Folding Rate Research Articles

Geologic evolution of the Peña flexure, Southwestern Pyrenees mountain front, Spain

The sedimentary and tectonic evolution of Oligocene continental growth strata of the Peña flexure records the development of a thrust-related anticlinorium and foreland-dipping molasse along the Southwestern Pyrenees mountain front. We exploit the coupling of uplift, the production of accommodation space, and resultant synorogenic sedimentation to explore the interplay of processes accompanying thrust faulting. A new magnetic polarity strata through ~3400 m of continuous pre- and syn-growth strata near the town of Sos del Rey Católico, Spain shows coordinated deposition and deformation. The ChRM data reside mostly in magnetite. Correlation to the Geomagnetic Polarity Time Scale suggests our measured stratigraphic section spans from 33.2 Ma (Chron C12r) in the Middle Campodarbe Formation to 23.4 Ma (chron C6C) at the top of the Bernués Formation. Shallowing in dip of the growth strata up-section is related to coeval slip on a mountain front backthrust. The onset of growth strata deposition and fault slip at this position along the Pyrenees mountain front occurred at about 28.6 ± 0.1 Ma. Folding rates calculated from the growth strata at the resolution of the polarity chrons (0.15–1.12 Myr), varies from 03°/Myr to 35°/Myr.

NMR Chemical Exchange Measurements Reveal That N6-Methyladenosine Slows RNA Annealing.

N6-Methyladenosine (m6A) is an abundant epitranscriptomic modification that plays important roles in many aspects of RNA metabolism. While m6A is thought to mainly function by recruiting reader proteins to specific RNA sites, the modification can also reshape RNA-protein and RNA-RNA interactions by altering RNA structure mainly by destabilizing base pairing. Little is known about how m6A and other epitranscriptomic modifications might affect the kinetic rates of RNA folding and other conformational transitions that are also important for cellular activity. Here, we used NMR R1ρ relaxation dispersion and chemical exchange saturation transfer to noninvasively and site-specifically measure nucleic acid hybridization kinetics. The methodology was validated on two DNA duplexes and then applied to examine how a single m6A alters the hybridization kinetics in two RNA duplexes. The results show that m6A minimally impacts the rate constant for duplex dissociation, changing koff by ∼1-fold but significantly slows the rate of duplex annealing, decreasing kon by ∼7-fold. A reduction in the annealing rate was observed robustly for two different sequence contexts at different temperatures, both in the presence and absence of Mg2+. We propose that rotation of the N6-methyl group from the preferred syn conformation in the unpaired nucleotide to the energetically disfavored anti conformation required for Watson-Crick pairing is responsible for the reduced annealing rate. The results help explain why in mRNA m6A slows down tRNA selection and more generally suggest that m6A may exert cellular functions by reshaping the kinetics of RNA conformational transitions.

Non-conservation of folding rates in the thioredoxin family reveals degradation of ancestral unassisted-folding.

Evolution involves not only adaptation, but also the degradation of superfluous features. Many examples of degradation at the morphological level are known (vestigial organs, for instance). However, the impact of degradation on molecular evolution has been rarely addressed. Thioredoxins serve as general oxidoreductases in all cells. Here, we report extensive mutational analyses on the folding of modern and resurrected ancestral bacterial thioredoxins. Contrary to claims from recent literature, in vitro folding rates in the thioredoxin family are not evolutionarily conserved, but span at least a ∼100-fold range. Furthermore, modern thioredoxin folding is often substantially slower than ancestral thioredoxin folding. Unassisted folding, as probed in vitro, thus emerges as an ancestral vestigial feature that underwent degradation, plausibly upon the evolutionary emergence of efficient cellular folding assistance. More generally, our results provide evidence that degradation of ancestral features shapes, not only morphological evolution, but also the evolution of individual proteins.

Folding of the β-Barrel Membrane Protein OmpA into Nanodiscs

Nanodiscs (NDs) are an excellent alternative to small unilamellar vesicles (SUVs) for studies of membrane protein structure, but it has not yet been shown that membrane proteins are able to spontaneously fold and insert into a solution of freely diffusing NDs. In this article, we present SDS-PAGE differential mobility studies combined with fluorescence, circular dichroism, and ultraviolet resonance Raman spectroscopy to confirm the spontaneous folding of outer membrane protein A (OmpA) into preformed NDs. Folded OmpA in NDs was incubated with Arg-C protease, resulting in the digestion of OmpA to membrane-protected fragments with an apparent molecular mass of ∼26 kDa (major component) and ∼24 kDa (minor component). The OmpA folding yields were greater than 88% in both NDs and SUVs. An OmpA adsorbed intermediate on NDs could be isolated at low temperature and induced to fold via an increase in temperature, analogous to the temperature-jump experiments on SUVs. The circular dichroism spectra of OmpA in NDs and SUVs were similar and indicated β-barrel secondary structure. Further evidence of OmpA folding into NDs was provided by ultraviolet resonance Raman spectroscopy, which revealed the intense 785 cm−1 structural marker for folded OmpA in NDs. The primary difference between folding in NDs and SUVs was the kinetics; the rate of folding was two- to threefold slower in NDs compared to in SUVs, and this decreased rate can tentatively be attributed to the properties of NDs. These data indicate that NDs may be an excellent alternative to SUVs for folding experiments and offer benefits of optical clarity, sample homogeneity, control of ND:protein ratios, and greater stability.

Considerations on the Rational Design of Covalently Conjugated Cell-Penetrating Peptides (CPPs) for Intracellular Delivery of Proteins: A Guide to CPP Selection Using Glucarpidase as the Model Cargo Molecule.

Access of proteins to their intracellular targets is limited by a hydrophobic barrier called the cellular membrane. Conjugation with cell-penetrating peptides (CPPs) has been shown to improve protein transduction into the cells. This conjugation can be either covalent or non-covalent, each with its unique pros and cons. The CPP-protein covalent conjugation may result in undesirable structural and functional alterations in the target protein. Therefore, we propose a systematic approach to evaluate different CPPs for covalent conjugations. This guide is presented using the carboxypeptidase G2 (CPG2) enzyme as the target protein. Seventy CPPs —out of 1155— with the highest probability of uptake efficiency were selected. These peptides were then conjugated to the N- or C-terminus of CPG2. Translational efficacy of the conjugates, robustness and thermodynamic properties of the chimera, aggregation possibility, folding rate, backbone flexibility, and aspects of in vivo administration such as protease susceptibility were predicted. The effect of the position of conjugation was evaluated using unpaired t-test (p < 0.05). It was concluded that N-terminal conjugation resulted in higher quality constructs. Seventeen CPP-CPG2/CPG2-CPP constructs were identified as the most promising. Based on this study, the bioinformatics workflow that is presented may be universally applied to any CPP-protein conjugate design.

Formation of a β-barrel membrane protein is catalyzed by the interior surface of the assembly machine protein BamA.

The β-barrel assembly machine (Bam) complex in Gram-negative bacteria and its counterparts in mitochondria and chloroplasts fold and insert outer membrane β-barrel proteins. BamA, an essential component of the complex, is itself a β-barrel and is proposed to play a central role in assembling other barrel substrates. Here, we map the path of substrate insertion by the Bam complex using site-specific crosslinking to understand the molecular mechanisms that control β-barrel folding and release. We find that the C-terminal strand of the substrate is stably held by BamA and that the N-terminal strands of the substrate are assembled inside the BamA β-barrel. Importantly, we identify contacts between the assembling β-sheet and the BamA interior surface that determine the rate of substrate folding. Our results support a model in which the interior wall of BamA acts as a chaperone to catalyze β-barrel assembly.

MuLiMs-MCoMPAs: A Novel Multiplatform Framework to Compute Tensor Algebra-Based Three-Dimensional Protein Descriptors.

This report introduces the MuLiMs-MCoMPAs software (acronym for Multi-Linear Maps based on N-Metric and Contact Matrices of 3D Protein and Amino-acid weightings), designed to compute tensor-based 3D protein structural descriptors by applying two- and three-linear algebraic forms. Moreover, these descriptors contemplate generalizing components such as novel 3D protein structural representations, (dis)similarity metrics, and multimetrics to extract geometrical related information between two and three amino acids, weighting schemes based on amino acid properties, matrix normalization procedures that consider simple-stochastic and mutual probability transformations, topological and geometrical cutoffs, amino acid, and group-based MD calculations, and aggregation operators for merging amino acidic and group MDs. The MuLiMs-MCoMPAs software, which belongs to the ToMoCoMD-CAMPS suite, was developed in Java (version 1.8) using the Chemistry Development Kit (CDK) (version 1.4.19) and the Jmol libraries. This software implemented a divide-and-conquer strategy to parallelize the computation of the indices as well as modules for data preprocessing and batch computing functionalities. Furthermore, it consists of two components: (i) a desktop-graphical user interface (GUI) and (ii) an API library. The relevance of this novel approach is demonstrated through two analyses that considered Shannon's entropy-based variability and a principal component analysis. These studies showed that the MuLiMs-MCoMPAs' three-linear descriptor family contains higher informational entropy than several other descriptors generated with available computation tools. Moreover, the MuLiMs-MCoMPAs indices capture additional orthogonal information to the one codified by the available calculation approaches. As a result, two sets of suggested theoretical configurations that contain 13648 two-linear indices and 20263 three-linear indices are available for download at tomocomd.com . Furthermore, as a demonstration of the applicability and easy integration of the MuLiMs library into a QSAR-based expert system, a software application (ProStAF) was generated to predict SCOP protein structural classes and folding rate. It can thus be anticipated that the MuLiMs-MCoMPAs framework will turn into a valuable contribution to the chem- and bioinformatics research fields.

Protein folding while chaperone bound is dependent on weak interactions

It is generally assumed that protein clients fold following their release from chaperones instead of folding while remaining chaperone-bound, in part because binding is assumed to constrain the mobility of bound clients. Previously, we made the surprising observation that the ATP-independent chaperone Spy allows its client protein Im7 to fold into the native state while continuously bound to the chaperone. Spy apparently permits sufficient client mobility to allow folding to occur while chaperone bound. Here, we show that strengthening the interaction between Spy and a recently discovered client SH3 strongly inhibits the ability of the client to fold while chaperone bound. The more tightly Spy binds to its client, the more it slows the folding rate of the bound client. Efficient chaperone-mediated folding while bound appears to represent an evolutionary balance between interactions of sufficient strength to mediate folding and interactions that are too tight, which tend to inhibit folding.

LEGO-based generalized set of two linear algebraic 3D bio-macro-molecular descriptors: Theory and validation by QSARs

Novel 3D protein descriptors based on bilinear, quadratic and linear algebraic maps in Rn are proposed. The latter employs the kth 2-tuple (dis) similarity matrix to codify information related to covalent and non-covalent interactions in these biopolymers. The calculation of the inter-amino acid distances is generalized by using several dis-similarity coefficients, where normalization procedures based on the simple stochastic and mutual probability schemes are applied. A new local-fragment approach based on amino acid-types and amino acid-groups is proposed to characterize regions of interest in proteins. Topological and geometric macromolecular cutoffs are defined using local and total indices to highlight non-covalent interactions existing between the side-chains of each amino acid. Moreover, local and total indices calculations are generalized considering a LEGO approach, by using several aggregation operators. Collinearity and variability analyses are performed to evaluate every generalizing component applied to the definition of these novel indices. These experiments are oriented to reduce the number of MDs obtained for performing prediction models. The predictive power of the proposed indices was evaluated using two benchmark datasets, folding rate and secondary structural classification of proteins. The proposed MDs are modeled using the following strategies: Multiple Linear Regression (MLR) and Support Vector Machine (SVM), respectively. The best regression model developed for the folding rate of proteins yields a cross-validation coefficient of 0.875 (Test Set) and the best model developed for secondary structural classification obtained 98% of instances correctly classified (Test Set). These statistical parameters are superior to the ones obtained with existing MDs reported in the literature. Overall, the new theoretical generalization enhanced the information extraction into the MDs, allowing a better correlation between these two evaluated benchmark datasets and the proposed indices. The optimal theoretical configurations defined for the calculation of these MDs consider low collinearity and less information redundancy among them. These theoretical configurations and the software are available at http://tomocomd.com/mulims-mcompas.

Supercoiling in a Protein Increases its Stability.

The supercoiling motif is the most complex type of nontrivial topology found in proteins with at least one disulfide bond and, to the best of our knowledge, it has not been studied before. We show that a protein from extremophilic species with such a motif can fold; however, the supercoiling changes a smooth landscape observed in reduced conditions into a two-state folding process in the oxidative conditions, with a deep intermediate state. The protein takes advantage of the hairpinlike motif to overcome the topological barrier and thus to supercoil. We find that the depth of the supercoiling motif, i.e., the length of the threaded terminus, has a crucial impact on the folding rates of the studied protein. We show that fluctuations of the minimal surface area can be used to measure local stability, and we find that supercoiling introduces stability into the protein. We suggest that the supercoiling motif enables the studied protein to live in physically extreme conditions, which are detrimental to most life on Earth.

Diverse Folding Pathways of HIV-1 Protease Monomer on a Rugged Energy Landscape

The modern energy landscape theory of protein folding predicts multiple folding pathways connecting a myriad of unfolded conformations and a well-defined folded state. However, direct experimental observation of heterogeneous folding pathways is difficult. Naturally evolved proteins typically exhibit a smooth folding energy landscape for fast and efficient folding by avoiding unfavorable kinetic traps. In this case, rapid fluctuations between unfolded conformations result in apparent two-state behavior and make different pathways indistinguishable. However, the landscape roughness can be different, depending on the selection pressures during evolution. Here, we characterize the unusually rugged folding energy landscape of human immunodeficiency virus-1 protease monomer using single-molecule Förster resonance energy transfer spectroscopy. Our data show that fluctuations between unfolded conformations are slow, which enables the experimental observation of heterogeneous folding pathways as predicted by the landscape theory. Although the landscape ruggedness is sensitive to the mutations and fluorophore locations, the folding rate is similar for various protease constructs. The natural evolution of the protease to have a rugged energy landscape likely results from intrinsic pressures to maintain robust folding when human immunodeficiency virus-1 mutates frequently, which is essential for its survival.

Calculation of the Hinge Moments of a Folding Wing Aircraft during the Flight-Folding Process

A folding wing morphing aircraft should complete the folding and unfolding process of its wings while in flight. Calculating the hinge moments during the morphing process is a critical aspect of a folding wing design. Most previous studies on this problem have adopted steady-state or quasi-steady-state methods, which do not simulate the free-flying morphing process. In this study, we construct an aeroelastic flight simulation platform based on the secondary development of ADAMS software to simulate the flight-folding process of a folding wing aircraft. A flexible multibody dynamic model of the folding wing structure is established in ADAMS using modal neutral files, and the doublet lattice method is developed to generate aerodynamic influence coefficient matrices that are suitable for the flight-folding process. The user subroutine is utilized, aerodynamic loading is realized in ADAMS, and an aeroelastic flight simulation platform of a folding wing aircraft is built. On the basis of this platform, the flight-folding process of the aircraft is simulated, the hinge moments of the folding wings are calculated, and the influences of the folding rate and the aircraft’s center of gravity (c.g.) position on the results are investigated. Results show that the steady-state method is applicable to the slow folding process. For the fast folding process, the steady-state simulation errors of the hinge moments are substantially large, and a transient method is required to simulate the flight-folding process. In addition, the c.g. position considerably affects the hinge moments during the folding process. Given that the c.g. position moves aft, the maximum hinge moments of the inner and outer wings constantly increase.

The effect of indwelling catheter care risk assessment on unplanned extubation and nursing outcome in patients undergoing hepatobiliary surgery

Objective To explore the effect of indwelling catheter care risk assessment on unplanned extubation and nursing effects in patients undergoing hepatobiliary surgery. Methods A total of seventy-two patients undergoing routine care for liver and gallbladder surgery from September 2015 to September 2016 in this hospital were selected as the control group.In addition, 72 cases of liver and gallbladder surgery patients admitted by the hospital from October 2016 to October 2017 for nursing risk assessment were selected as the observation group.The unplanned extubation and nursing effect were compared between the two groups. Results The rate of T tube, gastric tube, abdominal drainage tube, deep venous catheter and unplanned catheter extubation in the observation group was lower than that in the control group, and the difference was statistically significant (P<0.05). The rate of catheter blockage, catheter folding, catheter slip and complaint in the observation group was lower than that in the control group, and the nursing satisfaction was higher than that in the control group, the difference was statistically significant (P<0.05). Conclusions The application of indent catheter nursing risk assessment in patients with hepatobiliary surgery can effectively reduce the rate of unplanned extubation and improve the nursing effect, which is worthy of clinical promotion. Key words: Indwelling catheter; Nursing risk assessment; Hepatobiliary surgery; Unplanned extubation; Nursing effect

Tensor Algebra-based Geometrical (3D) Biomacro-Molecular Descriptors for Protein Research: Theory, Applications and Comparison with other Methods

In this report, a new type of tridimensional (3D) biomacro-molecular descriptors for proteins are proposed. These descriptors make use of multi-linear algebra concepts based on the application of 3-linear forms (i.e., Canonical Trilinear (Tr), Trilinear Cubic (TrC), Trilinear-Quadratic-Bilinear (TrQB) and so on) as a specific case of the N-linear algebraic forms. The definition of the kth 3-tuple similarity-dissimilarity spatial matrices (Tensor’s Form) are used for the transformation and for the representation of the existing chemical information available in the relationships between three amino acids of a protein. Several metrics (Minkowski-type, wave-edge, etc) and multi-metrics (Triangle area, Bond-angle, etc) are proposed for the interaction information extraction, as well as probabilistic transformations (e.g., simple stochastic and mutual probability) to achieve matrix normalization. A generalized procedure considering amino acid level-based indices that can be fused together by using aggregator operators for descriptors calculations is proposed. The obtained results demonstrated that the new proposed 3D biomacro-molecular indices perform better than other approaches in the SCOP-based discrimination and the prediction of folding rate of proteins by using simple linear parametrical models. It can be concluded that the proposed method allows the definition of 3D biomacro-molecular descriptors that contain orthogonal information capable of providing better models for applications in protein science.

Impact of bacterial chaperonin GroEL\u2013GroES on bacteriorhodopsin folding and membrane integration

Our understanding of molecular chaperone function in membrane protein biogenesis lags far behind that in soluble protein biogenesis. Through a combined approach including isothermal titration calorimetry, UV–Vis spectroscopy, and fluorescence spectroscopy, the behavior of ATP-dependent chaperonin GroEL–GroES, a paradigmatic chaperone of soluble protein folding, was investigated in the refolding of membrane protein bacteriorhodopsin (BR) and its membrane insertion. We found that BR bound asymmetrically to the double-ring GroEL, with a much higher affinity when it was partially denatured. GroEL alone showed a clear influence on BR refolding, but the presence of ATP was necessary to significantly enhance both the rate and yield of the GroEL-mediated folding, in contrast to the adverse effect of GroES on the folding yield. However, synergy between ATP and GroES was shown to be required not only for releasing high-affinity BR species from GroEL, but also for unfolding and rescuing the misfolded conformers complexed to GroEL. This is consistent with the observation that maximum rate enhancement of BR refolding or assembly with the prepared inverted membrane vesicles was achieved when the complete chaperonin system was used. Our results support the iterative unfolding mechanism of GroEL activity previously proposed for soluble proteins, whereby GroEL might perform repeated unfolding and release of BR, thus offering additional opportunities for timely folding or membrane integration. This work provides important information on the convergence of folding of membrane and soluble proteins in light of folding pathways and the role of molecular chaperones.

Molecular Modelling and Evaluation of Hidden Information in ABCB11 Gene Mutations.

Background:Cholestatic disorders are divided in the extra and intra-hepatic that created due to the severe liver diseases. ABCB11 encodes the bile salt export pump and this gene is mutated in several forms of intrahepatic cholestasis. So far, some molecular features of this gene was studies. Objective: Using a developed web server, we identified high number of rare codons in this gene, and four cases were related to BSEP-deficient patients which can be used for drug design. Material and Methods:By in-silico modelling of ABCB11, some of rare codons in different locations of ATP8b1 gene were identified and evaluated. Using several web servers a number of mutations that converted non-rare codons to rare codon in these patients were identified. Results: Some of these rare Codons were located at special positions by mutation of which, the new side chains do not seem suitable for protein structure and function. Furthermore, this mutation changed the protein folding rate that may have a critical role in proper folding. Thus, primary change of these codons contributes to BSEP deficiency. Conclusion:This work is a comprehensive analysis of rare codons of ABCB11 and assessment of a number of these rare codon in protein levels. Rare codons evaluation can enhance our understanding of ABCB11 structural protein of ABCB11, and help us to develop mutation-specific therapies in design of new drugs.

Ultrafast folding kinetics of WW domains reveal how the amino acid sequence determines the speed limit to protein folding

Protein (un)folding rates depend on the free-energy barrier separating the native and unfolded states and a prefactor term, which sets the timescale for crossing such barrier or folding speed limit. Because extricating these two factors is usually unfeasible, it has been common to assume a constant prefactor and assign all rate variability to the barrier. However, theory and simulations postulate a protein-specific prefactor that contains key mechanistic information. Here, we exploit the special properties of fast-folding proteins to experimentally resolve the folding rate prefactor and investigate how much it varies among structural homologs. We measure the ultrafast (un)folding kinetics of five natural WW domains using nanosecond laser-induced temperature jumps. All five WW domains fold in microseconds, but with a 10-fold difference between fastest and slowest. Interestingly, they all produce biphasic kinetics in which the slower phase corresponds to reequilibration over the small barrier (<3 RT) and the faster phase to the downhill relaxation of the minor population residing at the barrier top [transition state ensemble (TSE)]. The fast rate recapitulates the 10-fold range, demonstrating that the folding speed limit of even the simplest all-β fold strongly depends on the amino acid sequence. Given this fold's simplicity, the most plausible source for such prefactor differences is the presence of nonnative interactions that stabilize the TSE but need to break up before folding resumes. Our results confirm long-standing theoretical predictions and bring into focus the rate prefactor as an essential element for understanding the mechanisms of folding.

Breakdown of the scallop theorem for an asymmetrical folding molecular motor in soft matter.

We use molecular dynamic simulations to investigate the motion of a folding molecular motor inside soft matter. Purcell's scallop theorem forbids the displacement of the motor due to time symmetrical hydrodynamic laws at low Reynolds numbers whatever the asymmetry of the folding and unfolding rates. However, the fluctuation theorems imply a violation of the time symmetry of the motor's trajectories due to the entropy generated by the motor, suggesting a breakdown of the scallop theorem at the nanoscale. To clarify this picture, we study the predicted violation of time reversibility of the motor's trajectories, using two reverse asymmetric folding mechanisms. We actually observe this violation of time reversibility of the motor's trajectories. We also observe the previously reported fluidization of the medium induced by the motor's folding, but find that this induced diffusion is not enough to explain the increase of the motor's displacement. As a result, the motor is not carried by the medium in our system but moves by its own, in violation of the scallop theorem. The observed violation of the scallop theorem opens a route to create very simple molecular motors moving in soft matter environments.

Hydrophobic Moieties Bestow Fast-Folding and Hyperstability on Collagen Triple Helices.

Trans amide bonds and fast cis- trans isomerization of Xaa-Pro bonds are crucial for the stability and folding rate of collagen, the most abundant protein in mammals. Here, we explored the effect of pendant hydrophobic moieties on the folding and stability of collagen triple helices. Kinetic studies with a series of collagen model peptides showed that a local hydrophobic environment accelerates cis- trans isomerization to an extent that thermally induced unfolding and folding of the collagen triple helix take place at the same speed. Thermal denaturation studies revealed that the hydrophobic appendages provide hyperstable collagen triple helices ( Tm = 70 °C).

Reversing the typical pH stability profile of the Trp-cage.

The Trp-cage, an 18-20 residue miniprotein, has emerged as a primary test system for evaluating computational fold prediction and folding rate determination efforts. As it turns out, a number of stabilizing interactions in the Trp-cage folded state have a strong pH dependence; all prior Trp-cage mutants have been destabilized under carboxylate-protonating conditions. Notable among the pH dependent stabilizing interactions within the Trp-cage are: (1) an Asp as the helix N-cap, (2) an H-bonded Asp9/Arg16 salt bridge, (3) an interaction between the chain termini which are in close spatial proximity, and (4) additional side chain interactions with Asp9. In the present study, we have prepared Trp-cage species that are significantly more stable at pH 2.5 (rather than 7) and quantitated the contribution of each interaction listed above. The Trp-cage structure remains constant with the pH change. The study has also provided measures of the stabilizing contribution of indole ring shielding from surface exposure and the destabilizing effects of an ionized Asp at the C-terminus of an α-helix.