Early Stages Of Folding Research Articles

Knotting and unknotting proteins in the chaperonin cage: Effects of the excluded volume.

Molecular dynamics simulations are used to explore the effects of chaperonin-like cages on knotted proteins with very low sequence similarity, different depths of a knot but with a similar fold, and the same type of topology. The investigated proteins are VirC2, DndE and MJ0366 with two depths of a knot. A comprehensive picture how encapsulation influences folding rates is provided based on the analysis of different cage sizes and temperature conditions. Neither of these two effects with regard to knotted proteins has been studied by means of molecular dynamics simulations with coarse-grained structure-based models before. We show that encapsulation in a chaperonin is sufficient to self-tie and untie small knotted proteins (VirC2, DndE), for which the equilibrium process is not accessible in the bulk solvent. Furthermore, we find that encapsulation reduces backtracking that arises from the destabilisation of nucleation sites, smoothing the free energy landscape. However, this effect can also be coupled with temperature rise. Encapsulation facilitates knotting at the early stage of folding and can enhance an alternative folding route. Comparison to unknotted proteins with the same fold shows directly how encapsulation influences the free energy landscape. In addition, we find that as the size of the cage decreases, folding times increase almost exponentially in a certain range of cage sizes, in accordance with confinement theory and experimental data for unknotted proteins.

The Folding Pathway of the KIX Domain.

The KIX domain is an 89-residues globular domain with an important role in mediating protein-protein interactions. The presence of two distinct binding sites in such a small domain makes KIX a suitable candidate to investigate the effect of the potentially divergent demands between folding and function. Here, we report an extensive mutational analysis of the folding pathway of the KIX domain, based on 30 site-directed mutants, which allow us to assess the structures of both the transition and denatured states. Data reveal that, while the transition state presents mostly native-like interactions, the denatured state is somewhat misfolded. We mapped some of the non-native contacts in the denatured state using a second round of mutagenesis, based on double mutant cycles on 15 double mutants. Interestingly, such a misfolding arises from non-native interactions involving the residues critical for the function of the protein. The results described in this work appear to highlight the diverging demands between folding and function that may lead to misfolding, which may be observed in the early stages of folding.

Using Molecular Dynamics Simulations to Understand Pattern Formation in Polymers

We use Molecular Dynamics simulations to study single chain polymer dynamics and collapse to imitate the early stages of protein folding, as well as polymer brush collapse, to understand the physics and morphology of collapsing polymer brushes. Simulations of this nature can help us understand complex phenomena such as glass transition, molecular crowding and complex higher order organisation. As an example, we demonstrate a study of the physical origin of the polymer glass transition from the point of view of marginal rigidity, which is achieved above a certain number of intermolecular contacts. We find that when the average number of contacts per monomer (covalent and non-covalent) exceeds the critical value z∗=4, the system becomes solid and the dynamics arrested - a state that we declare the glass. We also look at polymer brushes and describe spinodal decomposition-like processes that take place upon brush collapse. It is often impossible to simulate large systems without a certain level of sophisticated coarse graining. We introduce a possible model for studying higher order organisation of chromatin, a biological complex of DNA and histone proteins within the nucleus of a cell. Chromatin organisation is a fascinating topic that resembles protein folding, however, it is much more complex due to multiple levels at which chromatin folding and dynamics have to be understood. Using a simplified force field, we also study helical structures formed by peptoids, small peptidomimetic molecules that are of importance to the pharmacological industry.

The fracture patterns of the Tin Tin anticline: Fracturing process during the foreland evolution in the Calchaquí Valley, northwestern Argentina

We present a field-based work which illustrates the fracture patterns of the carbonate-silicoclastic Yacoraite Formation in the Tin Tin anticline, a basement fault-related fold located in southern part of the Eastern Cordillera, northwestern Argentina. The fracture patterns include small-scale strike-slip faults (vertical shear fractures and en echelon arrays), thrust faults, extension fractures (joints, veins and normal faults) and stylolites. Extensional mesostructures were formed by along-foreland stretching, prior to the contractional ones that were formed by the layer-parallel shortening mechanism. Furthermore, all fractures are interpreted to be formed before or at the early stages of folding and thrusting during the Andean contraction, all of them belonging to the Eocene thrust belt-foreland system at the Calchaquí Valley of northwestern Argentina.

Imprint of Ancient Evolution on rRNA Folding.

In a model describing the origin and evolution of the translation system, ribosomal RNA (rRNA) grew in size by accretion [Petrov, A. S., et al. (2015) History of the Ribosome and the Origin of Translation. Proc. Natl. Acad. Sci. U.S.A. 112, 15396-15401]. Large rRNAs were built up by iterative incorporation and encasement of small folded RNAs, in analogy with addition of new LEGOs onto the surface of a preexisting LEGO assembly. In this model, rRNA robustness in folding arises from inherited autonomy of local folding. We propose that rRNAs can be decomposed at various granularities, retaining folding mechanism and folding competence. To test these predictions, we disassembled Domain III of the large ribosomal subunit (LSU). We determined whether local rRNA structure, stability, and folding pathways are autonomous. Thermal melting, chemical footprinting, and circular dichroism were used to infer rules that govern folding of rRNA. We deconstructed Domain III of the LSU rRNA by mapping out its complex multistep melting pathway. We studied Domain III and two equal-size "sub-Domains" of Domain III. The combined results are consistent with a model in which melting transitions of Domain III are conserved upon cleavage into sub-Domains. Each of the eight melting transitions of Domain III corresponds in Tm and ΔH with a transition observed in one of the two isolated sub-Domains. The results support a model in which structure, stability, and folding mechanisms are dominated by local interactions and are unaffected by separation of the sub-Domains. Domain III rRNA is distinct from RNAs that form long-range cooperative interaction networks at early stages of folding or that do not fold reversibly.

Fracture system evolution within the Cardium sandstone, central Alberta Foothills folds

The natural fracture system developed in the Cardium sandstone is examined in four outcropping structures that represent different stages of fold development. At the incipient stage of folding, the fracture system is dominated by large, widely spaced hybrid fractures that have very small displacements and are aligned in the regional shortening direction (type I orientation). These fractures are naturally propped open by asperities along the fracture surfaces. A lesser number of small thrust faults (type III orientation) are also developed. Extension fractures aligned parallel to the fold axis (type II orientation) begin to develop in the early stage of folding. Through the intermediate stage of folding, there is a progressive increase in the intensity of both type I and type II orientation fractures. Incremental increases in shear displacement on new or reactivated fractures create a gouge of comminuted sandstone grains along the fracture interface. As folding progresses to an advanced stage, there is major increase in the amount of shear displacement on both type I and type II orientation fractures. Many existing fractures coalesce into connected fracture zones and small faults that have shear offsets ranging from several centimeters to several meters. A breccia can result from intense fracturing in the rock within and marginal to these shear features. Slickensides on type I orientation features consistently indicate slip in a subhorizontal direction, even as bed dip increases. Multiple slickenside patterns record reactivation of these features. Type II orientation fractures and small faults consistently undergo bed-perpendicular slip. Type I and type II features both serve to stretch the Cardium sandstone beds but in different directions. Only type III features, which are a minor component of the fracture population, result in bed thickening.

The structural history and mineralization controls of the world-class Geita Hill gold deposit, Geita Greenstone Belt, Tanzania

The Geita Hill gold deposit is located in the Archean Geita Greenstone Belt and is one of the largest gold deposits in East Africa. The Geita Greenstone Belt experienced a complex deformation and intrusive history that is well illustrated and preserved in and around the Geita Hill gold deposit. Deformation involved early stages of ductile shearing and folding (D1 to D5), during which episodic emplacement of large diorite intrusive complexes, sills, and dykes occurred. These ductile deformation phases were followed by the development of brittle-ductile shear zones and faults (D6 to D8). The last stages of deformation were accompanied by voluminous felsic magmatism involving the intrusion of felsic porphyry dykes, within the greenstone belt, and the emplacement of large granitic bodies now forming the margins of the greenstone belt. Early, folded lamprophyre dykes, and later lamprophyre dykes, crosscutting the folded sequence are common, although volumetrically insignificant. The gold deposit formed late during the tectonic history of the greenstone belt, post-dating ductile deformation and synchronous with the development of brittle-ductile shear zones that overprinted earlier structural elements. The main mineralizing process involved sulfide replacement of magnetite-rich layers in ironstone and locally the replacement of ferromagnesian phases and magnetite in the diorite intrusions. The intersection between the brittle-ductile (D6) Geita Hill Shear Zone and different structural elements of ductile origin (e.g., fold hinges), and the contact between banded ironstone and folded diorite dykes and sills provided the optimal sites for gold mineralization.

Nanomechanical z-shape folding of graphene on flat substrate

Graphene folding is an essential process in the design and manufacturing of graphene origami. Here we report the nanomechanical z-shape (accordion) folding of single graphene sheets on flat substrates by using atomic force microscopy techniques. The quantitative nanomechanical measurements in conjunction with nonlinear mechanics modeling and molecular dynamics simulations reveal that a reversible out-of-plane buckling delamination of graphene occurs in its early-stage folding process, which enables graphene to deform into a stable self-folded z-shape conformation. The research findings are useful to the study of active and controllable folding of graphene and in the pursuit of graphene origami with intricate geometries.

Early Vendian stage of folding in the Patom folded zone: Syn-folded clastic dikes in the Dalnetaiginsky Group, Central Siberia

This work describes clastic dikes which intruded along cleavage planes into Lower Vendian clay shales of the Barakun Formation (the eastern part of the Patom folded zone). Dikes are composed of sandstones, which are common in other parts of the Barakun Formation, or more often by thin intercalations of finely oolitic limestones in surrounding shales. It is evident that dikes were formed as a result of elision processes synchronous to the folding, the major system of cleavage planes, and dehydration of clayey rocks of the Dalnetaiginsky Group at the end of Early Vendian. This stage of folding can be considered as one of the first phases of the Baikal folding by N.S. Shatsky.

Frustration Sculpts the Early Stages of Protein Folding

AbstractThe funneled energy landscape theory implies that protein structures are minimally frustrated. Yet, because of the divergent demands between folding and function, regions of frustrated patterns are present at the active site of proteins. To understand the effects of such local frustration in dictating the energy landscape of proteins, here we compare the folding mechanisms of the two alternative spliced forms of a PDZ domain (PDZ2 and PDZ2as) that share a nearly identical sequence and structure, while displaying different frustration patterns. The analysis, based on the kinetic characterization of a large number of site‐directed mutants, reveals that although the late stages for folding are very robust and biased by native topology, the early stages are more malleable and dominated by local frustration. The results are briefly discussed in the context of the energy‐landscape theory.

Frustration Sculpts the Early Stages of Protein Folding.

The funneled energy landscape theory implies that protein structures are minimally frustrated. Yet, because of the divergent demands between folding and function, regions of frustrated patterns are present at the active site of proteins. To understand the effects of such local frustration in dictating the energy landscape of proteins, here we compare the folding mechanisms of the two alternative spliced forms of a PDZ domain (PDZ2 and PDZ2as) that share a nearly identical sequence and structure, while displaying different frustration patterns. The analysis, based on the kinetic characterization of a large number of site-directed mutants, reveals that although the late stages for folding are very robust and biased by native topology, the early stages are more malleable and dominated by local frustration. The results are briefly discussed in the context of the energy-landscape theory.

The Kinetics of Nascent Protein Folding upon Release from the Ribosome

Very little is known about the way proteins attain their native structure within the context of the living cell. In addition to the ribosome's well-established role in peptide bond formation, recent studies suggest that ribosomes play an important role in the early stages of protein folding in the cell and may be crucial for the production of folded bioactive proteins. Importantly, little is known about the impact of the mechanism of protein release from the ribosome on the attainment of a correctly folded conformation. Here, we present a kinetic study on the release time-course of fully synthesized ribosome-bound nascent proteins upon addition of the antibiotic puromycin. We focus these studies on the E. coli globin ApoHmpH. By time-resolved gel electrophoresis, we are able to follow puromycin's hydrolysis of the ester bond linking nascent polypeptides to the 3’ end of tRNA. Steady-state fluorescence anisotropy allows us to follow the escape and folding of ApoHmpH from the ribosome. Finally, time decay fluorescence anisotropy analysis in the frequency domain complements the above techniques by providing insights into the local motions experienced by the nascent protein before and after release from the ribosome. Under experimental conditions where puromycin reacts at rates comparable to the naturally occurring release factors, we show that protein release from the ribosome is rate-limited by the C-terminal ester bond cleavage, and that escape from the ribosome and completion of folding occur quickly following this step. This result shows that the ribosomal context promotes a particularly “temporally efficient” folding upon nascent protein release. An important consequence of this phenomenon is the prevention of undesirable diffusion- and concentration-dependent phenomena such as aggregation.

정전기적 상호작용이 단백질 폴딩 반응에 끼치는 영향

The contribution of electrostatic interactions to protein folding reaction was studied by using mutant ubiquitin with lysine to alanine mutation at residue position 29. Based on the three dimensional structure of ubiquitin, lysine 29 is located close to negatively charged glutamate 16 and aspartate 21 and considered to stabilize the native state of ubiquitin by electrostatic interactions between these residues. The equilibrium unfolding experiment showed that the native stability was decreased by about ~20% upon mutation. This observation indicates lysine 29 indeed forms electrostatic interactions with nearby residues. Folding kinetics measurements using stopped-flow device and quantitative analysis of kinetics data indicate that ubiquitin folds from unfolded state to native state via intermediate state as observed previously. This intermediate state was observed to form immediately after the initiation of folding reaction. The folding intermediate was shown to be destabi- lized considerably upon lysine to alanine mutation. These observations indicate that electrostatic interactions can form early stage of protein folding and hence lead the folding reaction.

Kinetics study on the HIV-1 ectodomain protein quaternary structure formation reveals coupling of chain folding and self-assembly in the refolding cascade.

Entry of HIV-1 into the target cell is mediated by the envelope glycoprotein consisting of noncovalently associated surface subunit gp120 and transmembrane subunit gp41. To form a functional gp41 complex, the protein undergoes hairpin formation and self-assembly. The fusion event can be inhibited by gp41-derived peptides at nanomolar concentration and is highly dependent on the time of addition, implying a role of folding kinetics on the inhibitory action. Oligomerization of the gp41 ectodomain was demonstrated by light scattering measurements. Kinetic study by stopped-flow fluorescence and absorption measurements (i) revealed a multistate folding pathway and stable intermediates; (ii) showed a dissection of fast and slow components for early and late stages of folding, respectively, with 3 orders of magnitude difference in the time scale; (iii) showed the slow process was attributed to misfolding and unzipping of the hairpin; and (iv) showed retardation of the native hairpin formation is assumed to lead to coupling of the correctly registered hairpin and self-assembly. This coupling allows the deduction on the time scale of intrachain folding (0.1-1 s) for the protein. The folding reaction was illustrated by a free energy profile to explain the temporal dichotomy of fast and slow steps of folding as well as effective inhibition by gp41-derived peptide.

Protein folding: Understanding the role of water and the low Reynolds number environment as the peptide chain emerges from the ribosome and folds

The mechanism of protein folding during early stages of the process has three determinants. First, moving water molecules obey the rules of low Reynolds number physics without an inertial component. Molecular movement is instantaneous and size insensitive. Proteins emerging from the ribosome move and rotate without an external force if they change shape, forming and propagating helical structures that increases translocational efficiency. Forward motion ceases when the shape change or propelling force ceases. Second, application of quantum field theory to water structure predicts the spontaneous formation of low density coherent units of fixed size that expel dissolved atmospheric gases. Structured water layers with both coherent and non-coherent domains, form a sheath around the new protein. The surface of exposed hydrophobic amino acids is protected from water contact by small nanobubbles of dissolved atmospheric gases, 5 or 6 molecules on average, that vibrate, attracting even widely separated resonating nanobubbles. This force results from quantum effects, appearing only when the system is within and interacts with an oscillating electromagnetic field. The newly recognized quantum force sharply bends the peptide and is part of a dynamic field determining the pathway of protein folding. Third, the force initiating the tertiary folding of proteins arises from twists at the position of each hydrophobic amino acid, that minimizes surface exposure of the hydrophobic amino acids and propagates along the protein. When the total bend reaches 360°, the leading segment of water sheath intersects the trailing segment. This steric self-intersection expels water from overlapping segments of the sheath and by Newton׳s second law moves the polypeptide chain in an opposite direction. Consequently, with very few exceptions that we enumerate and discuss, tertiary structures are absent from proteins without hydrophobic amino acids, which control the early stages of protein folding and the overall shape of protein. Consequently, proteins only adopt a limited number of forms. The formation of quaternary structures is not necessarily prevented by the absence of hydrophobic amino acids.

Effect of non-native helix destabilization on the folding of equine β-lactoglobulin.

β-lactoglobulin forms a non-native α-helix during an early stage of folding. To address the role of the non-native structure in the folding process, we designed several mutants of equine β-lactoglobulin with reduced helical propensity in the non-native helix region. One of them, A123T, showed a similar structure to that of the wild-type protein; its folding kinetics was investigated by stopped-flow circular dichroism (CD) and fluorescence. Although A123T showed a reduced burst-phase CD intensity, its folding rate was similar to that of the wild-type protein, which indicated that the formation of the non-native helix does not accelerate or decelerate the folding reaction.

First Passage Analysis of the Folding of a β-Sheet Miniprotein: Is it More Realistic Than the Standard Equilibrium Approach?

Simulations of first-passage folding of the antiparallel β-sheet miniprotein beta3s, which has been intensively studied under equilibrium conditions by A. Caflisch and co-workers, show that the kinetics and dynamics are significantly different from those for equilibrium folding. Because the folding of a protein in a living system generally corresponds to the former (i.e., the folded protein is stable and unfolding is a rare event), the difference is of interest. In contrast to equilibrium folding, the Ch-curl conformations become very rare because they contain unfavorable parallel β-strand arrangements, which are difficult to form dynamically due to the distant N- and C-terminal strands. At the same time, the formation of helical conformations becomes much easier (particularly in the early stage of folding) due to short-range contacts. The hydrodynamic descriptions of the folding reaction have also revealed that while the equilibrium flow field presented a collection of local vortices with closed ”streamlines”, the first-passage folding is characterized by a pronounced overall flow from the unfolded states to the native state. The flows through the locally stable structures Cs-or and Ns-or, which are conformationally close to the native state, are negligible due to detailed balance established between these structures and the native state. Although there are significant differences in the general picture of the folding process from the equilibrium and first-passage folding simulations, some aspects of the two are in agreement. The rate of transitions between the clusters of characteristic protein conformations in both cases decreases approximately exponentially with the distance between the clusters in the hydrogen bond distance space of collective variables, and the folding time distribution in the first-passage segments of the equilibrium trajectory is in good agreement with that for the first-passage folding simulations.

Relationship between chain collapse and secondary structure formation in a partially folded protein.

Chain collapse and secondary structure formation are frequently observed during the early stages of protein folding. Is the chain collapse brought about by interactions between secondary structure units or is it due to polymer behavior in a poor solvent (coil-globule transition)? To answer this question, we measured small-angle X-ray scattering for a series of β-lactoglobulin mutants under conditions in which they assume a partially folded state analogous to the folding intermediates. Mutants that were designed to disrupt the secondary structure units showed the gyration radii similar to that of the wild type protein, indicating that chain collapse is due to coil-globule transitions.

Transient non-native helix formation during the folding of β-lactoglobulin.

In ideal proteins, only native interactions are stabilized step-by-step in a smooth funnel-like energy landscape. In real proteins, however, the transient formation of non-native structures is frequently observed. In this review, the transient formation of non-native structures is described using the non-native helix formation during the folding of β-lactoglobulin as a prominent example. Although β-lactoglobulin is a predominantly β-sheet protein, it has been shown to form non-native helices during the early stage of folding. The location of non-native helices, their stabilization mechanism, and their role in the folding reaction are discussed.

Ultrafast Hydrogen Exchange Reveals Specific Structural Events during the Initial Stages of Folding of Cytochrome c

Many proteins undergo a sharp decrease in chain dimensions during early stages of folding, prior to the rate-limiting step in folding. However, it remains unclear whether compact states are the result of specific folding events or a general hydrophobic collapse of the poly peptide chain driven by the change in solvent conditions. To address this fundamental question, we extended the temporal resolution of NMR-detected H/D exchange labeling experiments into the microsecond regime by adopting a microfluidics approach. By observing the competition between H/D exchange and folding as a function of labeling pH, coupled with direct measurement of exchange rates in the unfolded state, we were able to monitor hydrogen-bond formation for over 50 individual backbone NH groups within the initial 140 microseconds of folding of horse cytochrome c. Clusters of solvent-shielded amide protons were observed in two α-helical segments in the C-terminal half of the protein, while the N-terminal helix remained largely unstructured, suggesting that proximity in the primary structure is a major factor in promoting helix formation and association at early stages of folding, while the entropically more costly long-range contacts between the N- and C-terminal helices are established only during later stages. Our findings clearly indicate that the initial chain condensation in cytochrome c is driven by specific interactions among a subset of α-helical segments rather than a general hydrophobic collapse.