Kekule dissected and performed extensive chemical tests on amyloid-rich segments of spleen that they found that amyloid is protein in nature (Westermark, 2005). In 1907, Alois Alzheimer reported senile plaques and neurofibrillary tangles in the neocortex and hippocampus of a middle-aged woman with memory deficits and progressive loss of cognitive function, a disorder later known as Alzheimers disease (AD) (Forman et al., 2004). Five years after, in 1912, Lewy bodies, neuronal cytoplasmic aggregations containing misfolded fibrillar -synuclein (Jelliger, 2007) that was the pathological hallmark of Parkinsons disease (PD) was described by Friedrich Lewy (Forman et al., 2004). Spielmeyer in 1922 used the term Creutzfeldt-Jakob disease to describe a human neurodegenerative disease described in earlier reports by Hans Gerhard Creudtzfeldt and Alfons Maria Jakob (McKintosh et al., 2003). This term later became used to describe the human form of bovine spongiform encephalopathy (BSE). Both BSE and CJD and its variants belong to the group of disease known as transmissible spongiform encephalopathy (TSE). Creudzfelt-Jakob disease, Gerstmann-Strussler-Scheinker disease (GSS), kuru, and 5

6 fatal familial insomnia in humans, scrappie in sheep and goats, chronic wasting disease in elk and deer, mad cow disease in cows, and feline spongiform encephalopathy in cats are all forms of mammalian TSE (Jonathan DF Wadsworth, 1999, Pawe P. Liberski, 2004). Since the 18th century, cases of these livestock diseases have been reported, and the infectious agent was initially believed to be viruses due to its ability to pass through filters with small pore size. However, the lack of DNA prompted John Stanley Griffith in 1967 to suggest that it is, in fact, protein (McKintosh et al., 2003). Stanley Prusiner further characterized and described this infectious agent, eventually calling it prions to describe its protein nature and infectivity (Aguzzi et al., 2008). His studies on prions eventually won him the Nobel Prize for Medicine in 1997 (Prusiner, 1997). In 1996, following the government of United Kingdoms announcement that BSE, more popularly known as mad cow disease, can be transmitted to humans in the form of variant CJD (vCJD), a world-wide crisis on beef industry ensued, sparking public terror and market collapse on various beef and beef-related products (Dumble, 2001). Hundreds of vCJD cases were reported as the disease spread to various countries in Europe and North America (CDC, 2006) due to contaminated livestock feeds and importation of asymptomatic infected cattle. Prions and Prionoids As described in the previous chapter, protein misfolding diseases (PMD) encompass a wide range of diseases that share a common feature: the aggregation of misfolded proteins into amyloid fibrils and plaques (Csizmok and Tompa, 2009, Estrada et al., 2006, Menzies et al., 2011, Miller, 2004). These deposits accumulating in cells cause dysfunction and eventually, leads to death (Miller, 2004). The insoluble, highly ordered deposits called

7 amyloids has been described in the case of amyloid- peptide in AD, -synuclein in PD, the polyglutamine stretch of huntingtin in Huntingtons disease and prion proteins in prion diseases. Prions are self-templating elements known to be transmissible between individuals and even cross the specie barrier via the food chain (King et al., 2012). The prion hypothesis (also known as the protein-only hypothesis) postulates that infectious agents that cause TSE contain no nucleic acids, but are instead a post-translationally modified form of the native protein, possibly of different structural conformation. Upon introduction to the host, the misfolded protein form induces the conversion of the normal protein to the misfolded form (Aguzzi et al., 2008). However, not all PMD exhibit the infectivity shown by prion diseases, with its ability to cause widespread epidemics by transmission between individuals, and even with members of different species as with the case of BSE. Amyloid-forming proteins that exhibit infectivity with neighboring proteins or with neighboring cells, but not between individuals are called prionoids. (Aguzzi and Rajendran, 2009). In other words, prionoids are proteins that exhibit the misfolded and self-templating capability of bonafide prions but the transmission of prionoids, unlike prions that can cause widespread epidemic and even cross specie barrier, are restricted within the tissue or the individual. Natural transmission of prionoids between individuals is yet to be observed but it is possible in experimental models (King et al., 2012). Regardless of these differences, both the amyloid-forming prionoid and highly infectious prion proteins propagate through their -sheet-rich structure, forming nucleates and subsequently growing and polymerizing by recruiting and converting their soluble protein conformers (Alberti et al., 2009).

8 Studies on yeast prions led to the observation that these proteins share a common prion domain of about 60 amino acid in length rich in uncharged polar residues asparagine, glutamine and tyrosine as well as glycine (Alberti et al., 2009, King et al., 2012). These yeast prion domains, when over-expressed in proteins, increased priongenicity of the resulting protein while deletion of this domain renders the protein incapable of accessing its prion state (King et al., 2012). Furthermore, this domain is shown to be portable; for instance, appending the prion domain of the yeast prion prtein Sup35 to a reporter gene such as galactosidase or green fluorescent protein (GFP) confers prion behavior (King et al., 2012). Furthermore, closer analysis of these prion domains showed that certain key amino acids play a significant role in protein behavior (Alberti et al., 2009, King et al., 2012). It was previously believed that asparagine and glutamine residues equally contribute to prion behavior (King et al., 2012), but a study made by Alberti and colleagues (2009) showed that aggregation-prone prions have asparigine-rich prion domains while glutamine, proline and charged residues were abundant in non-aggregating prion domains. Furthermore, the spacing of amyloid breaking prolines and charged amino acids contributes significantly to prion behavior (Alberti et al., 2009). This and other studies pointed to the importance of asparagines in the formation of toxic oligomeric species and glutamines in the formation of self-templating prions (King et al., 2012). Mechanism of Protein Folding and Misfolding Central to PMDs are the change in protein conformation and aggregation. But how do proteins attain such conformations? Christian Anfinsen, based on his studies of ribonuclease A, introduced the thermodynamic hypothesis, stating that the three dimensional structure of a protein in its physiological environment, that is, in consideration with the solvent, pH, ionic

9 strength, temperature and presence of other solutes, is the one which the Gibbs free energy of the system is the lowest (Anfinsen, 1973), and that the proteins conformation is encoded in the physicochemical properties of the amino acid sequence. Cyrus Levinthal pointed out that a distinct, predetermined pathway must be in play, or else the native state of protein would not be found within reasonable time by any random search through conformational space, known as Levinthals paradox. Furthermore, it is also implied that the proteins native conformation may not necessarily by its lowest energy state, and many proteins adopt a still lower energy conformations as a molecular aggregate, such as amyloids (Englander et al., 2008). According to Levinthal, a protein folds by following an energetic funnel a funnelshaped energy landscape that allows protein to fold to its most stable conformation (Figure 2. 1). The funnel-shaped landscape is brought about by initial local interactions of a proteins amino acids, limiting the conformational space a protein must explore to fold properly (Renaud, 2010).

10

Figure 2. 1 Levinthal's Energetic Funnel

Using Levinthals concept of the energetic funnel, specific folding intermediates have been successfully found and observed in real time, most notably by Robert Baldwin and T. E. Creighton. (Englander et al., 2008). Baldwin characterized two types of folding patterns for proteins. The hierarchic model describes folding as a process beginning with structures that are local in sequence and marginal in stability. These local structures interact to produce intermediates of ever increasing complexity that ultimately grow to its native conformation. On the other hand, the non-hierarchic model is a process where tertiary structures stabilizes and determines local interactions (Baldwin and Rose, 1999). The hierarchic model eventually became known as the framework model. The hydrophobic collapse model stems from the

11 non-hierarchic model, postulating that the hydrophobic effect is the main driving force of folding, starting with the collapse of the chain and eventual formations of secondary structures (Szilagyi et al., 2007). In an attempt to unify the two models, the nucleationcondensation model was proposed. This model postulates that the overall structure condenses around an element of structure, the nucleus, that itself consolidates during the condensation (Itzhaki et al., 1995). Long range and other native hydrophobic interaction form in the transition state to stabilize the weak secondary structure, taking a route that is somewhere between the framework and hydrophobic collapse models (Szilagyi et al., 2007). Studies to detect prion domains The existence of prion domain in yeast proteins prompted the scouring of the human genome for proteins bearing the same or similar domains. Studies of prions in yeast showed that these proteins tend to have common distinctive domains in the amino acid sequence rich in asparagines, glutamine and glycine (King et al., 2012). A scan of the human genome made light of several RNA-binding proteins that tend to have these domains and rich in the amino acids suspected to be involved in prion formation. The study made by King et al. (2012) yielded four RNA-binding proteins with particularly high susceptibility to prion and other misfolding diseases diseases. FUS FUS is a multifunctional protein component of the nuclear ribonucleoprotein complex involved in pre-mRNA splicing and export of mature mRNA in the cytoplasm. The function of this particular protein is to bind both single-stranded and double stranded DNA and promote ATP-independent annealing of complementary single-stranded DNA. It is believed to play a role in maintenance of genomic integrity. Mutations in this protein is shown to be

12 involved in familial as well as sporadic Amyotrophic Lateral Schlerosis (ALS) and FrontoTemporal Lobar Degeneration with Ubiquitin-positive inclusions (FTLD-U), and aggragation of this protein is linked to Huntingtons disease and spinocerebellar ataxia. TDP-43 TDP-43, also referred to as TRDBP (TAR DNA binding protein) is a DNA and RNA binding protein that acts as a repressor. It is also associated with ALS and other neurodegenerative diseases. It can adopt specific isfolded forms that are highly toxic, and recently, it has been surmised that different RNA molecules can induce TDP-43 to take on different conformations or strains. Its aggregates might also sequester essential RNA molecules and further promote neurodegeneration. EWSR1 EWSR1 is a multifunctional protein that is involved in various cellular processes, including gene expression, cell signaling, and RNA processing and transport. The protein includes an N-terminal transcriptional activation domain and a C-terminal RNA-binding domain. Recently, it has also been linked to FTLD-U. it forms cytoplasmic aggregates and is toxic in yeast. Intrinsic disorder in proteins All of these models rely on the specific physicochemical properties of the proteins primary structure to dictate its final conformation. However, proteins are flexible macromolecular structures, and this flexibility allows them to perform their specific tasks within the cell. Some proteins, especially those involved in the regulatory pathways of higher eukaryotes display these characteristics. Known as intrinsically disordered proteins (IDPs), these proteins exists as an ensemble of rapidly interconverting conformations that resemble

13 denatured states (Tompa and Han, 2012). Some of IDPs have been implicated in PMD, such as -synuclein and the prion protein (Csizmok and Tompa, 2009, Tompa and Han, 2012). Since amyloid formation is mainly characterized by an extended hydrogen bonding interaction between the backbone amides in a cross -sheet formation, IDPs, with their unstructured state and exposed backbone, are especially prone to aggregation. Indeed, in studies of protein aggregation of a range of mutants under conditions favoring the unfolded states of globular proteins, it was found that amyloidogeneicity shows a significant positive correlation with hydrophobicity and -sheet forming potential, and negative correlation with total charge. Furthermore, IDPs are also found to be depleted in order-promoting amino acids WCFIYVL and enriched in dirsoder-promoting amino acids KESPQRA (Csizmok and Tompa, 2009). Aggregation and amyloid formation There are three existing models that describe the molecular mechanism of protein misfolding and aggregation based on kinetic modeling of protein aggregation (Estrada et al., 2006, Soto, 2001). The polymerization hypothesis postulates that protein oligomers that act as seeds that induce protein misfolding (Soto, 2001). First, there is a slow formation of an ordered nucleus due to the unfavorable interaction between the native monomeric conformation followed by polymerization and a growth phase, where the growing amyloid recruits native protein and induces it to misfold (Estrada et al., 2006). Hence, in this model, misfolding is a consequence of aggregation. Recent publications cited the template-directed (heterodimer) model (Apetri, 2004) developed from studies of prioon proteins which closely resembles the polymerization hypothesis of protein aggregation (Figure 2. 2). As such, both models stressed the presence of a misfolded seed as a necessary requirement for protein

14 misfolding and aggregation. It further postulates that there is a high activation energy barrier preventing a spontaneous conformation change. Instead, the misfolded prion protein forms a heteromeric complex with the functional protein. Subsequently, the complexation promotes the conformational change in the functional protein, resulting to two misfolded proteins homodimers. The dissociation of the homodimer to two misfolded prion monomer that can catalyze conformational changes in native proteins is the final and rate-limiting step in this model (Aguzzi et al., 2008, Apetri, 2004). Their difference lies in the function of the amyloid. For the polymerization hypothesis, the amyloid induces the misfolding whereas the template-directed refolding model describes it merely a by-product of misfolding.

Figure 2. 2 Template- directed refolding (top) and polymerization hypothesis (bottom)

An alternative to the polymerization hypothesis, the conformational hypothesis, postulates that the protein spontaneously converts from its native conformation and its aggregation-prone form without the need for an inducer or outside interference. The

15 aggregation-prone forms then interact with one another to form amyloid. Hence, aggregation is a consequence rather than the cause of the disease (Soto, 2001). Likewise, this is mirrored in the more recent paper by Apetri (2004) and Aguzzi (2008). Called the seeding (nucleated crystallization) model, it presents a thermodynamically-controlled conversion of the native functional protein to the misfolded prion fom. The conformational change is a reversible process and the native functional form is favored. The prion form is favored only when it forms a complex with other misfolded form, leading to aggregation. Once the seed is established, the formation of the misfolded conformation is accelerated. Supporting evidence for the spontaneous interconversion of the folded protein and its misfolded conformation is suggested by the discovery of the IDPs (Section 3.2).

Figure 2. 3 Seeding (top) and conformational hypothesis (bottom) The third model is intermediate between these two forms. Slight conformational changes result in the formation of an amyloidogenic intermediate, which is unstable in an aqueous environment because of exposure of hydrophobic segments to the solvent. This unstable intermediate is stabilized by intermolecular interactions with other molecules

16 forming small -sheet oligomers, which by further growth produce amyloid fibrils. In this model the conversion of the folded protein into the pathological form is triggered by structural changes, but complete misfolding is dependent upon oligomerization (Soto, 2001). This is termed the conformation/oligomerization hypotheis (Figure 2. 4).

Figure 2. 4 Conformation/Oligomerization Hypothesis

Molecular Dynamics Experimental determination of the folding mechanism and early aggregation steps were difficult to obtain, hence computational approaches were used to obtain an insight into these mechanisms. This is due to the metastable and short-lived nature of soluble pre-fibril oligomers at the early step of fibril formation (Jiang et al., 2009). Hence, computer simulations are carried out in the hopes of understanding the properties of molecules in terms of their structure and the microscopic interactions among them (Allen, 2004). The general process of describing complex chemical systems in terms of realistic atomic models, known as molecular modeling, has the ultimate goal of understanding and predicting macroscopic properties based on detailed knowledge on the atomic scale (Spoel et al., 2010). There are two main methods in molecular modeling: Monte Carlo simulations and Molecular Dynamics simulations. Molecular Dynamics, which is the focus of this study, is appropriate for generation of non-equilibrium ensembles for the analysis of dynamic events. It consists of

17 numerical, step-by-step solution of Newtons classical equation of motion for a system with N interacting atoms:
2 2

The forces F are the negatve derivatives of a potential function V (r1, r2, , rN) =

= , = 1

(2.1)

(2.2)

The coordinates of the atoms as a function of time represents the trajectory of the system. The average of the equilibrium trajectory gives an idea as to the macroscopic properties of the system (Spoel et al., 2010). In reality, atoms, consisting of the nucleus, electrons and photons that interact through electromagnetic and gravitational forces, and obey Dirac and Schrodingers equations (Berendsen, 2004). In order to simulate a large number of atoms in a system, molecular dynamic simulations had to employ several assumptions. First is the application of classical mechanics to describe atomic motion (Spoel et al., 2010). Although most atoms, especially the heavier ones, behave classically at normal temperatures, hydrogen and deuterium atoms behave quantum-mechanically at 300 K while electrons are fully quantum-mechanical in all cases (Berendsen, 2004). Furthermore, there were suggestions from literature (Berendsen, 2004, Berendsen et al., 1995, Spoel et al., 2010, Spoel et al., 2005) that hydrogen bonding occurs by quantum tunneling. Equations of classical harmonic oscillators also vary appreciable from the real quantum oscillation that occurs in atomic bond vibrations. This limitation has been corrected by the use of corrections in the classical harmonic oscillator or the use of bond constraints. Specifically treating bonds and bond angles as constraints in equations of motions is advantageous since quantum oscillators in ground state resemble

18 bond constraints more than they do classical harmonic oscillators. Furthermore, the application of this allows the algorithm to use larger time steps without the system blowing. Another assumption is that the electrons are in the ground state (Spoel et al., 2010). Hence, the force field accounts for the atoms only and no reactions take place. This is based on he Born-Oppenheimer Approximation stating that the wave function of the electron is affected by the location of the nucleus, hence the computation of the wave function of the electron is limited by the position of the nucleus (Berendsen, 2004). Molecular dynamics also rely on the use of force fields. The calculations of bonded and non-bonded forces and interactions as well as the parameters used are dictated by the type of force field implemented. Also, force fields have the capacity of being pair-additive (Spoel et al., 2010); all non-bonded forces result from the sum of all non-bonded pair interactions. This, however, pose a problem for large systems, as non-bonded energies tend to hit off the charts. Therefore, long-range interactions are cut-off and periodic boundary conditions are implemented to prevent unrealistic ballooning of the energy of the system. GROMACS 4.5.4 especially uses a cut-off radius for Lennard-Jones interactions as well as for Coulombic interactions. Force field Force field is a set of terms describing various forces and interactions in a molecule, namely, bond-stretching, bond-bending, electrostatic attraction and repulsion, along with a set of parameters obtained from experimental data. (Spoel et al., 2005). The force field relevant to this study is the Optimized Potential for Liquid Simulations all-atom (OPLSAA) force field. OPLS was developed from the earlier AMBER-united atom force field but was parametrized to produce experimental thermodynamic and structural data on fluids.

19 Based on the rationale that proteins and peptides consist of organic functional groups alcohols, esters, thioesters, amides, hydrocarbons, the OPLS force field was built up from parameters that model organic liquids (Jorgensen and Tirado-Rives, 1988). The non-bonded interactions are computationally represented through Coulomb and Lenard-Jones terms interacting between sites centered on the nuclei. The intermolecular interaction energy between molecules a and b is the sum of interactions between the sites on the two molecules (2.3).
2 = + 12 6

(2.3)

The non-bonded contribution to the intermolecular energy is evaluated with the same expression for all pairs of sites separated by more than three bonds. In the 1988 version of the OPLS force field, each atomic nucleus has an interaction site except for hydrocarbon (CHn) groups which are treated as united atom centered on the carbon atom. No special functions were used to describe the hydrogen bonding and there were no additional interaction sites for lone pairs. Standard geometries were used for the molecules with fixed bond lengths and bond angles, though torsional motion was included. Particular emphasis was placed on reproducing the experimental densities and heats of vaporization for the liquids (Jorgensen and Tirado-Rives, 1988). However computationally inexpensive united-atom models are, allatom models allow more flexibility for charged distributions and torsional energetic. Hence, the OPLS all-atom force field was developed (Jorgensen et al., 1996). However, amidst all these changes, there are still inaccuracies in the force fields developed. A major problem persisting is the functional form of the potential energy, which has several restrictions, namely, the use of atom-centered charges rather than the explicit

20 representations of the lone pairs on atoms which is a more accurate description of molecular charge distribution, and a failure to explicitly treat electronic charge distribution. To make up for these shortcomings, the parameters are adjusted accordingly, hence the continuing improvements in the OPLS force field series (Kaminski et al., 2001). Algorithms In order to solve equations of motion, molecular dynamics make use of algorithms to perform step-by step numerical integrations of the systems function. The simplest algorithm for solving these equations of motion is the Euler solution (Berendsen, 2004). However, it is unstable and inaccurate for MD purposes so several sophisticated predictor-corrector algorithms were developed. But the best and simplest algorithm that is widely-used is the Verlet Algorithm, and its other form, the Leap-Frog algorithm. These algorithms are favored in MD simulations because they are time-reversible, stable, symplectic (conserves volume in phase space) and simple (Allen, 2004). In GROMACS ver. 4.5.5, the leap-frog algorithm is the default integrator used in MD simulations: + 2 = 2 +
1 1 1

+ 2 = () + + 2

()

(2.4) (2.5)

Figure 2. 5 Visualization of the Leap-Frog Algorithm

21 As seen in Figure 2.5, the Leap-frog algorithm computes the position x and velocity v over a time t in alternating steps resembling two frogs leaping over each others backs. Also an important part of molecular dynamics simulation is the energy minimization. It ensures that there are no stearic clashes and inappropriate geometry that might cause the system to explode. There are several algorithms that are used in energy minimization, the most common of all is the steepest descent. It makes use of derivative information and takes a step in the direction of the negative gradient without considering the previous steps. It is fast, simple and easy to implement but like all other energy minimization mthods, it can only find the local minima of the system. It calculates the forces F and potential energy first followed by the new positions given as:

This equation defines the vector r as the vector of all 3N coordinates and its new position n +1 as defined by its maximum displacement hn, the negative gradient of the potential V or the force Fn, its previous position rn and the largest absolute value of the force components max|Fn|. If (Vn+1 < Vn) the new positions are accepted and hn+1 = 1:2hn. If (Vn+1 Vn) the new positions are rejected and hn = 0:2hn. The algorithm stops when either a userspecified value has been reached, the maximum nuber of steps has been performed or the value of F has converged to machine precision.

+1 = +

(| |)

(2.6)

22

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