Folding screening assayed by proteolysis: application to various cystine deletion mutants of vascular endothelial growth factor

The production of recombinant proteins in Escherichia coli oftenleads to the formation of inclusion bodies. Although this hasa number of advantages, a major disadvantage is the need todevelop folding protocols for the renaturing of the proteins.However, the systematic screening of folding conditions is oftenhampered by the lack of convenient assays to detect correctlyfolded proteins. To address this problem we present a simpleprotocol, which combines folding screens and limited proteolysisto rapidly assess and optimize folding conditions. The efficacyof this method, termed FSAP (folding screening assayed by proteolysis),is demonstrated by the large-scale folding, purification andcrystallization of various cystine deletion mutants of the cystineknot family member: vascular endothelial growth factor (VEGF).These mutants are particularly difficult to fold as the cystineknot is believed to make major contributions to the stabilityof the protein and this family of proteins lacks extensive hydrophobiccore regions.     

Coarse-Grained and Atomistic MD Simulations of RNA and DNA Folding

Although the main features of the protein folding problem are coming into clearer focus, the microscopic viewpoint of nucleic acid folding mechanisms is only just beginning to be addressed. Experiments, theory, and simulations are pointing to complex thermodynamic and kinetic mechanisms. As is the case for proteins, molecular dynamics (MD) simulations continue to be indispensable tools for providing a molecular basis for nucleic acid folding mechanisms. In this review, we provide an overview of biomolecular folding mechanisms focusing on nucleic acids. We outline the important interactions that are likely to be the main determinants of nucleic acid folding energy landscapes. We discuss recent MD simulation studies of empirical force field and Go-type MD simulations of RNA and DNA folding mechanisms to outline recent successes and the theoretical and computational challenges that lie ahead.     

Helix stability and hydrophobicity in the folding mechanism of the bacterial immunity protein Im9

Recent models suggest that the mechanism of protein folding is determined by the balance between the stability of secondary structural elements and the hydrophobicity of the sequence. Here we determine the role of these factors in the folding kinetics of Im9* by altering the secondary structure propensity or hydrophobicity of helices I, II or IV by the substitution of residues at solvent exposed sites. The folding kinetics of each variant were measured at pH 7.0 and 10 degrees C, under which conditions wild-type Im9* folds with two-state kinetics. We show that increasing the helicity of these sequences in regions known to be structured in the folding intermediate of Im7*, switches the folding of Im9* from a two- to three-state mechanism. By contrast, increasing the hydrophobicity of helices I or IV has no effect on the kinetic folding mechanism. Interestingly, however, increasing the hydrophobicity of solvent-exposed residues in helix II stabilizes the folding intermediate and the rate-limiting transition state, consistent with the view that this helix makes significant non-native interactions during folding. The results highlight the generic importance of intermediates in folding and show that such species can be populated by increasing helical propensity or by stabilizing inter-helix contacts through non-native interactions.     

Conservation of mechanism, variation of rate: folding kinetics of three homologous four-helix bundle proteins

The amino acid sequence of a protein determines both its final folded structure and the folding mechanism by which this structure is attained. The differences in folding behaviour between homologous proteins provide direct insights into the factors that influence both thermodynamic and kinetic properties. Here, we present a comprehensive thermodynamic and kinetic analysis of three homologous homodimeric four-helix bundle proteins. Previous studies with one member of this family, Rop, revealed that both its folding and unfolding behaviour were interesting and unusual: Rop folds (k(0)(f) = 29 s(-1)) and unfolds (k(0)(u) = 6 x 10(-7) s(-1)) extremely slowly for a protein of its size that contains neither prolines nor disulphides in its folded structure. The homologues we discuss have significantly different stabilities and rates of folding and unfolding. However, the rate of protein folding directly correlates with stability for these homologous proteins: proteins with higher stability fold faster. Moreover, in spite of possessing differing thermodynamic and kinetic properties, the proteins all share a similar folding and unfolding mechanism. We discuss the properties of these naturally occurring Rop homologues in relation to previously characterized designed variants of Rop.     

Myosin Assembly, Maintenance and Degradation in Muscle: Role of the Chaperone UNC-45 in Myosin Thick Filament Dynamics

Myofibrillogenesis in striated muscle cells requires a precise ordered pathway to assemble different proteins into a linear array of sarcomeres. The sarcomere relies on interdigitated thick and thin filaments to ensure muscle contraction, as well as properly folded and catalytically active myosin head. Achieving this organization requires a series of protein folding and assembly steps. The folding of the myosin head domain requires chaperone activity to attain its functional conformation. Folded or unfolded myosin can spontaneously assemble into short myosin filaments, but further assembly requires the short and incomplete myosin filaments to assemble into the developing thick filament. These longer filaments are then incorporated into the developing sarcomere of the muscle. Both myosin folding and assembly require factors to coordinate the formation of the thick filament in the sarcomere and these factors include chaperone molecules. Myosin folding and sarcomeric assembly requires association of classical chaperones as well as folding cofactors such as UNC-45. Recent research has suggested that UNC-45 is required beyond initial myosin head folding and may be directly or indirectly involved in different stages of myosin thick filament assembly, maintenance and degradation.     

Oxidative Folding in the Mitochondrial Intermembrane Space in Human Health and Disease

Oxidative folding in the mitochondrial intermembrane space (IMS) is a key cellular event associated with the folding and import of a large and still undetermined number of proteins. This process is catalyzed by an oxidoreductase, Mia40 that is able to recognize substrates with apparently little or no homology. Following substrate oxidation, Mia40 is reduced and must be reoxidized by Erv1/Alr1 that consequently transfers the electrons to the mitochondrial respiratory chain. Although our understanding of the physiological relevance of this process is still limited, an increasing number of pathologies are being associated with the impairment of this pathway; especially because oxidative folding is fundamental for several of the proteins involved in defense against oxidative stress. Here we review these aspects and discuss recent findings suggesting that oxidative folding in the IMS is modulated by the redox state of the cell.     

Protein Folding and Mechanisms of Proteostasis

Highly sophisticated mechanisms that modulate protein structure and function, which involve synthesis and degradation, have evolved to maintain cellular homeostasis. Perturbations in these mechanisms can lead to protein dysfunction as well as deleterious cell processes. Therefore in recent years the etiology of a great number of diseases has been attributed to failures in mechanisms that modulate protein structure. Interconnections among metabolic and cell signaling pathways are critical for homeostasis to converge on mechanisms associated with protein folding as well as for the preservation of the native structure of proteins. For instance, imbalances in secretory protein synthesis pathways lead to a condition known as endoplasmic reticulum (ER) stress which elicits the adaptive unfolded protein response (UPR). Therefore, taking this into consideration, a key part of this paper is developed around the protein folding phenomenon, and cellular mechanisms which support this pivotal condition. We provide an overview of chaperone protein function, UPR via, spatial compartmentalization of protein folding, proteasome role, autophagy, as well as the intertwining between these processes. Several diseases are known to have a molecular etiology in the malfunction of mechanisms responsible for protein folding and in the shielding of native structure, phenomena which ultimately lead to misfolded protein accumulation. This review centers on our current knowledge about pathways that modulate protein folding, and cell responses involved in protein homeostasis.     

Folding rate prediction based on neural network model

One of the most important challenges in biology is to understand the relationship between the folded structure of a protein and its primary amino acid sequence. A related and challenging task is to understand the relationship between sequences and folding rates of proteins. Previous studies found that one of contact order (CO), long-range order (LRO), and total contact distance (TCD) has a significant correlation with folding rate of protein. Although the predicted results from TCD can provide better results, the deviation is also large for some proteins. In this paper, we adopt back-propagation neural network to study the relationship between folding rate and protein structure. In our model, the input nodes are CO, LRO, and TCD, and the output node is folding rate. The number of nodes in the hidden layer is seven. Our results show that the relative errors for the predicted results are even lower than other methods in the literature. We also observe a best excellent correlation between the folding rate and contact parameters (including CO, LRO, and TCD), and find that the folding rate depends on CO, LRO and TCD simultaneously. This means that CO, LRO and TCD are similarly important in folding rate of protein. Some comparisons are made with other methods.     

Is Protein Folding a Thermodynamically Unfavorable,Active, Energy-Dependent Process?

The prevailing current view of protein folding is the thermodynamic hypothesis, under which the native folded conformation of a protein corresponds to the global minimum of Gibbs free energy G. We question this concept and show that the empirical evidence behind the thermodynamic hypothesis of folding is far from strong. Furthermore, physical theory-based approaches to the prediction of protein folds and their folding pathways so far have invariably failed except for some very small proteins, despite decades of intensive theory development and the enormous increase of computer power. The recent spectacular successes in protein structure prediction owe to evolutionary modeling of amino acid sequence substitutions enhanced by deep learning methods, but even these breakthroughs provide no information on the protein folding mechanisms and pathways. We discuss an alternative view of protein folding, under which the native state of most proteins does not occupy the global free energy minimum, but rather, a local minimum on a fluctuating free energy landscape. We further argue that ΔG of folding is likely to be positive for the majority of proteins, which therefore fold into their native conformations only through interactions with the energy-dependent molecular machinery of living cells, in particular, the translation system and chaperones. Accordingly, protein folding should be modeled as it occurs in vivo, that is, as a non-equilibrium, active, energy-dependent process.     

Global Folding of a Na+-Specific DNAzyme Studied by FRET

Recently, a few RNA-cleaving DNAzymes have been isolated with excellent specificity for Na⁺, and some of them contain a Na⁺-binding aptamer. This metal recognition mechanism is different from that of most previously reported DNAzymes. When using 2-aminopurine (2AP) as a probe, interesting local folding induced by Na⁺ was recently observed. In this work, FRET was used to probe the global folding of the Ce13d DNAzyme; one of the Na⁺-specific DNAzymes. FRET pairs were at different locations, which yielded a total of five constructs to probe the three-way junction structure with a large loop. With endlabeled DNAzymes, the global structure appears to be quite rigid with little folding upon adding up to 200 mm monovalent metal ions, although some minor differences were observed between Li⁺, Na⁺, and K⁺. This lack of significant conformational change is also consistent with circular dichroism spectroscopy data. The loop was then labeled with an internal tetramethylrhodamine fluorophore at the G14 position, and its cleavage activity was partially retained. A clear Na⁺-dependent folding was observed with spectral crossover. From a biosensing standpoint, global folding based sensors are unlikely to work due to the overall rigid structure of the DNAzyme. Therefore, the best way to use this DNAzyme to discriminate Na⁺ from K⁺ is based on cleavage activity, followed by probing local folding, whereas global folding is the least effective for metal discrimination.     

Demonstration by burst-phase analysis of a robust folding intermediate in the FF domain

The role of intermediates in the folding reaction of single-domainproteins is a controversial issue. It was previously shown bydifferent methods that an on-pathway intermediate is populatedin the presence of sodium sulphate during the folding of theFF domain from HYPA/FBP11. Here we demonstrate using analysisof the amplitudes of kinetic traces that this burst-phase foldingintermediate is present at different salt concentration andat various pH, and is also found in roughly 30 site-directedmutants. The intermediate appears robust to changing conditionsand thus fulfils an important criterion for a productive molecularspecies on the folding reaction pathway.     

The role of cotranslation in protein folding: a lattice model study

Computational studies of protein folding have implicitly assumed that folding occurs from a denatured state comprised of the entire protein. Cotranslational folding accounts for the linear production and release of a protein from the ribosome, allowing part of the protein to explore its conformation space before other parts have been synthesized. This gradual ‘extrusion’ from the ribosome can yield different folding kinetics than direct folding from the denatured state, for a lattice folding model. First, in model proteins containing chiefly short-ranged (local in sequence) contacts, cotranslational folding is shown to be significantly faster than direct folding from the denatured state. Secondly, for model proteins with two competing native states, cotranslational folding tilts the apparent equilibrium toward the state with a more local-contact dominant topology.     

表面活性剂辅助蛋白质体外折叠：分子模拟

采用分子模拟方法考察表面活性剂与蛋白质分子之间的相互作用及其对蛋白质折叠过程热力学特性的影响，蛋白质分子构建采用HP模型并引入了方阱类势函数.模拟结果显示：模型蛋白分子的某些折叠中间态会陷入局部能量最低状态而无法完成折叠;弱疏水性表面活性剂对模型蛋白的稳定性影响小，但可有效地帮助处于局部能量最低状态的蛋白折叠中间态通过能量壁垒而实现折叠;强疏水性表面活性剂则可与蛋白质形成高稳定性的复合物而阻止折叠的进行，需将其脱除才能使折叠过程重新开始.模拟结果还显示：表面活性剂的加入会使蛋白质折叠中间态更加丰富，从而能够光滑折叠过程中的能量阱;表面活性剂与变性环境对于蛋白质的折叠具有协同效应.模拟结果与文献报道的实验结果具有一致性，显示分子模拟的方法在揭示蛋白质折叠过程的微观机理以及表面活性剂类折叠助剂的分子设计方面有很好的应用前景.     

Impact of the C‐terminal Disulfide Bond on the Folding and Stability of Onconase

The two homologous proteins ribonuclease A and onconase fold through conserved initial contacts but differ significantly in their thermodynamic stability. A disulfide bond is located in the folding initiation site of onconase (the C‐terminal part of the protein molecule) that is missing in ribonuclease A, whereas the other three disulfide bonds of onconase are conserved in ribonuclease A. Consequently, the deletion of this C‐terminal disulfide bond (C87–C104) allows the impact of the contacts in this region on the folding of onconase to be studied. We found the C87A/C104A‐onconase variant to be less active and less stable than the wild‐type protein, whereas the tertiary structure, which was determined by both X‐ray crystallography and NMR spectroscopy, was only marginally affected. The folding kinetics of the variant, however, were found to be changed considerably in comparison to wild‐type onconase. Proton exchange experiments in combination with two‐dimensional NMR spectroscopy revealed differences in the native‐state dynamics of the two proteins in the folding initiation site, which are held responsible for the changed folding mechanism. Likewise, the molecular dynamics simulation of the unfolding reaction indicated disparities for both proteins. Our results show that the high stability of onconase is based on the efficient stabilization of the folding initiation site by the C‐terminal disulfide bond. The formation of the on‐pathway intermediate, which is detectable during the folding of the wild‐type protein and promotes the fast and efficient refolding reaction, requires the presence of this covalent bond.     

Cunning simplicity of protein folding landscapes

Funnel-like landscapes are widely used to visualize proteinfolding. It might seem that any funnel-like energy landscapehelps to avoid the `Levinthal paradox', i.e. to avoid samplingthe impossibly large number of conformations for a folding protein.This cunning suggestion, reinforced by beautiful drawings ofthe energy funnels, stimulated some simple models of proteinfolding; one of them [D.J. Bicout and A. Szabo (2000) ProteinSci., 9, 452–465] is especially straightforward and instructive.A thorough analysis of this strict funnel model (which doesnot consider a nucleation of phase separation in the courseof folding) shows that it cannot provide a simultaneous explanationfor both major features observed for protein folding: (i) foldingwithin non-astronomical time, and (ii) co-existence of the nativeand the unfolded states during the folding process. On the contrary,the nucleation mechanism of protein folding can account forboth these major features simultaneously.     

Folding core predictions from network models of proteins

Two different computational methods are employed to predict protein folding nuclei from native state structures, one based on an elastic network (EN) model and the other on a constraint network model of freely rotating rods. Three sets of folding cores are predicted with these models, and their correlation against the slow exchange folding cores identified by native state hydrogen-deuterium exchange (HX) experiments is used to test each method. These three folding core predictions rely on differences in the underlying models and relative importance of global or local motions for protein unfolding/folding reactions. For non-specific residue interactions, we use the Gaussian Network Model (GNM) to identify folding cores in the limits of two classes of motions, shortly referred to as global and local. The global mode minima from GNM represent the residues with the greatest potential for coordinating collective motions and are explored as potential folding nuclei. Additionally, the fast mode peaks that have previously been labeled as the kinetically hot residues are identified as a second folding core set dependent on local interactions. Finally, a third folding core set is defined by the most stable residues in a simulated thermal denaturation procedure of the FIRST software. This method uses an all-atomic analysis of the rigidity and flexibility of protein structures, which includes specific hydrophobic, polar and charged interactions. Comparison of the three folding core sets to HX data indicate that the fast mode peak residues determined by the GNM and the rigid folding cores of FIRST provide statistically significant enhancements over random correlation. The role of specific interactions in protein folding is also investigated by contrasting the differences between these two network-based computational methods.     

The folding pathway of an engineered circularly permuted PDZ domain

To understand the role of sequence connectivity in the folding pathway of a multi-state protein, we have analysed the folding kinetics of an engineered circularly permuted PDZ domain. This variant has been designed with the specific aim of posing two of the strands participating in the stabilisation of an early folding nucleus as contiguous elements in the primary structure. Folding of the circularly permuted PDZ2 has been explored by a variety of different experimental approaches including stopped-flow and continuous-flow kinetics, as well as ligand-induced folding experiments. Data reveal that although circular permutation introduces a significant destabilisation of the native state, a folding intermediate is stabilised and accumulated prior folding. Furthermore, quantitative analysis of the observed kinetics indicates an acceleration of the early folding events by more than two orders of magnitude. The results support the importance of sequence connectivity both in the mechanism and the speed of protein folding.     

Binding and folding: in search of intramolecular chaperone-like building block fragments

We propose an intramolecular chaperone which catalyzes foldingand neither dissociates nor is cleaved. This uncleaved foldaseis an intramolecular chain-linked chaperone, which constitutesa critical building block of the structure. Macroscopically,all molecular chaperones facilitate folding reactions and manifestsimilar energy landscapes. However, microscopically they differ.While intermolecular chaperones catalyze folding by unfoldingmisfolded conformations or prevent misfolding, the chain-linkedcleaved (proregion) and uncleaved intramolecular chaperone-likebuilding blocks suggested here, catalyze folding by bindingto, stabilizing and increasing the populations of native conformationsof adjacent building block fragments. In both, the more stablethe intramolecular chaperone fragment region, the faster isthe folding rate. Hence, mechanistically, intramolecular chaperonesand chaperone-like segments are similar. Both play a dual role,in folding and in protein function. However, while the functionalrole of the proregions is inhibitory, necessitating their cleavage,the function of the uncleaved intramolecular chaperone-likebuilding blocks does not require their subsequent removal. Onthe contrary, it requires that they remain in the structure.This may lead to the difference in the type of control theyare under: proteins folding with the assistance of the proregionhave been shown to be under kinetic control. It has been suggestedthat kinetically controlled folding reactions, with the proregioncatalyst removed, lend longevity under harsh conditions. Onthe other hand, proteins with uncleaved intramolecular chaperone-likebuilding blocks, with their `foldases' still attached, are largelyunder thermodynamic control, consistent with the control observedin most protein folding reactions. We propose that an uncleavedintramolecular chaperone-like fragment occurs frequently inproteins. We further propose that such proteins would be proneto changing conditions and in particular, to mutations in thiscritical building block region. We describe the features qualifyingit for its proposed chaperone-like role, compare it with inter-and intramolecular chaperones and review current literaturein this light. We further propose a mechanism showing how itlowers the barrier heights, leading to faster folding reactionrates. Since these fragments constitute an intergal part ofthe protein structure, we call these critical building blocksintramolecular, chaperone-like fragments, to clarify, distinguishand adhere to the definition of the transiently associatingchaperones. The new mechanism presented here differs from theconcept of `folding nuclei'. While the concept of folding nucleifocuses on a non-sequential distribution of the folding informationalong the entire protein chain, the chaperone-like buildingblock fragments proposition focuses on a segmental distributionof the folding information. This segmental distribution controlsthe distributions of the populations throughout the hierarchicalfolding processes.     

An in vitro peptide folding model suggests the presence of the molten globule state during nascent peptide folding

Although molten globules have been widely accepted as a generalintermediate in protein folding, there is no clear evidenceto show their presence during nascent peptide folding. Thispaper concentrates on whether the molten globule state occurs,and if it does, when does it form during nascent peptide folding,by comparing the changes in conformation during peptide chainextension of staphylococcal nuclease R. The results show thata large N-terminal fragment of staphylococcal nuclease, SNR121,which already contains more than 80% amino acid sequence ofthe nuclease, is found to fulfill all the criteria for the moltenglobule state, suggesting that the molten globule should occurat a later stage of peptide elongation. At this stage the hydrophobiccollapse of the polypeptide chain occurs driven by the hydrophobicforce, which leads to the formation of a solvent-accessiblenon-polar core, characterized by the high ANS-binding fluorescence.The nascent peptide folding of the nuclease is a hierarchicalprocess that at the very least includes the following steps:secondary structure accumulation, pre-molten globule state,molten globule state, post-molten globule state and finallythe native state. Constant conformation adjustment is necessaryfor correct folding and active expression of the protein.