Ca2+ Binding Enhanced Mechanical Stability of an Archaeal Crystallin

Structural topology plays an important role in protein mechanical stability. Proteins with β-sandwich topology consisting of Greek key structural motifs, for example, I27 of muscle titin and ¹⁰FNIII of fibronectin, are mechanically resistant as shown by single-molecule force spectroscopy (SMFS). In proteins with β-sandwich topology, if the terminal strands are directly connected by backbone H-bonding then this geometry can serve as a “mechanical clamp”. Proteins with this geometry are shown to have very high unfolding forces. Here, we set out to explore the mechanical properties of a protein, M-crystallin, which belongs to β-sandwich topology consisting of Greek key motifs but its overall structure lacks the “mechanical clamp” geometry at the termini. M-crystallin is a Ca²⁺ binding protein from Methanosarcina acetivorans that is evolutionarily related to the vertebrate eye lens β and γ-crystallins. We constructed an octamer of crystallin, (M-crystallin)₈, and using SMFS, we show that M-crystallin unfolds in a two-state manner with an unfolding force ∼90 pN (at a pulling speed of 1000 nm/sec), which is much lower than that of I27. Our study highlights that the β-sandwich topology proteins with a different strand-connectivity than that of I27 and ¹⁰FNIII, as well as lacking “mechanical clamp” geometry, can be mechanically resistant. Furthermore, Ca²⁺ binding not only stabilizes M-crystallin by 11.4 kcal/mol but also increases its unfolding force by ∼35 pN at the same pulling speed. The differences in the mechanical properties of apo and holo M-crystallins are further characterized using pulling speed dependent measurements and they show that Ca²⁺ binding reduces the unfolding potential width from 0.55 nm to 0.38 nm. These results are explained using a simple two-state unfolding energy landscape.     

Octarellin VI: Using Rosetta to Design a Putative Artificial (β/α)8 Protein

The computational protein design protocol Rosetta has been applied successfully to a wide variety of protein engineering problems. Here the aim was to test its ability to design de novo a protein adopting the TIM-barrel fold, whose formation requires about twice as many residues as in the largest proteins successfully designed de novo to date. The designed protein, Octarellin VI, contains 216 residues. Its amino acid composition is similar to that of natural TIM-barrel proteins. When produced and purified, it showed a far-UV circular dichroism spectrum characteristic of folded proteins, with α-helical and β-sheet secondary structure. Its stable tertiary structure was confirmed by both tryptophan fluorescence and circular dichroism in the near UV. It proved heat stable up to 70°C. Dynamic light scattering experiments revealed a unique population of particles averaging 4 nm in diameter, in good agreement with our model. Although these data suggest the successful creation of an artificial α/β protein of more than 200 amino acids, Octarellin VI shows an apparent noncooperative chemical unfolding and low solubility.     

Classification of α-Helical Membrane Proteins Using Predicted Helix Architectures

Despite significant methodological advances in protein structure determination high-resolution structures of membrane proteins are still rare, leaving sequence-based predictions as the only option for exploring the structural variability of membrane proteins at large scale. Here, a new structural classification approach for α-helical membrane proteins is introduced based on the similarity of predicted helix interaction patterns. Its application to proteins with known 3D structure showed that it is able to reliably detect structurally similar proteins even in the absence of any sequence similarity, reproducing the SCOP and CATH classifications with a sensitivity of 65% at a specificity of 90%. We applied the new approach to enhance our comprehensive structural classification of α-helical membrane proteins (CAMPS), which is primarily based on sequence and topology similarity, in order to find protein clusters that describe the same fold in the absence of sequence similarity. The total of 151 helix architectures were delineated for proteins with more than four transmembrane segments. Interestingly, we observed that proteins with 8 and more transmembrane helices correspond to fewer different architectures than proteins with up to 7 helices, suggesting that in large membrane proteins the evolutionary tendency to re-use already available folds is more pronounced.     

Single-Molecule Studies on PolySUMO Proteins Reveal Their Mechanical Flexibility

Proteins with β-sandwich and β-grasp topologies are resistant to mechanical unfolding as shown by single-molecule force spectroscopy studies. Their high mechanical stability has generally been associated with the mechanical clamp geometry present at the termini. However, there is also evidence for the importance of interactions other than the mechanical clamp in providing mechanical stability, which needs to be tested thoroughly. Here, we report the mechanical unfolding properties of ubiquitin-like proteins (SUMO1 and SUMO2) and their comparison with those of ubiquitin. Although ubiquitin and SUMOs have similar size and structural topology, they differ in their sequences and structural contacts, making them ideal candidates to understand the variations in the mechanical stability of a given protein topology. We observe a two-state unfolding pathway for SUMO1 and SUMO2, similar to that of ubiquitin. Nevertheless, the unfolding forces of SUMO1 (∼130 pN) and SUMO2 (∼120 pN) are lower than that of ubiquitin (∼190 pN) at a pulling speed of 400 nm/s, indicating their lower mechanical stability. The mechanical stabilities of SUMO proteins and ubiquitin are well correlated with the number of interresidue contacts present in their structures. From pulling speed-dependent mechanical unfolding experiments and Monte Carlo simulations, we find that the unfolding potential widths of SUMO1 (∼0.51 nm) and SUMO2 (∼0.33 nm) are much larger than that of ubiquitin (∼0.19 nm), indicating that SUMO1 is six times and SUMO2 is three times mechanically more flexible than ubiquitin. These findings might also be important in understanding the functional differences between ubiquitin and SUMOs.     

Substrate recognition by the POTRA domains of TpsB transporter FhaC

Widespread in Gram-negative bacteria, the two-partner secretion (TPS) pathway mediates the secretion of large, β-helical 'TpsA' proteins with various functions. TpsA proteins harbour a conserved, N-proximal TPS domain essential for secretion. TpsB transporters specifically recognize their TpsA partners in the periplasm and mediate their translocation across the outer membrane through a hydrophilic channel. The FHA/FhaC pair of Bordetella pertussis represents a model TPS system. FhaC is composed of a β barrel preceded by two periplasmic POTRA domains in tandem. Here we show that both POTRAs are involved in FHA recognition. Surface plasmon resonance analyses indicated an interaction of micromolar affinity between the POTRAs and the TPS domain with fast association and dissociation steps, consistent with the transient character of this interaction in vivo. Major interaction sites in POTRAs correspond to hydrophobic grooves formed by a β sheet edge and the flanking α helix, well-suited to accommodate extended, amphipathic strands of the substrate and consistent with β augmentation. The initial recruitment of the TPS domain to POTRAs appears to be facilitated by electrostatic attractions. A domain corresponding to the first part of the repeat-rich central region of FHA is also recognized by the POTRAs, suggesting successive interactions in the course of secretion.     

Protein Stability and Dynamics Modulation: The Case of Human Frataxin

Frataxin (FXN) is an α/β protein that plays an essential role in iron homeostasis. Apparently, the function of human FXN (hFXN) depends on the cooperative formation of crucial interactions between helix α1, helix α2, and the C-terminal region (CTR) of the protein. In this work we quantitatively explore these relationships using a purified recombinant fragment hFXN90–195. This variant shows the hydrodynamic behavior expected for a monomeric globular domain. Circular dichroism, fluorescence, and NMR spectroscopies show that hFXN90–195 presents native-like secondary and tertiary structure. However, chemical and temperature induced denaturation show that CTR truncation significantly destabilizes the overall hFXN fold. Accordingly, limited proteolysis experiments suggest that the native-state dynamics of hFXN90–195 and hFXN90–210 are indeed different, being the former form much more sensitive to the protease at specific sites. The overall folding dynamics of hFXN fold was further explored with structure-based protein folding simulations. These suggest that the native ensemble of hFXN can be decomposed in at least two substates, one with consolidation of the CTR and the other without consolidation of the CTR. Explicit-solvent all atom simulations identify some of the proteolytic target sites as flexible regions of the protein. We propose that the local unfolding of CTR may be a critical step for the global unfolding of hFXN, and that modulation of the CTR interactions may strongly affect hFXN physiological function.     

Transmembrane Protein Alignment and Fold Recognition Based on Predicted Topology

Background

Although Transmembrane Proteins (TMPs) are highly important in various biological processes and pharmaceutical developments, general prediction of TMP structures is still far from satisfactory. Because TMPs have significantly different physicochemical properties from soluble proteins, current protein structure prediction tools for soluble proteins may not work well for TMPs. With the increasing number of experimental TMP structures available, template-based methods have the potential to become broadly applicable for TMP structure prediction. However, the current fold recognition methods for TMPs are not as well developed as they are for soluble proteins.

Methodology

We developed a novel TMP Fold Recognition method, TMFR, to recognize TMP folds based on sequence-to-structure pairwise alignment. The method utilizes topology-based features in alignment together with sequence profile and solvent accessibility. It also incorporates a gap penalty that depends on predicted topology structure segments. Given the difference between α-helical transmembrane protein (αTMP) and β-strands transmembrane protein (βTMP), parameters of scoring functions are trained respectively for these two protein categories using 58 αTMPs and 17 βTMPs in a non-redundant training dataset.

Results

We compared our method with HHalign, a leading alignment tool using a non-redundant testing dataset including 72 αTMPs and 30 βTMPs. Our method achieved 10% and 9% better accuracies than HHalign in αTMPs and βTMPs, respectively. The raw score generated by TMFR is negatively correlated with the structure similarity between the target and the template, which indicates its effectiveness for fold recognition. The result demonstrates TMFR provides an effective TMP-specific fold recognition and alignment method.     

Unfolding of the amyloid β-peptide central helix: mechanistic insights from molecular dynamics simulations

Alzheimer's disease (AD) pathogenesis is associated with formation of amyloid fibrils caused by polymerization of the amyloid β-peptide (Aβ), which is a process that requires unfolding of the native helical structure of Aβ. According to recent experimental studies, stabilization of the Aβ central helix is effective in preventing Aβ polymerization into toxic assemblies. To uncover the fundamental mechanism of unfolding of the Aβ central helix, we performed molecular dynamics simulations for wild-type (WT), V18A/F19A/F20A mutant (MA), and V18L/F19L/F20L mutant (ML) models of the Aβ central helix. It was quantitatively demonstrated that the stability of the α-helical conformation of both MA and ML is higher than that of WT, indicating that the α-helical propensity of the three nonpolar residues (18, 19, and 20) is the main factor for the stability of the whole Aβ central helix and that their hydrophobicity plays a secondary role. WT was found to completely unfold by a three-step mechanism: 1) loss of α-helical backbone hydrogen bonds, 2) strong interactions between nonpolar sidechains, and 3) strong interactions between polar sidechains. WT did not completely unfold in cases when any of the three steps was omitted. MA and ML did not completely unfold mainly due to the lack of the first step. This suggests that disturbances in any of the three steps would be effective in inhibiting the unfolding of the Aβ central helix. Our findings would pave the way for design of new drugs to prevent or retard AD.     

Engineered Bi-Histidine Metal Chelation Sites Map the Structure of the Mechanical Unfolding Transition State of an Elastomeric Protein Domain GB1

Determining the structure of the transition state is critical for elucidating the mechanism behind how proteins fold and unfold. Due to its high free energy, however, the transition state generally cannot be trapped and studied directly using traditional structural biology methods. Thus, characterizing the structure of the transition state that occurs as proteins fold and unfold remains a major challenge. Here, we report a novel (to our knowledge) method that uses engineered bi-histidine (bi-His) metal-binding sites to directly map the structure of the mechanical unfolding transition state of proteins. This method is adapted from the traditional ψ-value analysis, which uses engineered bi-His metal chelation sites to probe chemical (un)folding transition-state structure. The ϕ^M2+_U-value is defined as ΔΔG_‡-N/ΔΔG_U-N, which is the energetic effects of metal chelation by the bi-His site on the unfolding energy barrier (ΔG_‡-N) relative to its thermodynamic stability (ΔG_U-N) and can be used to obtain information about the transition state in the mutational site. As a proof of principle, we used the small protein GB1 as a model system and set out to map its mechanical unfolding transition-state structure. Using single-molecule atomic force microscopy and spectrofluorimetry, we directly quantified the effect of divalent metal ion binding on the mechanical unfolding free energy and thermodynamic stability of GB1, which allowed us to quantify ϕ^M2+_U-values for different sites in GB1. Our results enabled us to map the structure of the mechanical unfolding transition state of GB1. Within GB1’s mechanical unfolding transition state, the interface between force-bearing β-strands 1 and 4 is largely disrupted, and the first β-hairpin is partially disordered while the second β-hairpin and the α-helix remain structured. Our results demonstrate the unique application of ψ-value analysis in elucidating the structure of the transition state that occurs during the mechanical unfolding process, offering a potentially powerful new method for investigating the design of novel elastomeric proteins.     

Hierarchical Cascades of Instability Govern the Mechanics of Coiled Coils: Helix Unfolding Precedes Coil Unzipping

Coiled coils are a fundamental emergent motif in proteins found in structural biomaterials, consisting of α-helical secondary structures wrapped in a supercoil. A fundamental question regarding the thermal and mechanical stability of coiled coils in extreme environments is the sequence of events leading to the disassembly of individual oligomers from the universal coiled-coil motifs. To shed light on this phenomenon, here we report atomistic simulations of a trimeric coiled coil in an explicit water solvent and investigate the mechanisms underlying helix unfolding and coil unzipping in the assembly. We employ advanced sampling techniques involving steered molecular dynamics and metadynamics simulations to obtain the free-energy landscapes of single-strand unfolding and unzipping in a three-stranded assembly. Our comparative analysis of the free-energy landscapes of instability pathways shows that coil unzipping is a sequential process involving multiple intermediates. At each intermediate state, one heptad repeat of the coiled coil first unfolds and then unzips due to the loss of contacts with the hydrophobic core. This observation suggests that helix unfolding facilitates the initiation of coiled-coil disassembly, which is confirmed by our 2D metadynamics simulations showing that unzipping of one strand requires less energy in the unfolded state compared with the folded state. Our results explain recent experimental findings and lay the groundwork for studying the hierarchical molecular mechanisms that underpin the thermomechanical stability/instability of coiled coils and similar protein assemblies.     

Alternative function for the mitochondrial SAM complex in biogenesis of alpha-helical TOM proteins

The mitochondrial outer membrane contains two preprotein translocases: the general translocase of outer membrane (TOM) and the β-barrel–specific sorting and assembly machinery (SAM). TOM functions as the central entry gate for nuclear-encoded proteins. The channel-forming Tom40 is a β-barrel protein, whereas all Tom receptors and small Tom proteins are membrane anchored by a transmembrane α-helical segment in their N- or C-terminal portion. Synthesis of Tom precursors takes place in the cytosol, and their import occurs via preexisting TOM complexes. The precursor of Tom40 is then transferred to SAM for membrane insertion and assembly. Unexpectedly, we find that the biogenesis of α-helical Tom proteins with a membrane anchor in the C-terminal portion is SAM dependent. Each SAM protein is necessary for efficient membrane integration of the receptor Tom22, whereas assembly of the small Tom proteins depends on Sam37. Thus, the substrate specificity of SAM is not restricted to β-barrel proteins but also includes the majority of α-helical Tom proteins.     

Analysis of the LAGLIDADG interface of the monomeric homing endonuclease I-DmoI

The general structural fold of the LAGLIDADG endonuclease family consists of two similar α/β domains (αββαββα) that assemble either as homodimers or monomers with the domains related by pseudo-two-fold symmetry. At the center of this symmetry is the closely packed LAGLIDADG two-helix bundle that forms the main inter- or intra-molecular contact region between the domains of single- or double-motif proteins, respectively. In this work, we further examine the role of the LAGLIDADG residues involved in the helix–helix interaction. The interchangeability of the LAGLIDADG helix interaction was explored by grafting interfacial residues from the homodimeric I-CreI into the corresponding positions in the monomeric I-DmoI. The resulting LAGLIDADG exchange mutant is partially active, preferring to nick dsDNA rather than making the customary double-strand break. A series of partial revertants within the mutated LAGLIDADG region are shown to restore cleavage activity to varying degrees resulting in one I-DmoI mutant that is more active than wild-type I-DmoI. The phenotype of some of these mutants was reconciled on the basis of similarity to the GxxxG helix interaction found in transmembrane proteins. Additionally, a split variant of I-DmoI was created, demonstrating that the LAGLIDADG helices of I-DmoI are capable of forming and maintaining the protein–protein interface in trans to create an active heterodimer.     

SLLISWD Sequence in the 10FNIII Domain Initiates Fibronectin Fibrillogenesis

Fibronectin (FN) assembly into extracellular matrix is tightly regulated and essential to embryogenesis and wound healing. FN fibrillogenesis is initiated by cytoskeleton-derived tensional forces transmitted across transmembrane integrins onto RGD binding sequences within the tenth FN type III (10FNIII) domains. These forces unfold 10FNIII to expose cryptic FN assembly sites; however, a specific sequence has not been identified in 10FNIII. Our past steered molecular dynamics simulations modeling 10FNIII unfolding by force at its RGD loop predicted a mechanical intermediate with a solvent-exposed N terminus spanning the A and B β-strands. Here, we experimentally confirm that the predicted 23-residue cryptic peptide 1 (CP1) initiates FN multimerization, which is mediated by interactions with 10FNIII that expose hydrophobic surfaces that support 8-anilino-1-napthalenesulfonic acid binding. Localization of multimerization activity to the C terminus led to the discovery of a minimal 7-amino acid “multimerization sequence” (SLLISWD), which induces polymerization of FN and the clotting protein fibrinogen in addition to enhancing FN fibrillogenesis in fibroblasts. A point mutation at Trp-6 that reduces exposure of hydrophobic sites for 8-anilino-1-napthalenesulfonic acid binding and β-structure formation inhibits FN multimerization and prevents physiological cell-based FN assembly in culture. We propose a model for cell-mediated fibrillogenesis whereby cell traction force initiates a cascade of intermolecular exchange starting with the unfolding of 10FNIII to expose the multimerization sequence, which interacts with strand B of another 10FNIII domain via a Trp-mediated β-strand exchange to stabilize a partially unfolded intermediate that propagates FN self-assembly.     

Pivotal Role of Extended Linker 2 in the Activation of Gα by G Protein-coupled Receptor

G protein-coupled receptors (GPCRs) relay extracellular signals mainly to heterotrimeric G-proteins (Gαβγ) and they are the most successful drug targets. The mechanisms of G-protein activation by GPCRs are not well understood. Previous studies have revealed a signal relay route from a GPCR via the C-terminal α5-helix of Gα to the guanine nucleotide-binding pocket. Recent structural and biophysical studies uncover a role for the opening or rotating of the α-helical domain of Gα during the activation of Gα by a GPCR. Here we show that β-adrenergic receptors activate eight Gα_s mutant proteins (from a screen of 66 Gα_s mutants) that are unable to bind Gβγ subunits in cells. Five of these eight mutants are in the αF/Linker 2/β2 hinge region (extended Linker 2) that connects the Ras-like GTPase domain and the α-helical domain of Gα_s. This extended Linker 2 is the target site of a natural product inhibitor of G_q. Our data show that the extended Linker 2 is critical for Gα activation by GPCRs. We propose that a GPCR via its intracellular loop 2 directly interacts with the β₂/β₃ loop of Gα to communicate to Linker 2, resulting in the opening and closing of the α-helical domain and the release of GDP during G-protein activation.     

The solution structure of the amino-terminal domain of human DNA polymerase epsilon subunit B is homologous to C-domains of AAA+ proteins

DNA polymerases α, δ and ε are large multisubunit complexes that replicate the bulk of the DNA in the eukaryotic cell. In addition to the homologous catalytic subunits, these enzymes possess structurally related B subunits, characterized by a carboxyterminal calcineurin-like and an aminoproximal oligonucleotide/oligosaccharide binding-fold domain. The B subunits also share homology with the exonuclease subunit of archaeal DNA polymerases D. Here, we describe a novel domain specific to the N-terminus of the B subunit of eukaryotic DNA polymerases ε. The N-terminal domain of human DNA polymerases ε (Dpoe2NT) expressed in Escherichia coli was characterized. Circular dichroism studies demonstrated that Dpoe2NT forms a stable, predominantly α-helical structure. The solution structure of Dpoe2NT revealed a domain that consists of a left-handed superhelical bundle. Four helices are arranged in two hairpins and the connecting loops contain short β-strand segments that form a short parallel sheet. DALI searches demonstrated a striking structural similarity of the Dpoe2NT with the α-helical subdomains of ATPase associated with various cellular activity (AAA+) proteins (the C-domain). Like C-domains, Dpoe2NT is rich in charged amino acids. The biased distribution of the charged residues is reflected by a polarization and a considerable dipole moment across the Dpoe2NT. Dpoe2NT represents the first C-domain fold not associated with an AAA+ protein.     

Surface Tensiometry of Apolipoprotein B Domains at Lipid Interfaces Suggests a New Model for the Initial Steps in Triglyceride-rich Lipoprotein Assembly

Apolipoprotein B (apoB) is the principal protein component of triacylglyceride (TAG)-rich lipoproteins, including chylomicrons and very low density lipoprotein, which is the precursor to LDL (the “bad cholesterol”). TAG-rich lipoprotein assembly is initiated by the N-terminal βα1 superdomain of apoB, which co-translationally binds and remodels the luminal leaflet of the rough endoplasmic reticulum. The βα1 superdomain contains four domains and is predicted to interact directly with lipids. Using drop tensiometry, we examined the interfacial properties of the α-helical and C-sheet domains and several subdomains to establish a detailed structure-function relationship at the lipid/water interface. The adsorption, stress response, exchangeability, and pressure (Π)-area relationship were studied at both triolein/water and triolein/1-palmitoyl, 2-oleoylphosphatidylcholine/water interfaces that mimic physiological environments. The α-helical domain spontaneously adsorbed to a triolein/water interface and formed a viscoelastic surface. It was anchored to the surface by helix 6, and the other helices were ejected and/or remodeled on the surface as a function of surface pressure. The C-sheet instead formed an elastic film on a triolein/water interface and was irreversibly anchored to the lipid surface, which is consistent with the behavior of amphipathic β-strands. When both domains were adsorbed together on the surface, the C-sheet shielded a portion of the α-helical domain from the surface, which retained its globular structure. Overall, the unique secondary and tertiary structures of the N-terminal domains of apoB support the intrinsic capability of co-translational lipid recruitment. The evidence presented here allows the construction of a detailed model of the initiation of TAG-rich lipoprotein assembly.     

Stability of bacteriorhodopsin alpha-helices and loops analyzed by single-molecule force spectroscopy

The combination of high-resolution atomic force microscopy imaging and single-molecule force spectroscopy allows the identification, selection, and mechanical investigation of individual proteins. In a recent paper we had used this technique to unfold and extract single bacteriorhodopsins (BRs) from native purple membrane patches. We show that subsets of the unfolding spectra can be classified and grouped to reveal detailed insight into the individualism of the unfolding pathways. We have further developed this technique and analysis to report here on the influence of pH effects and local mutations on the stability of individual structural elements of BR against mechanical unfolding. We found that, although the seven transmembrane alpha-helices predominantly unfold in pairs, each of the helices may also unfold individually and in some cases even only partially. Additionally, intermittent states in the unfolding process were found, which are associated with the stretching of the extracellular loops connecting the alpha-helices. This suggests that polypeptide loops potentially act as a barrier to unfolding and contribute significantly to the structural stability of BR. Chemical removal of the Schiff base, the covalent linkage of the photoactive retinal to the helix G, resulted in a predominantly two-step unfolding of this helix. It is concluded that the covalent linkage of the retinal to helix G stabilizes the structure of BR. Trapping mutant D96N in the M state of the proton pumping photocycle did not affect the unfolding barriers of BR.     

Mechanical Properties of β-Catenin Revealed by Single-Molecule Experiments

β-catenin is a central component of the adaptor complex that links cadherins to the actin cytoskeleton in adherens junctions and thus, it is a good candidate to sense and transmit mechanical forces to trigger specific changes inside the cell. To fully understand its molecular physiology, we must first investigate its mechanical role in mechanotransduction within the cadherin system. We have studied the mechanical response of β-catenin to stretching using single-molecule force spectroscopy and molecular dynamics. Unlike most proteins analyzed to date, which have a fixed mechanical unfolding pathway, the β-catenin armadillo repeat region (ARM) displays low mechanostability and multiple alternative unfolding pathways that seem to be modulated by its unstructured termini. These results are supported by steered molecular dynamics simulations, which also predict its mechanical stabilization and unfolding pathway restrictions when the contiguous α-helix of the C-terminal unstructured region is included. Furthermore, simulations of the ARM/E-cadherin cytosolic tail complex emulating the most probable stress geometry occurring in vivo show a mechanical stabilization of the interaction whose magnitude correlates with the length of the stretch of the cadherin cytosolic tail that is in contact with the ARM region.     

The presequence pathway is involved in protein sorting to the mitochondrial outer membrane

The mitochondrial outer membrane contains integral α-helical and β-barrel proteins that are imported from the cytosol. The machineries importing β-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane. We show that import of Om45 occurs via the translocase of the outer membrane and the presequence translocase of the inner membrane. Assembly of Om45 in the outer membrane involves the MIM machinery. Om45 thus follows a new mitochondrial biogenesis pathway that uses elements of the presequence import pathway to direct a protein to the outer membrane.     

The N-terminal Helix Controls the Transition between the Soluble and Amyloid States of an FF Domain

Background

Protein aggregation is linked to the onset of an increasing number of human nonneuropathic (either localized or systemic) and neurodegenerative disorders. In particular, misfolding of native α-helical structures and their self-assembly into nonnative intermolecular β-sheets has been proposed to trigger amyloid fibril formation in Alzheimer’s and Parkinson’s diseases.

Methods

Here, we use a battery of biophysical techniques to elucidate the conformational conversion of native α-helices into amyloid fibrils using an all-α FF domain as a model system.

Results

We show that under mild denaturing conditions at low pH this FF domain self-assembles into amyloid fibrils. Theoretical and experimental dissection of the secondary structure elements in this domain indicates that the helix 1 at the N-terminus has both the highest α-helical and amyloid propensities, controlling the transition between soluble and aggregated states of the protein.

Conclusions

The data illustrates the overlap between the propensity to form native α-helices and amyloid structures in protein segments.

Significance

The results presented contribute to explain why proteins cannot avoid the presence of aggregation-prone regions and indeed use stable α-helices as a strategy to neutralize such potentially deleterious stretches.