Intermolecular Beta-sheets Research Articles

In silico protein dynamics in the human cytoplasm: Partial folding, misfolding, fold switching, and non-native interactions.

We examine the influence of cellular interactions in all-atom models of a section of the Homo sapiens cytoplasm on the early folding events of the three-helix bundle protein B (PB). While genetically engineered PB is known to fold in dilute water box simulations in three microseconds, the three initially unfolded PB copies in our two cytoplasm models using a similar force field did not reach the native state during 30-microsecond simulations. We did however capture the formation of all three helices in a compact native-like topology. Folding in vivo is delayed because intramolecular contact formation within PB is in direct competition with intermolecular contacts between PB and surrounding macromolecules. In extreme cases, intermolecular beta-sheets are formed. Interactions with other macromolecules are also observed to promote structure formation, for example when a PB helix in our simulations is shielded from solvent by macromolecular crowding. Sticking and crowding in our models initiate sampling of helix/sheet structural plasticity of PB. Relatedly, in past in vitro experiments, similar GA domains were shown to switch between two different folds. Finally, we also observed that stickiness between PB and the cellular environment can be modulated in our simulations through the reduction in protein hydrophobicity when we reversed PB back to the wild-type sequence. This study demonstrates that even fast-folding proteins can get stuck in non-native states in the cell, making them useful models for protein-chaperone interactions and early stages of aggregate formation relevant to cellular disease.

Catalytically Active Amyloids as Future Bionanomaterials.

Peptides and proteins can aggregate into highly ordered and structured conformations called amyloids. These supramolecular structures generally have convergent features, such as the formation of intermolecular beta sheets, that lead to fibrillary architectures. The resulting fibrils have unique mechanical properties that can be exploited to develop novel nanomaterials. In recent years, sequences of small peptides have been rationally designed to self-assemble into amyloids that catalyze several chemical reactions. These amyloids exhibit reactive surfaces that can mimic the active sites of enzymes. In this review, I provide a state-of-the-art summary of the development of catalytically active amyloids. I will focus especially on catalytic activities mediated by hydrolysis, which are the most studied examples to date, as well as novel types of recently reported activities that promise to expand the possible repertoires. The combination of mechanical properties with catalytic activity in an amyloid scaffold has great potential for the development of future bionanomaterials aimed at specific applications.

EFFECTS OF ISOMETRIC EXERCISE ON THE SPECTROMETRIC PROFILE OF OLDER ADULTS WITH HIGH BLOOD PRESSURE

Objective: A decline in protein homeostasis is associated with the development and progression of several cardiovascular diseases, including hypertension. Physical exercise is a promising intervention to counter defects in protein quality control. Isometric resistance training (IRT) promotes clinically relevant reductions in blood pressure (BP), however, its effects on protein homeostasis are not clearly defined. We determined the general proteomic profile in human plasma of older adults with high normal BP to hypertension and how it changes in response to IRT. Design and method: Older adults (n = 11, age: 73 ± 3 years) with high normal BP to hypertension participated in 8-weeks of IRT. IRT consisted of 4x45 sec contractions, alternatively with both hands, at an intensity of 50% of the maximum voluntary contraction, with a 1-min interval between sets, performed 3 times per week. Fourier-transform infrared (FTIR) spectroscopy assessed changes in the plasma after versus before IRT in the region of the spectra attributed to the secondary structure of proteins (1700–1600 cm-1). Results: Samples showed an excellent discrepancy before versus after IRT in the 1700–1600 cm-1 region. IRT induced a shift in the peak in the in the spectra region of 1622 cm-1; this region is attributed to intermolecular beta-sheets, which are common structures found in mature fibrils. Mature fibrils are prone to form toxic protein aggregates, especially amyloid aggregates. After IRT, participants appeared to have lower levels of intermolecular beta-sheets and samples were enriched with antiparallel beta-sheets (1696 cm-1) and beta-turns (1665 cm-1). Conclusions: The decrease in intermolecular beta-sheets may represent an improvement in the protein turnover and a better protein quality control in hypertension as a result of IRT. The differences in the antiparallel beta-sheets and beta-turns after IRT indicate IRT is modifying the protein profile. Further studies are needed to confirm these results and to address how exercise is influencing protein homeostasis.

Polysorbate 80 controls Morphology, structure and stability of human insulin Amyloid-Like spherulites

Amyloid protein aggregates are not only associated with neurodegenerative diseases and may also occur as unwanted by-products in protein-based therapeutics. Surfactants are often employed to stabilize protein formulations and reduce the risk of aggregation. However, surfactants alter protein-protein interactions and may thus modulate the physicochemical characteristics of any aggregates formed. Human insulin aggregation was induced at low pH in the presence of varying concentrations of the surfactant polysorbate 80. Various spectroscopic and imaging methods were used to study the aggregation kinetics, as well as structure and morphology of the formed aggregates. Molecular dynamics simulations were employed to investigate the initial interaction between the surfactant and insulin. Addition of polysorbate 80 slowed down, but did not prevent, aggregation of insulin. Amyloid spherulites formed under all conditions, with a higher content of intermolecular beta-sheets in the presence of the surfactant above its critical micelle concentration. In addition, a denser packing was observed, leading to a more stable aggregate. Molecular dynamics simulations suggested a tendency for insulin to form dimers in the presence of the surfactant, indicating a change in protein-protein interactions. It is thus shown that surfactants not only alter aggregation kinetics, but also affect physicochemical properties of any aggregates formed.

SARS‐CoV‐2 ORF8 Accessory Protein Dimerization Domains and Protein‐Protein Host Interaction

As the world continues to fight the coronavirus pandemic caused by SARS‐CoV‐2, we are gaining valuable insights by comparing this virus to other human coronaviruses such as SARS‐CoV and MERS‐CoV that have also caused outbreaks. Coronaviruses infect many animal species, but disease severity varies between strains. Viral accessory proteins are generally implicated in increased infectivity, pathogenicity, and virulence. The accessory protein open reading frame 8 (ORF8), although not essential for viral replication, appears to play a critical role in disease severity by inhibiting multiple pathways of the immune response. Furthermore, the ORF8 gene is found within a highly variable portion of the genome, increasing the possibility of dangerous mutant forms arising. Structure‐based design of therapeutics holds great promise for effective targeting of such rapidly evolving threats. Thus, we designed physical models and computer representations of ORF8, based on the published structure 7JTL, to better evaluate the structural features responsible for viral pathogenicity. ORF8 exists as a homodimer held together by amino acid residues that interact through hydrophobic interactions, hydrogen bonding, a salt bridge, and a disulfide bond. These interactions occur in a region referred to as the covalent interface. Another interface, referred to as the noncovalent interface, exists between separate homodimers and results in oligomerization. The homodimers in the oligomer are held together by an intermolecular beta sheet that is stabilized by an extensive hydrophobic surface. These two dimerization interfaces are unique to SARS‐CoV‐2 and have been proposed to be involved in the protein's ability to evade and suppress the host immune response. The computer and physical representations allow for better visualization of the ORF8 structure to evaluate the role of the multimeric structure. ORF8 has been found to interact with various host proteins, including Interleukin‐17 Receptor A (IL17RA), a pro‐inflammatory cytokine. To further corroborate the existence of this interaction and to better understand the role oligomerization might play in pathogenicity, we assessed the possible interactions of ORF8 (7JTL) and IL17RA (5N9B) through theoretical modeling using available tools such as HDOCK. A better understanding of these inter‐subunit interactions may improve our understanding of the unique mechanism the virus uses to evade the immune system and may be instrumental in the development of effective therapeutics.

Influence of thermal processing on the physicochemical properties of bovine lactoferrin

Aqueous solutions of lactoferrin (LF) were subjected to thermal processing (72, 80, 85, 95 °C for 15 s) using a rheometer. Despite the low protein concentration and low ionic strength of the LF solutions, analyses post-processing revealed the presence of heat-induced, covalently-linked, protein aggregates. Aggregation was accompanied by increased surface hydrophobicity and zeta-potential and changes to the secondary structure of the protein, including reduction in the proportion of alpha helix domains coupled with increased intermolecular beta sheet structures. The native red colour of the protein was lost upon thermal processing, not attributable to the release of LF-bound iron. Micro-differential scanning calorimetry showed LF to be irreversibly denatured on heating with endotherms observed at 58.7 °C (apo-LF) and 89.6 °C (holo-LF). Thermal processing at 72–95 °C resulted in irreversible changes to the structural and physicochemical properties of bovine LF, whereby the lowest heating intensity had the least impact on LF structure.

Conformational properties of intrinsically disordered proteins bound to the surface of silica nanoparticles

BackgroundProtein-nanoparticle (NP) interactions dictate properties of nanoconjugates relevant to bionanotechnology. Non-covalent adsorption generates a protein corona (PC) formed by an inner and an outer layer, the hard and soft corona (HC, SC). Intrinsically disordered proteins (IDPs) exist in solution as conformational ensembles, whose response to the presence of NPs is not known. MethodsThree IDPs (α-casein, Sic1 and α-synuclein) and lysozyme are compared, describing conformational properties inside HC on silica NPs by circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy. ResultsIDPs inside HC are largely unstructured, but display small, protein-specific conformational changes. A minor increase in helical content is observed for α-casein and α-synuclein, reminiscent of membrane effects on α-synuclein. Frozen in their largely disordered conformation, bound proteins do not undergo folding induced by dehydration, as they do in their free forms. While HC thickness approaches the hydrodynamic diameter of the protein in solution for lysozyme, it is much below the respective values for IDPs. NPs boost α-synuclein aggregation kinetics in a dose-dependent manner. ConclusionsIDPs maintain structural disorder inside HC, experiencing minor, protein-specific, induced folding and stabilization against further conformational transitions, such as formation of intermolecular beta-sheets upon dehydration. The HC is formed by a single layer of protein molecules. SC likely plays a key role stabilizing amyloidogenic α-synuclein conformers. General significanceProtein-NP interactions can mimic those with macromolecular partners, allowing dissection of contributing factors by rational design of NP surfaces. Application of NPs in vivo should be carefully tested for amyloidogenic potential.

Silk Fibroin-Sophorolipid Gelation: Deciphering the Underlying Mechanism.

Silk fibroin (SF) protein, produced by silkworm Bombyx mori, is a promising biomaterial, while sophorolipid (SL) is an amphiphilic functional biosurfactant synthesized by nonpathogenic yeast Candida bombicola. SL is a mixture of two forms, acidic (ASL) and lactonic (LSL), which when added to SF results in accelerated gelation of silk fibroin. LSL is known to have multiple biological functionalities and hence hydrogels of these green molecules have promising applications in the biomedical sector. In this work, SANS, NMR, and rheology are employed to examine the assembling properties of individual and mixed SLs and their interactions with SF to understand the mechanism that leads to rapid gelation. SANS and NMR studies show that ASL assembles to form charged micelles, while LSL forms micellar assemblies and aggregates of a mass fractal nature. ASL and LSL together form larger mixed micelles, all of which interact differently with SF. It is shown that preferential binding of LSL to SF causes rapid unfolding of the SF chain leading to the formation of intermolecular beta sheets, which trigger fast gelation. Based on the observations, a mechanism for gelation of SF in the presence of different sophorolipids is proposed.

RAS/Effector Interactions from Structural and Biophysical Perspective.

RAS is a molecular switch that regulates a large number of pathways through interactions with many effector proteins. Most RAS/effector complexes are short-lived, demonstrating fast association and fast dissociation rate and Kds ranging from 10(-8)-10(-5) M, compatible with the signaling function of these interactions in the cell. RAS effectors share little sequence homology but all contain an RAS binding domain that exhibits ubiquitin fold. All effectors bind to the same epitope on RAS by forming an intermolecular beta sheet and creating a number of favorable hydrogen bonds and salt bridges across the binding interface. Several hot-spots on both RAS and effector molecules constitute a general recognition mode. RAS/effector interactions occur only when RAS is found in the active, GTP-bound state, and are disrupted upon GTP hydrolysis, most probably due to increased flexibility of the RAS molecule. Recent NMR studies demonstrate how in the presence of multiple binding partners, RAS prefers certain effectors to others. The hierarchy of these interactions could be altered for RAS oncogenic mutants, thus perturbing the network of the downstream signaling. Insights obtained through biophysical and structural studies of effectors interacting with RAS and its mutants establish the basic principles that could be used for designing drugs in RAS-associated diseases.

Global and Local Conformational Heterogeniety Governs the Pre-Nucleation Phase in Amyloidogenic Self-Assembly

The formation of amyloid fibrils has been found to be a nucleation-dependent reaction for which the emergence of oligomeric entities is an inevitable and thus presumably, a critical intermediate step in the filamentous growth process. So far it has been not straight-forward to translate the extensive structural knowledge gained for the stable, fibrillar and crystalline states to the soluble oligomers prevalent during the pre-nucleation phase of the multi-step aggregation pathways. In this contribution we investigate the dynamical and structural properties of pre-brillar oligomeric states at atomistic resolution for several amyloidogenic peptide and protein fragments using unbiased explicit solvent molecular dynamics (MD) simulations. We have examined in particular the following questions: Which common secondary and teriary structural characteristics can be found in low molecular weight amyloid oligomers? What role do self-complentary side chain interfaces of intermolecular beta-sheets play in the oligomerization process? We analyzed simulations using multiple force fields in a consensus approach, in addition the results are compared in the light of recent experimental measurements done on model systems for peptide oligomerization. An extensive collision cross section analysis revealed heterogeneous oligomeric aggregates that lack a single, defined tertiary structure during the pre-nucleation phase. To gain insight into the structural organization of the early aggregates spontaneously formed on the microsecond time scale, we probed the substructure of the oligomers and found an increased ordering of intra-molecular conformations and inter-molecular packing motifs with growing aggregate size. We furthermore found that the ensemble of ordered higher-order oligomers sampled a multitude of common beta-structure motifs and was composed of emerging beta-sheet subunit layers. Our results suggest that distinct fibril-like conformations emerge in the oligomeric state and underscore the notion that certain pre-nucleated oligomers serve as a critical intermediate step on-pathway to fibrils.

Cross-linking in the silks of bees, ants and hornets

Silk production is integral to the construction of nests or cocoons for many Aculeata, stinging Hymenopterans such as ants, bees and wasps. Here we report the sequences of new aculeate silk proteins and compare cross-linking among nine native silks from three bee species (Apis mellifera, Bombus terrestris and Megachile rotundata), three ant species (Myrmecia forficata, Oecophylla smaragdina and Harpegnathos saltator) and three hornets (Vespa analis, Vespa simillima and Vespa mandarinia). The well studied silks of spiders and silkworms are comprised of large proteins that are cross-linked and stabilized predominantly by intra and intermolecular beta sheet structure. In contrast, the aculeate silks are comprised of relatively small proteins that contain central coiled coil domains and comparatively reduced amounts of beta sheet structure. The hornet silks, which have the most beta sheet structure and the greatest amount of amino acid sequence outside the coiled-coil domains, dissolve in concentrated LiBr solution and appear to be stabilized predominantly by beta sheet structure like the classic silks. In contrast, the ant and bee silks, which have less beta sheet and less sequence outside the coiled-coil domains, could not be dissolved in LiBr and appear to be predominantly stabilized by covalent cross-linking. The iso-peptide cross-linker, ε-(γ-glutamyl)-lysine that is produced by transglutaminase enzymes, was demonstrated to be present in all silks by mass spectrometry, but at greater levels in silks of ants and bees. The bee silks and ant cocoons, but not the Oecophylla nest silks, appeared to be further stabilized by tanning reactions.

Structural Characterization of IgG1 mAb Aggregates and Particles Generated Under Various Stress Conditions

IgG1 mAb solutions were prepared with and without sodium chloride and subjected to different environmental stresses. Formation of aggregates and particles of varying size was monitored by a combination of size-exclusion chromatography, Nanoparticle Tracking Analysis, Micro-flow Imaging (MFI), turbidity, and visual assessments. Stirring and heating induced the highest concentration of particles. In general, the presence of NaCl enhanced this effect. The morphology of the particles formed from mAb samples exposed to different stresses was analyzed from transmission electron microscopy and MFI images. Shaking samples without NaCl generated the most fibrillar particles, whereas stirring created largely spherical particles. The composition of the particles was evaluated for covalent cross-linking by SDS-PAGE, overall secondary structure by FTIR microscopy, and surface apolarity by extrinsic fluorescence spectroscopy. Freeze-thaw and shaking led to particles containing protein with native-like secondary structure. Heating and stirring produced IgG1-containing aggregates and particles with some non-native disulfide cross-links, varying levels of intermolecular beta sheet content, and increased surface hydrophobicity. These results highlight the importance of evaluating protein particle morphology and composition, in addition to particle number and size distributions, to better understand the effect of solution conditions and environmental stresses on the formation of protein particles in mAb solutions.

Study of the denaturation/aggregation behaviour of whole porcine plasma and its protein fractions during heating under acidic pH by variable-temperature FTIR spectroscopy

Variable-temperature (VT) Fourier transform infrared (FTIR) spectroscopy was employed to examine changes in secondary structure of whole plasma proteins as well as of plasma protein fractions (serum, serum albumin and globulins) upon heating at pH 4.5 and to establish their kinetics of thermally induced protein aggregation through formation of non-native intermolecular beta-sheets. A detailed analysis of the amide I′ band in the VT-FTIR spectra indicated that plasma proteins were more thermally sensitive at pH 4.5 than at pH 7.5 both when found as mixtures and in monomolecular systems, with the thermal aggregation being strongly enhanced under acidic conditions, particularly in the case of serum albumin. Comparison of the spectral changes of plasma and serum (fibrinogen-depleted plasma) during heating indicated that fibrinogen has no role in protein aggregation under acidic conditions, in contrast to findings at pH 7.5. Considering the particular characteristics of the different plasma proteins, the strong predominance of positive charges in the plasma as a whole at pH 4.5 along with the effects of these pH conditions on the conformation of fibrinogen could be suggested as the main factors responsible for the lack of a contribution by fibrinogen to protein aggregation. Moreover, 2D correlation spectroscopy indicated that the sequence of structural changes occurring during heating was practically identical among the different protein fractions examined and completely different from that established at pH 7.5, with the native beta-sheets being now more heat-sensitive than the alpha-helical structures and with protein aggregation through the formation of intermolecular beta-sheets beginning after native beta-sheets started to unfold.

Disappearance of intermolecular beta-sheets upon adsorption of beta-lactoglobulin aggregates at the oil–water interfaces of emulsions

Native proteins usually undergo structural modification upon adsorption at interface. Heat treatments are commonly applied at the industrial scale and lead to aggregation of proteins. We characterized nanometric aggregates of β-lactoglobulin by infrared spectroscopy in solutions, in hexadecane oil-in-water emulsions and at the air–water interface at low and high (0.1 M) ionic strengths and at pH 7. In solutions, on the contrary to native β-lactoglobulin, all aggregates prepared with or without salt possessed intermolecular β-sheets evidenced by two strong absorption bands at 1614 cm−1 and 1682 cm−1. In emulsions, at low ionic strength, they lose their intermolecular β-sheets once they are adsorbed at the oil–water interface. At high ionic strength, most of aggregates are localized at the interfaces where they lose their intermolecular β-sheets in direct contact with the surface and only partially when they are farther from the interface. The loss of intermolecular β-sheets was similarly observed at the air–liquid interface.

Evaluating the Consequences of Adsorption-Induced Structural Perturbations of Proteins

Maintaining the stability of a biologically-active therapeutic agent throughout its lifecycle is a critical parameter in successful pharmaceutical formulation. Interfacial effects such as surface adsorption, which can occur during drug storage and delivery, may culminate in various forms of instabilities: protein unfolding, activity loss and population of non-native, aggregate-prone states. A central paradigm that underpins our understanding of proteins with solid surfaces is that protein adsorption leads to changes in secondary structure. Bound proteins tend to denature and these non-native, adsorbed structures are likely stabilized by loss of alpha-helices and concomitant formation of intermolecular beta-sheets. This research seeks to critically assess the impact this behavior has on protein desorption, where irreversible conformational change may lead to aggregation or other forms of instability. We employ a robust study design to examine the kinetics of adsorption, desorption, and structural transitions of lysozyme on fumed silica nanoparticles as a function of surface coverage. We use circular dichroism (CD) spectroscopy to monitor structural transitions on the surface, in situ. The results show that despite significant adsorption-induced structural loss, surprisingly, adsorption is reversible, and protein desorption is predictable in a coverage-dependent manner. We find evidence of a two-state model, involving exchange between a native-like dissolved and highly perturbed adsorbed state. Since the in situ circular dichroism (CD) derived secondary structure of the adsorbed proteins are essentially unaffected by changes in surface coverage, these results are not consistent with previous claims that surface-induced denaturation is coverage dependent. Inspired by results from homopolymer adsorption experiments, we speculate that more local descriptors, such as the number of amino acids per chain that are physically adsorbed on the surface, likely control the desorption process.

Unfolded annealing molecular dynamics conformers for wild-type and disease-associated variants of alpha-synuclein show no propensity for beta-sheetformation

Aggregation of alpha-synuclein leads to the formation of Lewy bodies in the brains of patients affected by Parkinson's disease (PD). Native human alpha-synuclein is unfolded in solution but assumes a partial alpha-helical conformation upon transient binding to lipid membranes. Annealing Molecular Dynamics (AMD) was used to generate a diverse set of unfolded conformers of free monomeric wild-type alpha-synuclein and PD-associated mutants A30P and A53T. The AMD conformers were compared in terms of secondary structure, hydrogen bond network, solvent-accessible surface per residue, and molecular volume. The objective of these simulations was to identify structural properties near mutation sites and the non-amyloid component (NAC) region that differ between wild- type and disease-associated variants and may be associated to aggregation of alpha- synuclein. Based on experimental evidence, a hypothesis exists that aggregation involves the formation of intermolecular beta sheets. According to our results, disease-associated mutants of alpha-synuclein are no more propense to contain extended beta regions than wild-type alpha-synuclein. Moreover, extended beta structures (necessary for beta sheet formation) were not found at or around positions 30 and 53, or the NAC region in any unfolded conformer of wild-type, A30P or A53T alpha-synuclein, under the conditions of the simulations. These results do not support the hypothesis that the mutant's higher propensity to aggregation results solely from changes in amino acid sequence leading to changes in secondary structure folding propensity.

Polycomb Group Targeting through Different Binding Partners of RING1B C-Terminal Domain

RING1B, a Polycomb Group (PcG) protein, binds methylated chromatin through its association with another PcG protein called Polycomb (Pc). However, RING1B can associate with nonmethylated chromatin suggesting an alternate mechanism for RING1B interaction with chromatin. Here, we demonstrate that two proteins with little sequence identity between them, the Pc cbox domain and RYBP, bind the same surface on the C-terminal domain of RING1B (C-RING1B). Pc cbox and RYBP each fold into a nearly identical, intermolecular beta sheet with C-RING1B and a loop structure which are completely different in the two proteins. Both the beta sheet and loop are required for stable binding and transcription repression. Further, a mutation engineered to disrupt binding on the Drosophila dRING1 protein prevents chromatin association and PcG function in vivo. These results suggest that PcG targeting to different chromatin locations relies, in part, on binding partners of C-RING1B that are diverse in sequence and structure.

Structural Basis of Selective Ubiquitination of TRF1 by SCFFbx4

TRF1 is a critical regulator of telomere length. As such, TRF1 levels are regulated by ubiquitin-dependent proteolysis via an SCF E3 ligase where Fbx4 contributes to substrate specification. Here, we report the crystal structure of the Fbx4-TRF1 complex at 2.4 A resolution. Fbx4 contains an unusual substrate-binding domain that adopts a small GTPase fold. Strikingly, this atypical GTPase domain of Fbx4 binds to a globular domain of TRF1 through an intermolecular beta sheet, instead of recognizing short peptides/degrons as often seen in other F-box protein-substrate complexes. Importantly, mutations in this interface abrogate Fbx4-dependent TRF1 binding and ubiquitination. Furthermore, the data demonstrate that recognition of TRF1 by SCF(Fbx4) is regulated by another telomere protein, TIN2. Our results reveal an atypical small GTPase domain within Fbx4 as a substrate-binding motif for SCF(Fbx4) and uncover a mechanism for selective ubiquitination and degradation of TRF1 in telomere homeostasis control.

Multiple Evolutionary Mechanisms Reduce Protein Aggregation

The folding of polypeptides into stable globular protein structures requires protein sequences with a relatively high hydrophobicity and secondary structure propensity. These biophysical properties, however, also favor protein aggregation via the formation of intermolecular beta-sheets and, as a result, globular structure and aggregation are inextricable properties of protein polypeptides. Aggregates that are enriched in beta-sheet structures have been found in diseased tissues in association with at least twenty different human disorders and the effect of aggregation on protein function include simple loss-of-function but also often a gain of toxicity. Given both the ubiquity and the potentially lethal consequences of protein aggregation, negative selective pressure strongly minimizes aggregation. Various evolutionary strategies keep aggregation in check, including (1) the optimisation of the thermodynamic stability of the protein, which precludes aggregation by burial of the aggregation prone regions in solvent inaccessible regions of the structure, (2) segregation between folding nuclei and aggregation nuclei within a protein sequence, (3) the placement of so-called gatekeeper residues at the flanks of aggregating segments, that reduce the aggregation rate of (partially) unfolded proteins, and (4) molecular chaperones that target aggregation nucleating sequences directly, thereby further suppressing aggregation in a cellular environment. In this review we describe the intrinsic features built into protein sequence and structure that protect against aggregation.

Conformational diversity in prion protein variants influences intermolecular β-sheet formation

A conformational transition of normal cellular prion protein (PrP(C)) to its pathogenic form (PrP(Sc)) is believed to be a central event in the transmission of the devastating neurological diseases known as spongiform encephalopathies. The common methionine/valine polymorphism at residue 129 in the PrP influences disease susceptibility and phenotype. We report here seven crystal structures of human PrP variants: three of wild-type (WT) PrP containing V129, and four of the familial variants D178N and F198S, containing either M129 or V129. Comparison of these structures with each other and with previously published WT PrP structures containing M129 revealed that only WT PrPs were found to crystallize as domain-swapped dimers or closed monomers; the four mutant PrPs crystallized as non-swapped dimers. Three of the four mutant PrPs aligned to form intermolecular beta-sheets. Several regions of structural variability were identified, and analysis of their conformations provides an explanation for the structural features, which can influence the formation and conformation of intermolecular beta-sheets involving the M/V129 polymorphic residue.