Prion proteins: folding and aggregation properties

The partial unfolding or alternative folding of a class of polypeptides is at the origin of fascinating events in living cells. In their non-native conformation, these constitutive polypeptides called prions are at the origin of a protein-based structural heredity. These polypeptides are closely associated to a class of fatal neurodegenerative illnesses in mammals and to the emergence and propagation of phenotypic traits in baker's yeasts. The structural transition from the correctly folded, native form of a prion protein to a persistent misfolded form that ultimately may cause cell death or the transmission of phenotypic traits is not yet fully understood. The mechanistic models accounting for this structure-based mode of inheritance and the extent of partial unfolding of prions or their alternative folding and the subsequent aggregation process are developed and discussed. Finally, the potential regulation of prion propagation by molecular chaperones is presented.     

Correlation between hydrophobicity sequence and accessibility sequence of membrane proteins

In this paper, we used correlation analysis to quantify the correlations between the hydrophobicity sequence and accessibility sequence of 26 alpha-helix bundle membrane proteins and 119 transmembrane helices. Statistical significances of these correlations were also assessed. A slightly positive correlation was found in the alpha-helix bundle membrane proteins due to the contribution of extra-membranous domains. No correlation was found in the transmembrane domains.     

Positioning of proteins in membranes: a computational approach

A new computational approach has been developed to determine the spatial arrangement of proteins in membranes by minimizing their transfer energies from water to the lipid bilayer. The membrane hydrocarbon core was approximated as a planar slab of adjustable thickness with decadiene-like interior and interfacial polarity profiles derived from published EPR studies. Applicability and accuracy of the method was verified for a set of 24 transmembrane proteins whose orientations in membranes have been studied by spin-labeling, chemical modification, fluorescence, ATR FTIR, NMR, cryo-microscopy, and neutron diffraction. Subsequently, the optimal rotational and translational positions were calculated for 109 transmembrane, five integral monotopic and 27 peripheral protein complexes with known 3D structures. This method can reliably distinguish transmembrane and integral monotopic proteins from water-soluble proteins based on their transfer energies and membrane penetration depths. The accuracies of calculated hydrophobic thicknesses and tilt angles were approximately 1 A and 2 degrees, respectively, judging from their deviations in different crystal forms of the same proteins. The hydrophobic thicknesses of transmembrane proteins ranged from 21.1 to 43.8 A depending on the type of biological membrane, while their tilt angles with respect to the bilayer normal varied from zero in symmetric complexes to 26 degrees in asymmetric structures. Calculated hydrophobic boundaries of proteins are located approximately 5 A lower than lipid phosphates and correspond to the zero membrane depth parameter of spin-labeled residues. Coordinates of all studied proteins with their membrane boundaries can be found in the Orientations of Proteins in Membranes (OPM) database:http://opm.phar.umich.edu/.     

Gating and conduction of nano-channel forming proteins: a computational approach

Monitoring conformational changes in ion channels is essential to understand their gating mechanism. Here, we explore the structural dynamics of four outer membrane proteins with different structures and functions in the slowest nonzero modes of vibration. Normal mode analysis was performed on the modified elastic network model of channel in the membrane. According to our results, when membrane proteins were analyzed in the dominant mode, the composed pores, TolC and α-hemolysin showed large motions at the intramembrane β-barrel region while, in other porins, OmpA and OmpF, largest motions observed in the region of external flexible loops. A criterion based on equipartition theorem was used to measure the possible amplitude of vibration in channel forming proteins. The current approach complements theoretical and experimental techniques including HOLE, Molecular Dynamics (MD), and voltage clamp used to address the channel’s structure and dynamics and provides the means to conduct a theoretical simultaneous study of the structure and function of the channel.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:3     

Different circular permutations produced different folding nuclei in proteins: a computational study

There have been many studies about the effect of circular permutation on the transition state/folding nucleus of proteins, with sometimes conflicting conclusions from different proteins and permutations. To clarify this important issue, we have studied two circular permutations of a lattice protein model with side-chains. Both permuted sequences have essentially the same native state as the original (wild-type) sequence. Circular permutant 1 cuts at the folding nucleus of the wild-type sequence. As a result, the permutant has a drastically different nucleus and folds more slowly than wild-type. In contrast, circular permutant 2 involves an incision at a site unstructured in the wild-type transition state, and the wild-type nucleus is largely retained in the permutant. In addition, permutant 2 displays both two-state and multi-state folding, with a native-like intermediate state occasionally populated. Neither the wild-type nor permutant 1 has a similar intermediate, and both fold in an apparently two-state manner. Surprisingly, permutant 2 folds at a rate identical with that of the wild-type. The intermediate in permutant 2 is stabilised by native and non-native interactions, and cannot be classified simply as on or off-pathway. So we advise caution in attributing experimental data to on or off-pathway intermediates. Finally, our work illuminates the results on alpha-spectrin SH3, chymotrypsin inhibitor 2 and beta-lactoglobulin, and supports a key assumption in the experimental efforts to locate potential nucleation sites of real proteins via circular permutations.     

Correlation between sequence hydrophobicity and surface-exposure pattern of database proteins

Hydrophobicity is thought to be one of the primary forces driving the folding of proteins. On average, hydrophobic residues occur preferentially in the core, whereas polar residues tend to occur at the surface of a folded protein. By analyzing the known protein structures, we quantify the degree to which the hydrophobicity sequence of a protein correlates with its pattern of surface exposure. We have assessed the statistical significance of this correlation for several hydrophobicity scales in the literature, and find that the computed correlations are significant but far from optimal. We show that this less than optimal correlation arises primarily from the large degree of mutations that naturally occurring proteins can tolerate. Lesser effects are due in part to forces other than hydrophobicity, and we quantify this by analyzing the surface-exposure distributions of all amino acids. Lastly, we show that our database findings are consistent with those found from an off-lattice hydrophobic-polar model of protein folding.     

Relative influence of hydrophobicity and net charge in the aggregation of two homologous proteins

A potentially amyloidogenic protein has to be at least partially unfolded to form amyloid aggregates. However, aggregation of the partially or totally unfolded state of a protein is modulated by at least three other factors: hydrophobicity, propensity to form secondary structure, and net charge of the polypeptide chain. We propose to evaluate the relative importance of net charge, as opposed to the other factors, on protein aggregation and amyloidogenicity. For this aim, we have used two homologous proteins that were previously shown to be able to form amyloid fibrils in vitro, the N-terminal domain of HypF from Escherichia coli (HypF-N) and human muscle acylphosphatase (AcP). The aggregation process from an ensemble of partially unfolded conformations is ca. 1000-fold faster for HypF-N than for AcP. This difference can mainly be attributed to a higher hydrophobicity and a lower net charge for HypF-N than for AcP. By using protein engineering methods, we have decreased the net charge of AcP to a value identical to that of wild-type HypF-N and increased the net charge of HypF-N to a value identical to that of wild-type AcP. Amino acid substitutions were selected to minimize changes in hydrophobicity and secondary structure propensities. We were able to estimate that the difference in net charge between the two wild-type proteins contributes to 20-25% of the difference in their aggregation rates. An understanding of the relative influences of these forces in protein aggregation has implications for elucidating the complexity of the aggregation process, for predicting the effect of natural mutations, and for accurate protein design.     

Prediction of the aggregation propensity of proteins from the primary sequence: aggregation properties of proteomes

In the cell, protein folding into stable globular conformations is in competition with aggregation into non-functional and usually toxic structures, since the biophysical properties that promote folding also tend to favor intermolecular contacts, leading to the formation of β-sheet-enriched insoluble assemblies. The formation of protein deposits is linked to at least 20 different human disorders, ranging from dementia to diabetes. Furthermore, protein deposition inside cells represents a major obstacle for the biotechnological production of polypeptides. Importantly, the aggregation behavior of polypeptides appears to be strongly influenced by the intrinsic properties encoded in their sequences and specifically by the presence of selective short regions with high aggregation propensity. This allows computational methods to be used to analyze the aggregation properties of proteins without the previous requirement for structural information. Applications range from the identification of individual amyloidogenic regions in disease-linked polypeptides to the analysis of the aggregation properties of complete proteomes. Herein, we review these theoretical approaches and illustrate how they have become important and useful tools in understanding the molecular mechanisms underlying protein aggregation.     

Combined sequence and sequence-structure-based methods for analyzing RAAS gene SNPs: a computational approach

Abstract

The renin–angiotensin–aldosterone system (RAAS) plays a key role in the regulation of blood pressure (BP). Mutations on the genes that encode components of the RAAS have played a significant role in genetic susceptibility to hypertension and have been intensively scrutinized. The identification of such probably causal mutations not only provides insight into the RAAS but may also serve as antihypertensive therapeutic targets and diagnostic markers. The methods for analyzing the SNPs from the huge dataset of SNPs, containing both functional and neutral SNPs is challenging by the experimental approach on every SNPs to determine their biological significance. To explore the functional significance of genetic mutation (SNPs), we adopted combined sequence and sequence-structure-based SNP analysis algorithm. Out of 3864 SNPs reported in dbSNP, we found 108 missense SNPs in the coding region and remaining in the non-coding region. In this study, we are reporting only those SNPs in coding region to be deleterious when three or more tools are predicted to be deleterious and which have high RMSD from the native structure. Based on these analyses, we have identified two SNPs of REN gene, eight SNPs of AGT gene, three SNPs of ACE gene, two SNPs of AT1R gene, three SNPs of CYP11B2 gene and three SNPs of CMA1 gene in the coding region were found to be deleterious. Further this type of study will be helpful in reducing the cost and time for identification of potential SNP and also helpful in selecting potential SNP for experimental study out of SNP pool.     

Local sequence patterns of hydrophobicity and solvent accessibility in soluble globular proteins

We examined the variation in the solvent accessibility and hydrophobicity of the amino acids along the sequences of 58 soluble globular proteins with known tertiary structure. We found that there is a significant tendency for the accessibilities to run in clusters along the sequence but that the hydrophobicities are distributed without such nonrandom clusters. Theseresults suggest severe limitations on the power of sequence analysis tools that use average hydrophobicity scores of overlapping subsequences to predict accessibility.     

Derivation of a solubility condition for proteins from an analysis of the competition between folding and aggregation

Failure in maintaining protein solubility in vivo impairs protein homeostasis and results in protein misfolding and aggregation, which are often associated with severe neurodegenerative and systemic disorders that include Alzheimer's and Parkinson's diseases and type II diabetes. In this work we formulate a model of the competition between folding and aggregation, and derive a condition on the solubility of proteins in terms of the stability of their folded states, their aggregation propensities and their degradation rates. From our model, the bistability between folding and aggregation emerges as an intrinsic aspect of protein homeostasis. The analysis of the conditions that determine such a bistability provides a rationalization of the recently observed relationship between the cellular abundance and the aggregation propensity of proteins. We then discuss how the solubility condition that we derive can help rationalise the correlation that has been reported between evolutionary rates and expression levels or proteins, as well as in vivo protein solubility and expression level measurements, and recently elucidated trends of proteome evolution.     

Characterization of the inhibition mechanism of a tissuefactor inhibiting single-chain variable fragment: a combined computational approach

Journal of Molecular Modeling - In order to predict the impact sensitivity of high explosives, we designed and evaluated several models based on the trigger linkage hypothesis and the Arrhenius...     

Reducing the computational complexity of protein folding via fragment folding and assembly

Understanding, and ultimately predicting, how a 1-D protein chain reaches its native 3-D fold has been one of the most challenging problems during the last few decades. Data increasingly indicate that protein folding is a hierarchical process. Hence, the question arises as to whether we can use the hierarchical concept to reduce the practically intractable computational times. For such a scheme to work, the first step is to cut the protein sequence into fragments that form local minima on the polypeptide chain. The conformations of such fragments in solution are likely to be similar to those when the fragments are embedded in the native fold, although alternate conformations may be favored during the mutual stabilization in the combinatorial assembly process. Two elements are needed for such cutting: (1) a library of (clustered) fragments derived from known protein structures and (2) an assignment algorithm that selects optimal combinations to "cover" the protein sequence. The next two steps in hierarchical folding schemes, not addressed here, are the combinatorial assembly of the fragments and finally, optimization of the obtained conformations. Here, we address the first step in a hierarchical protein-folding scheme. The input is a target protein sequence and a library of fragments created by clustering building blocks that were generated by cutting all protein structures. The output is a set of cutout fragments. We briefly outline a graph theoretic algorithm that automatically assigns building blocks to the target sequence, and we describe a sample of the results we have obtained.     

Hierarchical protein folding pathways: a computational study of protein fragments

We have previously presented a building block folding model. The model postulates that protein folding is a hierarchical top-down process. The basic unit from which a fold is constructed, referred to as a hydrophobic folding unit, is the outcome of combinatorial assembly of a set of "building blocks." Results obtained by the computational cutting procedure yield fragments that are in agreement with those obtained experimentally by limited proteolysis. Here we show that as expected, proteins from the same family give very similar building blocks. However, different proteins can also give building blocks that are similar in structure. In such cases the building blocks differ in sequence, stability, contacts with other building blocks, and in their 3D locations in the protein structure. This result, which we have repeatedly observed in many cases, leads us to conclude that while a building block is influenced by its environment, nevertheless, it can be viewed as a stand-alone unit. For small-sized building blocks existing in multiple conformations, interactions with sister building blocks in the protein will increase the population time of the native conformer. With this conclusion in hand, it is possible to develop an algorithm that predicts the building block assignment of a protein sequence whose structure is unknown. Toward this goal, we have created sequentially nonredundant databases of building block sequences. A protein sequence can be aligned against these, in order to be matched to a set of potential building blocks.     

Protein folding and aggregation: two sides of the same coin in the condensation of proteins revealed by pressure studies

Hydrostatic pressure can be considered as "thermodynamic tweezers" to approach the protein folding problem and to study the cases when folding goes wrong leading to the protein folding disorders. The main outcome of the use of high pressure in this field is the stabilization of folding intermediates such as partially folded conformations, thus allowing us to characterize their structural properties. Because partially folded intermediates are usually at the intersection between productive and off-pathway folding, they may give rise to misfolded proteins, aggregates and amyloids that are involved in many neurodegenerative diseases, such as transmissible spongiform encephalopathies, Alzheimer's disease, Parkinson's disease and Huntington's disease. Of particular interest is the use of hydrostatic pressure to unveil the structural transitions in prion conversion and to populate possible intermediates in the folding/unfolding pathway of the prion protein. The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform, the PrP(Sc) (from scrapie). It has been demonstrated that hydrostatic pressure affects the balance between the different prion species. The last findings on the application of high pressure on amyloidogenic proteins will be discussed here as regards to their energetic and volumetric properties. The use of high pressure promises to contribute to the identification of the underlying mechanisms of these neurodegenerative diseases and to develop new therapeutic approaches.