Rapid Folding Research Articles

The transition state for coupled folding and binding of a disordered DNA binding domain resembles the unbound state.

The basic zippers (bZIPs) are one of two large eukaryotic families of transcription factors whose DNA binding domains are disordered in isolation but fold into stable α-helices upon target DNA binding. Here, we systematically disrupt pre-existing helical propensity within the DNA binding region of the homodimeric bZIP domain of cAMP-response element binding protein (CREB) using Ala-Gly scanning and examine the impact on target binding kinetics. We find that the secondary structure of the transition state strongly resembles that of the unbound state. The residue closest to the dimerization domain is largely folded within both unbound and transition states; dimerization apparently propagates additional helical propensity into the basic region. The results are consistent with electrostatically-enhanced DNA binding, followed by rapid folding from the folded zipper outwards. Fly-casting theory suggests that protein disorder can accelerate binding. Interestingly however, we did not observe higher association rate constants for mutants with lower levels of residual structure in the unbound state.

Light- and Solvent-Responsive Bilayer Hydrogel Actuators with Reversible Bending Behaviors.

Light-responsive hydrogel systems have gained significant attention due to their unique ability to undergo controlled and reversible swelling behavior in response to light stimuli. Combining light-responsive hydrogels with nonresponsive polymers offers a unique self-folding feature that can be used in soft robotic actuator designs. However, simple formulation of such systems with rapid response time is still a challenging task. Herein, we demonstrate a simple but versatile bilayer polymeric design combining light-responsive spiropyran-polyacrylamide (SP-PAAm) with polyacrylamide (PAAm) hydrogels. The photochromic spiropyran in our polymer design is a closed-ring, hydrophobic compound and turns into an open-ring, hydrophilic merocyanine isomer under light irradiation. The swelling degree of SP-PAAm and PAAm hydrogels was evaluated using LED lights with different wavelengths and solvent media (e.g., water, ethanol, DMF, and DMSO). We observed that SP-PAAm hydrogels reached a swelling ratio of ∼370% with the illumination of the blue LED in the DMF medium. By combining light-responsive SP-PAAm hydrogels with nonresponsive PAAm, a proof-of-concept demonstration was performed to demonstrate the applicability of our fabricated platforms. Although fabricated one-armed bilayer hydrogels possessed self-folding ability with a folding angle of ∼40° in 30 min, the four-armed bilayer platforms demonstrated more efficient and rapid folding behavior and reached a folding angle of ∼75° in ∼15 min. Given their simplicity and efficiency, we believe that such polymeric designs may offer new avenues for the fields of polymeric actuators and soft robotic systems.

Watching ion-driven kinetics of ribozyme folding and misfolding caused by energetic and topological frustration one molecule at a time.

Folding of ribozymes into well-defined tertiary structures usually requires divalent cations. How Mg2+ ions direct the folding kinetics has been a long-standing unsolved problem because experiments cannot detect the positions and dynamics of ions. To address this problem, we used molecular simulations to dissect the folding kinetics of the Azoarcus ribozyme by monitoring the path each molecule takes to reach the folded state. We quantitatively establish that Mg2+ binding to specific sites, coupled with counter-ion release of monovalent cations, stimulate the formation of secondary and tertiary structures, leading to diverse pathways that include direct rapid folding and trapping in misfolded structures. In some molecules, key tertiary structural elements form when Mg2+ ions bind to specific RNA sites at the earliest stages of the folding, leading to specific collapse and rapid folding. In others, the formation of non-native base pairs, whose rearrangement is needed to reach the folded state, is the rate-limiting step. Escape from energetic traps, driven by thermal fluctuations, occurs readily. In contrast, the transition to the native state from long-lived topologically trapped native-like metastable states is extremely slow. Specific collapse and formation of energetically or topologically frustrated states occur early in the assembly process.

A study of a protein folding machine: transient rotation of the polypeptide backbone facilitates rapid folding of protein domains in all-atom molecular dynamics simulations

A study of a protein folding machine: transient rotation of the polypeptide backbone facilitates rapid folding of protein domains in all-atom molecular dynamics simulations

Small RNAs and Hfq capture unfolded RNA target sites during transcription

Small ribonucleoproteins (sRNPs) target nascent precursor RNAs to guide folding, modification, and splicing during transcription. Yet, rapid co-transcriptional folding of the RNA can mask sRNP sites, impeding target recognition and regulation. To examine how sRNPs target nascent RNAs, we monitored binding of bacterial Hfq⋅DsrA sRNPs to rpoS transcripts using single-molecule co-localization co-transcriptional assembly (smCoCoA). We show that Hfq⋅DsrA recursively samples the mRNA before transcription of the target site to poise it for base pairing with DsrA. We adapted smCoCoA to precisely measure when the target site is synthesized and revealed that Hfq⋅DsrA often binds the mRNA during target site synthesis close to RNA polymerase (RNAP). We suggest that targeting transcripts near RNAP allows an sRNP to capture a site before the transcript folds, providing a kinetic advantage over post-transcriptional targeting. We propose that other sRNPs may also use RNAP-proximal targeting to hasten recognition and regulation.

Configurational entropy, transition rates, and optimal interactions for rapid folding in coarse-grained model proteins.

Under certain conditions, the dynamics of coarse-grained models of solvated proteins can be described using a Markov state model, which tracks the evolution of populations of configurations. The transition rates among states that appear in the Markov model can be determined by computing the relative entropy of states and their mean first passage times. In this paper, we present an adaptive method to evaluate the configurational entropy and the mean first passage times for linear chain models with discontinuous potentials. The approach is based on event-driven dynamical sampling in a massively parallel architecture. Using the fact that the transition rate matrix can be calculated for any choice of interaction energies at any temperature, it is demonstrated how each state's energy can be chosen such that the average time to transition between any two states is minimized. The methods are used to analyze the optimization of the folding process of two protein systems: the crambin protein and a model with frustration and misfolding. It is shown that the folding pathways for both systems are comprised of two regimes: first, the rapid establishment of local bonds, followed by the subsequent formation of more distant contacts. The state energies that lead to the most rapid folding encourage multiple pathways, and they either penalize folding pathways through kinetic traps by raising the energies of trapping states or establish an escape route from the trapping states by lowering free energy barriers to other states that rapidly reach the native state.

Covalent Capture of a Collagen Mimetic Peptide with an Integrin-Binding Motif.

Collagen mimetic peptides (CMPs) are an excellent model to study the structural and biological properties of the extracellular matrix (ECM) due to ease of synthesis and variability in sequence. To ensure that synthetic materials accurately mimic the structure and function of natural collagen in the ECM, it is necessary to conserve the triple helix. However, CMP folding is subject to equilibrium, and frequently peptides exist in solution as both monomer and triple helix. Additionally, the stability of CMPs is highly dependent on peptide length and amino acid composition, leading to suboptimal performance. Here, we report the utility of covalent capture, a method to (a) direct the folding of a supramolecular triple helix and (b) form isopeptide bonds between the helix strands, in the design of an integrin-binding peptide with a GFOGER motif. Covalent capture effectively locked the triple helix and yielded a peptide with high thermal stability and a rapid folding rate. Compared to supramolecular triple helices bearing the same GFOGER-binding site, cell adhesion was substantially increased. In vitro assays using EDTA/Mg2+ and an anti-α2β1 antibody demonstrated the preservation of the high specificity of the binding event. This covalently captured integrin-binding peptide provides a template for the future design of bioactive ECM mimics, which can overcome limitations of supramolecular approaches for potential drug and biomaterial designs.

Connecting conformational stiffness of the protein with energy landscape by a single experiment.

The structure-function dynamics of a protein as a flexible polymer is essential to describe its biological functions. Here, using single-molecule magnetic tweezers, we have studied the effect of ionic strength on the folding mechanics of protein L, and probed its folding-associated physical properties by re-measuring the same protein in a range of ammonium sulfate concentrations from 150 mM to 650 mM. We observed an electrolyte-dependent conformational dynamics and folding landscape of the protein in a single experiment. Salt increases the refolding kinetics, while decreasing the unfolding kinetics under force, which in turn modifies the barrier heights towards the folded state. Additionally, salt enhances the molecular compaction by decreasing the Kuhn length of the protein polymer from 1.18 nm to 0.58 nm, which we have explained by modifying the freely jointed chain model. Finally, we correlated polymer chain physics to the folding dynamics, and thus provided an analytical framework for understanding compaction-induced folding mechanics across a range of ionic strengths from a single experiment.

Rapid detection of miRNA via development of consecutive adenines (polyA)-based electrochemical biosensors

Herein, we report rapid electrochemical detection of miRNA let-7a based on a DNA probe consisting of a polyA and Fc-co-labeled harpin structure (the polyA-H probe). The polyA-H probe could be facilely immobilized on Au surfaces through the interactions between polyA and Au, followed by its pre-hybridization with a single strand (S1). The probe's surface density could be optimized for minimizing steric hindrance via changing the polyA block length. The target let-7a could be rapidly amplified via loop-mediated isothermal amplification (LAMP) with four simplified primers, followed by inducing the formation of dimeric i-motif (DIM) structure via H+-induced rapid folding of two C-rich sequences of motif strand 1 and strand 2. It was found that, after introducing the as-formed DIM to hybridize the S1, the immobilized polyA20-H probe could rapidly revert to its hairpin structure, sending out a turn-on electrochemical signal of the Fc. The total time for detecting the let-7a was around 80 min, obviously less than that of most of electrochemical DNA sensors reported previously. The biosensor showed a linear relationship of the current response to the let-7a in the range of 10 fM to 50 nM with a limit of detection (LOD) of 5.1 fM. Our biosensors were further tested using human serum spiked with the let-7a and the extracts of the breast adenocarcinoma cells spiked with and without the let-7a, respectively. Satisfied results were obtained. This study shows a potential promising future of development of electrochemical biosensors for rapid detection of miRNAs in the application of clinical practice.

NanoRobotic Structures with Embedded Actuation via Ion Induced Folding.

4D structures are tridimensional structures with time-varying abilities that provide high versatility, sophisticated designs, and a broad spectrum of actuation and sensing possibilities. The downsizing of these structures below 100 μm opens up exceptional opportunities for many disciplines, including photonics, acoustics, medicine, and nanorobotics. However, it requires a paradigm shift in manufacturing methods, especially for dynamic structures. A novel fabrication method based on ion-induced folding of planar multilayer structures embedding their actuation is proposed-the planar structures are fabricated in bulk through batch microfabrication techniques. Programmable and accurate bidirectional foldings (-70°-+90°) of Silica/Chromium/Aluminium (SiO2 /Cr/Al) multilayer structures are modeled, experimentally demonstrated then applied to embedded electrothermal actuation of controllable and dynamic 4D nanorobotic structures. The method is used to produce high-performances case-study grippers for nanorobotic applications in confined environments. Once folded, a gripping task at the nano-scale is demonstrated. The proposed fabrication method is suitable for creating small-scale 4D systems for nanorobotics, medical devices, and tunable metamaterials, where rapid folding and enhanced dynamic control are required.

Insights into the nucleation and crystallization analysis of PHB-rubber toughened PLA biocomposites

An insightful understanding of the isothermal cold crystallization, melting behavior, and kinetics of the novel PHB-rubber copolymer (PHB-di-rub)-reinforced polylactide (PLA) were determined. Incorporating 10 wt% PHB-di-rub significantly reduces the crystallization half-times (t1/2) by 32-fold, lowers the kinetic parameter (Kg) and folding surface energy (σe), which show the rapid polymer chains folding and the super-nucleating capability due to simultaneous instigated nucleation and plasticization. Concurrently, the shear-thinning behavior verified the plasticization effect and the convergence of diffraction peak show development of primary α′-crystalline form that contributes to the PLAs' ultra-extensibility. This evocative finding could comprehend a valuable framework for the future development of high-performance PLA/PHB-based copolymer biocomposites with total green physiognomies.

Efficient integration of transmembrane domains depends on the folding properties of the upstream sequences

The topology of most membrane proteins is defined by the successive integration of α-helical transmembrane domains at the Sec61 translocon. The translocon provides a pore for the transfer of polypeptide segments across the membrane while giving them lateral access to the lipid. For each polypeptide segment of ∼20 residues, the combined hydrophobicities of its constituent amino acids were previously shown to define the extent of membrane integration. Here, we discovered that different sequences preceding a potential transmembrane domain substantially affect its hydrophobicity requirement for integration. Rapidly folding domains, sequences that are intrinsically disordered or very short or capable of binding chaperones with high affinity, allow for efficient transmembrane integration with low-hydrophobicity thresholds for both orientations in the membrane. In contrast, long protein fragments, folding-deficient mutant domains, and artificial sequences not binding chaperones interfered with membrane integration, requiring higher hydrophobicity. We propose that the latter sequences, as they compact on their hydrophobic residues, partially folded but unable to reach a native state, expose hydrophobic surfaces that compete with the translocon for the emerging transmembrane segment, reducing integration efficiency. The results suggest that rapid folding or strong chaperone binding is required for efficient transmembrane integration.

Molecular chaperones facilitate soluble expression of recombinant non-toxic mutant CRM197 of diphtheria toxin in Escherichia coli

Diphtheria toxin is an ADP-ribosyltransferase toxic to human cells. Mutation of the active site in its catalytic domain eliminates the toxicity, but retains its immunogenicity. A non-toxic mutant of diphtheria toxin known as CRM197 protein has become an ideal carrier protein for conjugate vaccines. CRM197 can further improve its immunogenicity by cross-linking with other antigens, so it has good potential to find broad applications. Unfortunately, inclusion bodies are easily formed during the expression of recombinant CRM197 protein in Escherichia coli, which greatly reduces its yield. In order to address this problem, pG-KJE8 vector carrying molecular chaperones and plasmid pET28a-CRM197, were co-expressed in Escherichia coli. The results showed that the recombinant CRM197 protein was successfully expressed and appeared largely in inclusion bodies. The molecular chaperones DnaK, DnaJ, GrpE, GroES and GroEL5 expressed can facilitate correct and rapid folding of CRM197. Furthermore, it can also improve the recovery rate of soluble CRM197 protein. The soluble expression of CRM197 was maximized upon addition of 1.0 mmol/L IPTG, 0.5 mg L-arabinose, 5.0 ng/mL tetracycline and induction at 20oC for 16 h. The soluble CRM197 protein shows good immunoreactivity, demonstrating the molecular chaperones expressed from pG-KJE8 facilitated the soluble expression of CRM197 protein in E. coli.

Reversible Supra-Folding of User-Programmed Functional DNA Nanostructures on Fuzzy Cationic Substrates.

We report that user-defined DNA nanostructures, such as two-dimensional (2D) origamis and nanogrids, undergo a rapid higher-order folding transition, referred to as supra-folding, into three-dimensional (3D) compact structures (origamis) or well-defined μm-long ribbons (nanogrids), when they adsorb on a soft cationic substrate prepared by layer-by-layer deposition of polyelectrolytes. Once supra-folded, origamis can be switched back on the surface into their 2D original shape through addition of heparin, a highly charged anionic polyelectrolyte known as an efficient competitor of DNA-polyelectrolyte complexation. Orthogonal to DNA base-pairing principles, this reversible structural reconfiguration is also versatile; we show in particular that 1) it is compatible with various origami shapes, 2) it perfectly preserves fine structural details as well as site-specific functionality, and 3) it can be applied to dynamically address the spatial distribution of origami-tethered proteins.

Experimental Performance Research of Vertical Axis Wind Turbine with Expandable Airfoil

In order to improve the wind energy utilization of the vertical axis wind turbine (VAWT), a new type of VAWT with expandable NACA0012 airfoil is proposed in this paper. The airfoil has opening and folding state. The airfoil is open in windward which is similar with curved blade in Savonius tubine. And it is folded in leeward which is similar with airfoil in H-type turbine. In this paper, a wind tunnel test system is designed to measure the aerodynamic performance of the new turbine and three main structural parameters of the turbine are analyzed. The structural parameters contain the maximum unilateral open angle which influence the flatten of the blade and the sweep wind area. The revolution angle of the rotor corresponding to the self-rotation open angle which influence the effective working time and the airfoil self-rotation effect on the turbine rotation. And the revolution angle of the rotor corresponding to the self-rotation folded angle of the airfoil which influence the wind resistance and impact on the turbine in rapid folding process. The results show that the maximum wind energy utilization of new turbine with appropriate airfoil structural parameters and tip speed ratio corresponding maximum wind energy utilization is higher than Savonius turbine which means the performance of the new turbine capturing wind power is better than Savonius turbine and can be more suitably used in areas with relatively barren wind resource.

Helix-Coil Transition at a Glycine Following a Nascent α-Helix: A Synergetic Guidance Mechanism for Helix Growth.

A detailed understanding of forces guiding the rapid folding of a polypeptide from an apparently random coil state to an ordered α-helical structure following the rate-limiting preorganization of the initial three residue backbones into helical conformation is imperative to comprehending and regulating protein folding and for the rational design of biological mimetics. However, several details of this process are still unknown. First, although the helix-coil transition was proposed to originate at the residue level (J. Chem. Phys. 1959, 31, 526-535; J. Chem. Phys. 1961, 34, 1963-1974), all helix-folding studies have only established it between time-averaged bulk states of a long-lived helix and several transiently populated random coils, along the whole helix model sequence. Second, the predominant thermodynamic forces driving either this two-state transition or the faster helix growth following helix nucleation are still unclear. Third, the conformational space of the random coil state is not well-defined unlike its corresponding α-helix. Here we investigate the restrictions placed on the conformational space of a Gly residue backbone, as a result of it immediately succeeding a nascent α-helical turn. Analyses of the temperature-dependent 1D-, 2D-NMR, FT-IR, and CD spectra and GROMACS MD simulation trajectory of a Gly residue backbone following a model α-helical turn, which is artificially rigidified by a covalent hydrogen bond surrogate, reveal that: (i) the α-helical turn guides the ϕ torsion of the Gly exclusively into either a predominantly populated entropically favored α-helical (α-ϕ) state or a scarcely populated random coil (RC-ϕ) state; (ii) the α-ϕ state of Gly in turn favors the stability of the preceding α-helical turn, while the RC-ϕ state disrupts it, revealing an entropy-driven synergetic guidance for helix growth in the residue following helix nucleation. The applicability of a current synergetic guidance mechanism to explain rapid helix growth in folded and unfolded states of proteins and helical peptides is discussed.

Dynamic folding of origami by exploiting asymmetric bi-stability

In this study, we examine a rapid and reversible origami folding method by exploiting a combination of resonance excitation, asymmetric bi-stability, and active control. The underlying idea is that, by harmonically exciting a bi-stable origami at its resonance frequencies, one can induce rapid folding between its different stable equilibria with a much smaller actuation magnitude than static folding. To this end, we use a bi-stable water-bomb base as an archetypal example to uncover the underlying principles of dynamic folding based on numerical simulation and experimental testing. If the water-bomb initially settles at its “weak” stable state, one can use a base excitation to induce the intra-well resonance. As a result, the origami would fold and remain at the other “strong” stable state even if the excitation does not stop. The origami dynamics starting from the strong state, on the other hand, is more complicated. The water-bomb origami is prone to show inter-well oscillation rather than a uni-directional switch due to a nonlinear relationship between the dynamic folding behavior, asymmetric potential energy barrier, the difference in resonance frequencies, and excitation amplitude. Therefore, we develop an active feedback control strategy, which cuts off the base excitation input at the critical moment to achieve robust and uni-directional folding from the strong stable state to the weak one. The results of this study can apply to many different kinds of origami and create a new approach for rapid and reversible (self-)folding, thus advancing the application of origami in shape morphing systems, adaptive structures, and reconfigurable robotics.

Elastically and Plastically Foldable Electrothermal Micro‐Origami for Controllable and Rapid Shape Morphing

AbstractIntegrating origami principles within traditional microfabrication methods can produce shape morphing microscale metamaterials and 3D systems with complex geometries and programmable mechanical properties. However, available micro‐origami systems usually have slow folding speeds, provide few active degrees of freedom, rely on environmental stimuli for actuation, and allow for either elastic or plastic folding but not both. This work introduces an integrated fabrication–design–actuation methodology of an electrothermal micro‐origami system that addresses the above‐mentioned challenges. Controllable and localized Joule heating from electrothermal actuator arrays enables rapid, large‐angle, and reversible elastic folding, while overheating can achieve plastic folding to reprogram the static 3D geometry. Because the proposed micro‐origami do not rely on an environmental stimulus for actuation, they can function in different atmospheric environments and perform controllable multi‐degrees‐of‐freedom shape morphing, allowing them to achieve complex motions and advanced functions. Combining the elastic and plastic folding enables these micro‐origami to first fold plastically into a desired geometry and then fold elastically to perform a function or for enhanced shape morphing. The proposed origami systems are suitable for creating medical devices, metamaterials, and microrobots, where rapid folding and enhanced control are desired.

Действие прямоугольных электрических импульсов низкой частоты на трихоплакса (тип Placozoa)

Влияние низкочастотного электромагнитного излучения (НЭМИ) на растения и животных, включая человека, достаточно спорно. Мало известно и о воздействии НЭМИ на гидробионтов. Мы изучили действие прямоугольных импульсов напряжения различной амплитуды, длительности и скважности, пропущенных через морскую воду, на трихоплакса (тип Placozoa) как на возможную тестовую лабораторную модель. В опытах использовали три штамма Placozoa, Trichoplax adhaerens (H1), Trichoplax sp. (H2) и Hoilungia hongkongensis (H13), отобранных на стационарной стадии роста культуры. Для генерации последовательности прямоугольных импульсов заданной длительности и скважности с частотой до 2 кГц применяли аппаратную платформу Arduino Uno. Среднее значение напряжения до 500 мВ регулировали с помощью схемы делителя напряжения. Для доказательства специфичности действия электрических импульсов на потенциалзависимые кальциевые каналы трихоплакса использовали ингибитор активности кальциевых каналов амлодипин. Животных стимулировали электрическим током под стереомикроскопом. Электроды располагали в непосредственной близости от животного. Исследовали сопутствующие изменения поведения и морфологии пластинки трихоплакса. Выделяли стимулирующие и подавляющие воздействия. Наблюдения документировали с помощью фото- и видеосъёмки. Отслеживали траектории движения отдельных особей. Увеличение напряжения на электродах при фиксированной частоте 20 Гц приводило к тому, что животные штамма H2 покидали «зону электродов» в течение нескольких минут при 25 мВ, однако теряли подвижность пропорционально росту напряжения и обездвиживались при 500 мВ. Именно поэтому в дальнейших опытах применяли напряжение 50 мВ. В экспериментах с двумя электродами, находящимися с одной стороны трихоплакса, у животного было больше возможностей перемещаться в разных направлениях, чем в случае расположения электродов по обеим сторонам пластинки. Направление движения использовали как характеристический признак. Отмечено, что трихоплаксы мигрируют в области с низкой плотностью линий электрического поля, которые расположены вдали или за электродами. Животные из старой культуры отличались меньшей чувствительностью к электрическому раздражителю. Штамм H2 был наиболее чувствительным и демонстрировал более выраженные физиологические реакции на частотах 2 Гц и 2 кГц с напряжением 50 мВ, чем штамм H1 и особенно штамм H13. В зависимости от длительности стимулирующих прямоугольных импульсов, их числа, амплитуды и варьирующей частоты менялись двигательные реакции и морфология животных: от направленной или стохастической миграции в сторону анода/катода или от него до обездвиживания животных, увеличения оптической плотности по периферии и в центре пластинки и до сворачивания трихоплакса и отделения его от субстрата. В дополнительных опытах на Trichoplax sp. H2 показано, что при длительности импульсов 35 мс и задержке импульсов от 1 мс до 10 с доля обездвиженных животных увеличивается до 80 % при минимальной задержке. Тем не менее в случае применения амлодипина в концентрации 25 нМ практически все трихоплаксы в течение нескольких минут сохраняли подвижность несмотря на обработку электрическими импульсами. Между тем при использовании амлодипина в концентрации 250 нМ животные двигались дискоординированно и не могли покинуть «электродную ловушку». Далее пластинка трихоплакса становилась ригидной, что выражалось в неизменности формы животного при движении. Наконец, амлодипин в концентрации 50 мкМ вызывал быстрое сворачивание краёв трихоплакса в розетку в вентрально-дорсальном направлении и последующую диссоциацию пластинки на отдельные клетки. В целом применяемое электрическое воздействие имело кумулятивный, но обратимый эффект, который, как предполагается, может быть связан с работой потенциалзависимых кальциевых каналов. Амлодипин в большой концентрации (50 мкМ) вызывал разрушение трихоплакса, в умеренной (250 нМ) он нарушал, вероятно, распространение волн активации, что приводило к дискоординации движений животного, а в малой (25 нМ) предотвращал электрошок.

Molecular origins of folding rate differences in the thioredoxin family.

Thioredoxins are a family of conserved oxidoreductases responsible for maintaining redox balance within cells. They have also served as excellent model systems for protein design and engineering studies particularly through ancestral sequence reconstruction methods. The recent work by Gamiz-Arco et al. [Biochem J (2019) 476, 3631-3647] answers fundamental questions on how specific sequence differences can contribute to differences in folding rates between modern and ancient thioredoxins but also among a selected subset of modern thioredoxins. They surprisingly find that rapid unassisted folding, a feature of ancient thioredoxins, is not conserved in the modern descendants suggestive of co-evolution of better folding machinery that likely enabled the accumulation of mutations that slow-down folding. The work thus provides an interesting take on the expected folding-stability-function constraint while arguing for additional factors that contribute to sequence evolution and hence impact folding efficiency.