Slow Folding Research Articles

The heat capacity decomposition method, a well-established analytical approach in polymer thermodynamics for elucidating thermal transitions in homogeneous polymers, is extended here to heterogeneous systems. We demonstrate that the decomposition of heat capacity based on partition function zeros allows the identification of transition-like crossovers originating from compact low-energy states, thereby enabling the evaluation of the foldability of HP sequences. The occurrence of significant crossovers between the collapse and folding transitions indicates slow folding behavior, whereas their absence characterizes good folders. This criterion is further validated through kinetic Monte Carlo simulations of two representative sequences.

Our current understanding of protein folding is based predominantly on studies of small (<150 aa) proteins that refold reversibly from a chemically denatured state. However, as protein length increases so does the competition between off-pathway misfolding and on-pathway folding, creating a more complex energy landscape ("folding funnel"). Little is known about how intermediates populated during the folding of larger proteins affect navigation of this more complex landscape. Previously, we reported extremely slow folding rates for the 539 aa β-helical passenger domain of pertactin (P.69T), including conditions that favor the formation of a kinetically trapped, off-pathway partially folded state (PFS). Existence of an on-pathway intermediate for P.69T folding was speculated but its characterization remained elusive. In this work, we exploited the extremely slow kinetics of PFS unfolding to develop a double-jump "denaturant challenge" assay. With this assay, we identified a transient unfolding intermediate, PFS*, that adopts a similar structure to PFS, including C-terminal folded structure and a disordered N terminus, yet unfolds much more quickly than PFS. Additional experiments revealed that PFS* also functions as an on-pathway intermediate for P.69T folding. Collectively, these results support a C-to-N-terminal model for P.69T folding, with folding initiated in the C-terminus with the rate-limiting formation of the transient on-pathway PFS* intermediate, which sits at the junction of the kinetic competition between folding and misfolding. Notably, processive folding from C-to-N terminus also occurs during C-to-N-terminal translocation of P.69T across the bacterial outer membrane. These results illuminate the crucial role of kinetics when navigating a complex energy landscape for protein folding.

It is hard to imagine how proteins can thread and form knots in their polypeptide chains, but they do. These topologically complex structures have challenged the traditional protein folding views of simple funnel-shaped energy landscapes. Previous experimental studies on the folding mechanisms of deeply knotted proteins with a single trefoil knot have yielded evidence that this topology has a more complicated folding landscape than other simpler proteins. However, to date, there have been no attempts to study the folding of any protein in which multiple threading events are needed to create more than one knot within a single polypeptide chain. Here, we report the construction and characterization of an artificial tandemly knotted protein. We find compelling evidence that both domains of the protein form trefoil knots with similar structures and stabilities to the parent single trefoil-knotted protein. In addition, we show that this tandemly knotted protein has a complex folding pathway in which there are additional very slow folding phases that we propose correspond to the formation of the second knot within the system. We also find evidence that during folding this protein gets transiently trapped in deep kinetic traps, however, the majority of protein chains (>90%) manage to partially unfold and acquire the native tandem-knot topology. This work highlights the fact that Nature can tolerate more complex protein topologies than we thought, and despite considerable misfolding during folding, protein chains can find their way to the native state even in the absence of molecular chaperones.

Many proteins have slow folding times in vitro that are physiologically untenable. To combat this challenge, ATP-dependent chaperonins are thought to possess the unique ability to catalyze protein folding. Performing quantitative model selection using protein folding and unfolding data, we here show that short nucleic acids containing G-quadruplex (G4) structure can also catalyze protein folding. Performing the experiments as a function of temperature demonstrates that the G4 reshapes the underlying driving forces of protein folding. As short nucleic acids can catalyze protein folding without the input of ATP, the ability of the cell to fold proteins is far higher than previously anticipated.

Riboswitches are ligand-responsive gene-regulatory RNA elements that perform key roles in maintaining cellular homeostasis. Understanding how riboswitch sensitivity to ligand (EC50) is controlled is critical to explain how highly conserved aptamer domains are deployed in a variety of contexts with different sensitivity demands. Here we uncover roles by which RNA folding dynamics control riboswitch sensitivity in cells. By investigating the Clostridium beijerinckii pfl ZTP riboswitch, we identify multiple mechanistic routes of altering expression platform sequence and structure to slow RNA folding, all of which enhance riboswitch sensitivity. Applying these methods to riboswitches with diverse aptamer architectures and regulatory mechanisms demonstrates the generality of our findings, indicating that any riboswitch that operates in a kinetic regime can be sensitized by slowing expression platform folding. Our results add to the growing suite of knowledge and approaches that can be used to rationally program cotranscriptional RNA folding for biotechnology applications, and suggest general RNA folding principles for understanding dynamic RNA systems in other areas of biology.

Force spectroscopy gives access to the underlying free energy landscape of protein folding. Proteins exhibit folding rates between microseconds and hours. To access slow folding rates, magnetic tweezers have shown to be a promising tool, yet it remained unclear if magnetic tweezers capture kinetics of ultra-fast folding proteins. Here, we study the folding mechanics and kinetics of λ6-85; a five-helix bundle protein with fast ~20 µs folding time in the thermal denaturation midpoint. We observed two-state folding of λ6-85 at ~ 6.2 pN and ~ 250 ms folding time in the mechanical midpoint. With optical tweezers, we found that λ6-85 folds at the mechanical midpoint with ~ 15 ms, 16-fold faster than in magnetic tweezers. To resolve the discrepancy between magnetic and optical tweezers, we developed a physics-based model taking into account the constant force condition of magnetic tweezers and the spacer mechanics. Using this model, we reach agreement between magnetic tweezers, optical tweezers and thermal denaturation experiments. In summary, we show that magnetic tweezers capture kinetics of ultrafast conformational changes, even at low forces. The model for extrapolation of the kinetics to force-free conditions provides opportunities of comparability for the growing community of magnetic tweezers force spectroscopy.

Our current understanding of protein folding is based predominantly on studies of small (<150 aa) proteins that refold reversibly from a chemically denatured state. As protein length increases, the competition between off-pathway misfolding and on-pathway folding likewise increases, creating a more complex energy landscape. Little is known about how intermediates populated during the folding of larger proteins affect navigation of this more complex landscape. Previously, we reported extremely slow folding rates for the 539 aa β-helical passenger domain of pertactin (P.69T), including conditions that favor the formation of a kinetically trapped, off-pathway partially folded state (PFS). The existence of an on-pathway intermediate for P.69T folding was speculated but its characterization remained elusive. In this work, we exploited the extremely slow kinetics of PFS unfolding to develop a double-jump “denaturant challenge” assay. With this assay, we identified a transient unfolding intermediate, PFS*, that adopts a similar structure to PFS, including C-terminal folded structure and a disordered N-terminus, yet unfolds much more quickly than PFS. Additional experiments revealed that PFS* also functions as an on-pathway intermediate for P.69T folding. Collectively, these results support a two-step, C-to-N-terminal model for P.69T folding: folding initiates in the C-terminus with the rate-limiting formation of the transient on-pathway PFS* intermediate, which sits at the junction of the kinetic competition between folding and misfolding. Notably, processive folding from C-to-N-terminus also occurs during C-to-N-terminal translocation of P.69T across the bacterial outer membrane. These results illuminate the crucial role of kinetics when navigating a complex energy landscape for protein folding.

ABSTRACT The embryonic development of marine ectotherms has been shown to be strongly temperature dependent across the world's oceans. However, at the coldest sites, in the polar regions, development is even slower than would be predicted on the basis of the temperature dependence of development in warmer waters, and this is thought to be a consequence of changes in physical characteristics of cytoplasm near 0 °C—such as viscosity and osmolyte packing that slow protein folding and increase the likelihood of interference by charged particles, and their effect on protein synthesis. The overwhelming majority of studies of rates of embryonic development have been laboratory-based, with animals either collected directly from the sea and spawned in the laboratory or held first in the laboratory and preconditioned to set environments before spawning. Few studies have assessed development from regularly collected samples and assessed field development, especially from polar latitudes. Here we present data for the Antarctic bivalve mollusc Aequiyoldia eightsii, tracking its development from spawning on 25/26 May to hatching of pelagic veligers on 12 June and the disappearance of pediveliger larvae from the water column at the end of September or early October, 108–114 days later. Larval dry mass was constant at 16.7 µg (SE = 0.19) across the pelagic phase, except for the initial few days after hatching when it was 9.55 µg (SE = 0.60). The difference was likely the calcification of the larval shell. The development time to trochophore was 189 h, and this was in line with previous studies showing larval development at temperatures around 0 °C is around 4–22 times slower than would be predicted from the general effect of temperature on development rates.

The most abundant natural collagens form heterotrimeric triple helices. Synthetic mimics of collagen heterotrimers have been found to fold slowly, even compared to the already slow rates of homotrimeric helices. These prolonged folding rates are not understood. Here we compare the stabilities, specificities and folding rates of three heterotrimeric collagen mimics designed through a computationally assisted approach. The crystal structure of one ABC-type heterotrimer verified a well-controlled composition and register and elucidated the geometry of pairwise cation-π and axial and lateral salt bridges in the assembly. This collagen heterotrimer folds much faster (hours versus days) than comparable, well-designed systems. Circular dichroism and NMR data suggest the folding is frustrated by unproductive, competing heterotrimer species and these species must unwind before refolding into the thermodynamically favoured assembly. The heterotrimeric collagen folding rate is inhibited by the introduction of preformed competing triple-helical assemblies, which suggests that slow heterotrimer folding kinetics are dominated by the frustration of the energy landscape caused by competing triple helices.

Collagenopathies are a group of clinically diverse disorders caused by defects in collagen folding and secretion. For example, mutations in the gene encoding collagen type-II, the primary collagen in cartilage, can lead to diverse chondrodysplasias. One example is the Gly1170Ser substitution in procollagen-II, which causes precocious osteoarthritis. Here, we biochemically and mechanistically characterize an induced pluripotent stem cell-based cartilage model of this disease, including both hetero- and homozygous genotypes. We show that Gly1170Ser procollagen-II is notably slow to fold and secrete. Instead, procollagen-II accumulates intracellularly, consistent with an endoplasmic reticulum (ER) storage disorder. Likely owing to the unique features of the collagen triple helix, this accumulation is not recognized by the unfolded protein response. Gly1170Ser procollagen-II interacts to a greater extent than wild-type with specific ER proteostasis network components, consistent with its slow folding. These findings provide mechanistic elucidation into the etiology of this disease. Moreover, the easily expandable cartilage model will enable rapid testing of therapeutic strategies to restore proteostasis in the collagenopathies.

Proteins are constantly undergoing folding and unfolding transitions, with rates that determine their homeostasis in vivo and modulate their biological function. The ability to optimize these rates without affecting overall native stability is hence highly desirable for protein engineering and design. The great challenge is, however, that mutations generally affect folding and unfolding rates with inversely complementary fractions of the net free energy change they inflict on the native state. Here we address this challenge by targeting the folding transition state (FTS) of chymotrypsin inhibitor 2 (CI2), a very slow and stable two-state folding protein with an FTS known to be refractory to change by mutation. We first discovered that the CI2's FTS is energetically taxed by the desolvation of several, highly conserved, charges that form a buried salt bridge network in the native structure. Based on these findings, we designed a CI2 variant that bears just four mutations and aims to selectively stabilize the FTS. This variant has >250-fold faster rates in both directions and hence identical native stability, demonstrating the success of our FTS-centric design strategy. With an optimized FTS, CI2 also becomes 250-fold more sensitive to proteolytic degradation by its natural substrate chymotrypsin, and completely loses its activity as inhibitor. These results indicate that CI2 has been selected through evolution to have a very unstable FTS in order to attain the kinetic stability needed to effectively function as protease inhibitor. Moreover, the CI2 case showcases that protein (un)folding rates can critically pivot around a few key residues-interactions, which can strongly modify the general effects of known structural factors such as domain size and fold topology. From a practical standpoint, our results suggest that future efforts should perhaps focus on identifying such critical residues-interactions in proteins as best strategy to significantly improve our ability to predict and engineer protein (un)folding rates.

Many folding enzymes use separate domains for the binding of substrate proteins and for the catalysis of slow folding reactions such as prolyl isomerization. FKBP12 is a small prolyl isomerase without a chaperone domain. Its folding activity is low, but it could be increased by inserting the chaperone domain from the homolog SlyD of E. coli near the prolyl isomerase active site. We inserted two other chaperone domains into human FKBP12: the chaperone domain of SlpA from E. coli, and the chaperone domain of SlyD from Thermococcus sp. Both stabilized FKBP12 and greatly increased its folding activity. The insertion of these chaperone domains had no influence on the FKBP12 and the chaperone domain structure, as revealed by two crystal structures of the chimeric proteins. The relative domain orientations differ in the two crystal structures, presumably representing snapshots of a more open and a more closed conformation. Together with crystal structures from SlyD-like proteins, they suggest a path for how substrate proteins might be transferred from the chaperone domain to the prolyl isomerase domain.

Kinks define boundaries between distinct configurations of a material. In the context of mechanical metamaterials, kinks have recently been shown to underpin logic, shape-changing and locomotion functionalities. So far such kinks propagate by virtue of inertia or of an external load. Here, we discover the emergence of propagating kinks in purely dissipative kirigami. To this end, we create kirigami that shape-change into different textures depending on how fast they are stretched. We find that if we stretch fast and wait, the viscoelastic kirigami can eventually snap from one texture to another. Crucially, such a snapping instability occurs in a sequence and a propagating diffusive kink emerges. As such, it mimics the slow sequential folding observed in biological systems, e.g., Mimosa Pudica. We finally demonstrate that diffusive kinks can be harnessed for basic machine-like functionalities, such as sensing, dynamic shape morphing, transport and manipulation of objects.

The cis/trans isomerization of peptidyl-prolyl peptide bonds is often the bottleneck of the refolding reaction for proteins containing cis proline residues in the native state. Proline (Pro) analogues, especially C4-substituted fluoroprolines, have been widely used in protein engineering to enhance the thermodynamic stability of peptides and proteins and to investigate folding kinetics. 4-thiaproline (Thp) has been shown to bias the ring pucker of Pro, to increase the cis population percentage of model peptides in comparison to Pro, and to diminish the activation energy barrier for the cis/trans isomerization reaction. Despite its intriguing properties, Thp has been seldom incorporated into proteins. Moreover, the impact of Thp on the folding kinetics of globular proteins has never been reported. In this study, we show that upon incorporation of Thp at cisPro76 into the thioredoxin variant Trx1P the half-life of the refolding reaction decreased from ~2 h to ~35 s. A dramatic acceleration of the refolding rate could be observed also for the protein pseudo wild-type barstar upon replacement of cisPro48 with Thp. Quantum chemical calculations suggested that the replacement of the Cγ H2 group by a sulfur atom in the pyrrolidine ring, might lower the barrier for cis/trans rotation due to a weakened peptide bond. The protein variants retained their thermodynamic stability upon incorporation of Thp, while the catalytic and enzymatic activities of the modified Trx1P remained unchanged. Our results show that the Pro isostere Thp might accelerate the rate of the slow refolding reaction for proteins containing cis proline residues in the native state, independent from the local structural environment.

The recent discovery of metamorphic proteins, which can switch between multiple conformations under native conditions, has challenged the well-established one sequence-one structure paradigm of protein folding. This is exemplified in the C-terminal domain of the multidomain transcription factor RfaH, which converts from an α-helical coiled-coil conformation in its autoinhibited state to a β-barrel conformation upon activation. Here, we use multisite line shape analysis of 19F NMR-monitored equilibrium chemical denaturation measurements of two 19F-labeled variants of full-length RfaH, to show that it folds/unfolds slowly on the NMR time scale, in an apparent all-or-none fashion at physiological pH and room temperature in the 3.3-4.8 M urea concentration range. The significant thermodynamic stability and slow unfolding rate (kinetic stability) are likely responsible for maintaining the closed autoinhibited state of RfaH, preventing spurious interactions with RNA polymerase (RNAP) when not functional. Our results provide a quantitative understanding of the folding-function relationship in the model fold-switching protein RfaH.

The extent and molecular basis of interdomain communicationinmultidomain proteins, central to understanding allostery and function,is an open question. One simple evolutionary strategy could involvethe selection of either conflicting or favorable electrostatic interactionsacross the interface of two closely spaced domains to tune the magnitudeof interdomain connectivity. Here, we study a bilobed domain FF34from the eukaryotic p190A RhoGAP protein to explore one such designprinciple that mediates interdomain communication. We find that whilethe individual structural units in wild-type FF34 are marginally coupled,they exhibit distinct intrinsic stabilities and low cooperativity,manifesting as slow folding. The FF3-FF4 interface harbors a frustratednetwork of highly conserved electrostatic interactions—a charge troika—that promotes the population of multiple,decoupled, and non-native structural modes on a rugged native landscape.Perturbing this network via a charge-reversal mutation not only enhancesstability and cooperativity but also dampens the fluctuations globallyand speeds up the folding rate by at least an order of magnitude.Our work highlights how a conserved but nonoptimal network of interfacialelectrostatic interactions shapes the native ensemble of a bilobedprotein, a feature that could be exploited in designing molecularsystems with long-range connectivity and enhanced cooperativity.

Secretory preproteins of the Sec pathway are targeted post-translationally and cross cellular membranes through translocases. During cytoplasmic transit, mature domains remain non-folded for translocase recognition/translocation. After translocation and signal peptide cleavage, mature domains fold to native states in the bacterial periplasm or traffic further. We sought the structural basis for delayed mature domain folding and how signal peptides regulate it. We compared how evolution diversified a periplasmic peptidyl-prolyl isomerase PpiA mature domain from its structural cytoplasmic PpiB twin. Global and local hydrogen-deuterium exchange mass spectrometry showed that PpiA is a slower folder. We defined at near-residue resolution hierarchical folding initiated by similar foldons in the twins, at different order and rates. PpiA folding is delayed by less hydrophobic native contacts, frustrated residues and a β-turn in the earliest foldon and by signal peptide-mediated disruption of foldon hierarchy. When selected PpiA residues and/or its signal peptide were grafted onto PpiB, they converted it into a slow folder with enhanced invivo secretion. These structural adaptations in a secretory protein facilitate trafficking.

The DNA G-quadruplex (GQ) displays structural polymorphisms, and interactions between its loops and flanking sequences critically determine which of the diverse GQ conformers is adopted. All-atom molecular dynamics (MD) simulations of GQs are computationally challenging due to slow folding times and force field (ff) artifacts. In an earlier study, a direct folding simulation of the simplest DNA GQ (TBA15) was first reported using a modified version of the AMBER bsc1 ff (bsc1_vdW ff). Despite this successful folding simulation, it was later found that the bsc1_vdW ff is somewhat limited in terms of describing loop structures of GQs, which is problematic because GQ loop regions play key roles in ligand binding to modulate GQ activities. In this study, we further modified the bsc1_vdW ff to enhance the GQ loop prediction by fine-tuning a limited number of van der Waals (vdW) parameters of the standard AMBER bsc1 ff to improve the GQ loop distribution of a target GQ system (three-layered antiparallel GQ; mHtel21). Test simulations of this newly generated ff (bsc1_vdWL ff) on DNA GQs with diverse topologies (hybrid1, hybrid2, and parallel propeller) revealed that loop structures were predicted more accurately than by the bsc1_vdW ff. We consider that enhanced sampling MD simulation methods in combination with bsc1_vdWL provide useful simulation protocols for resolving outstanding issues of DNA GQ folding and GQ/ligand binding at the all-atom level.

Acylphosphatase (AcP) is a small protein with 98 amino acid residues that catalyzes the hydrolysis of carboxyl-phosphate bonds. AcP is a typical two-state protein with slow folding rate due to its relatively large contact order in the native structure. The mechanical properties and unfolding behavior of AcP has been studied by atomic force microscope. Here using stable magnetic tweezers, we measured the force-dependent folding rates within a force range 1-3 pN, and unfolding rates 15-40 pN. The obtained unfolding rates show different force sensitivities at forces below and above ∼27 pN, which determines a free-energy landscape with two energy barriers. Our results indicate that the free-energy landscape of small globule proteins have general Bactrian camel shape, and large contact order of the native state produces a high barrier dominate at low forces.

In comparison to globular proteins, the spontaneous folding and insertion of β-barrel membrane proteins are surprisingly slow, typically occurring on the order of minutes. Using single-molecule Förster resonance energy transfer to report on the folding of fluorescently labeled outer membrane protein G we measured the real-time insertion of a β-barrel membrane protein from an unfolded state. Folding events were rare and fast (<20 ms), occurring immediately upon arrival at the membrane. This combination of infrequent, but rapid, folding resolves this apparent dichotomy between slow ensemble kinetics and the typical timescales of biomolecular folding.